Reduced risk tobacco products and methods of making same

The present application is a continuation of U.S. application Ser. No. 11/913,870, filed Mar. 25, 2011, which is 371 entry into the U.S. of Paris Convention Application No. PCT/US06/18065, filed May 10, 2006, which claims the benefit of priority to U.S. Provisional Application Ser. No. 60/680,283, filed May 11, 2005. Each of the above-mentioned priority documents is incorporated by reference in its entirety into the present application.

The invention relates to reduced risk tobacco and tobacco products and methods for detecting, identifying and evaluating such tobacco and tobacco products to determine the potential that these compositions have to contribute to a tobacco-related disease.

The leading preventable cause of death and disability in the United States is the chronic use of tobacco products, in particular, cigarettes. In addition to lung cancers, tobacco use plays important direct and indirect roles in the etiology of a wide range of other cancers, including those of the upper aerodigestive tract (i.e., oral cavity, pharynx, larynx, and esophagus), bladder, stomach, kidney, pancreas, uterine cervix, and blood (myeloid leukemia). Exposure to tobacco carcinogens and toxins is also a major cause of other diseases of the pulmonary system (e.g., bronchitis, emphysema, and chronic obstructive pulmonary disease), the cardiovascular system (e.g., stroke, atherosclerosis, and myocardial infarction), and the female reproductive system (e.g., increased risk of miscarriage, premature delivery, low birth weight, and stillbirth). While numerous studies have elucidated some of the biological effects of cigarette smoke that result in its ability to induce this range of pathologies in smokers, little is known about the nature and temporal association of molecular events that drive specific stages in the multi-step processes that result in clinically evident disease. This is due to the fact that cigarette smoke is a complex chemical mixture of gases and suspended particulate material that consists of a wide variety of condensed organic compounds (i.e., ‘tar’) that collectively contain a large number of toxins, carcinogens, co-carcinogens, mutagens, and reactive organic and inorganic molecules. Thus, there is a pressing need to decrease the health risk caused by tobacco products.

Embodiments described herein generally relate to tobacco and/or tobacco products having a reduced amount of a harmful compound, and methods of developing, screening and using such tobacco and tobacco products. For example, several approaches are provided to reduce the amount of one or more harmful compounds in tobacco by, for example, modifying the expression of a gene that is involved in the production of a harmful compound in tobacco. Also provided are methods of determining whether the removal of a harmful compound yields a tobacco and/or a tobacco product that has a reduced potential to contribute to a tobacco-related disease. Also provided are reduced-risk tobacco and tobacco products made in accordance with the methods provided herein. Also provided are methods of using the reduced-risk tobacco and tobacco products made in accordance with the methods provided herein.

As described in more detail below, provided herein are nucleic acid molecules and nucleic acid constructs that contain sequences that can be used to inhibit expression of a gene involved in the biosynthesis of a compound associated with a tobacco-related disease. Also provided herein are modified tobaccos and modified tobacco products that have been modified by composition and/or configuration in order to deliver to the user a reduced amount of a compound associated with a tobacco-related disease. Exemplary modified tobaccos are tobaccos that have been genetically modified to contain a reduced amount of a compound associated with a tobacco-related disease. Exemplary genetically modified tobaccos are those containing the nucleic acid molecules or constructs provided herein. Exemplary modified tobacco products are those containing modified tobacco or, those containing a modified filter, where the modification results in delivery to the user of a reduced amount of a compound associated with a tobacco-related disease.

Also provided herein are methods of analyzing tobacco products such as the modified tobacco and modified tobacco products described herein, so as to determine whether the tobacco product is a reduced risk product (e.g., a product that has a reduced propensity to modulate cellular homeostasis, or a reduced level of induction of a cellular marker for a tobacco-related disease). Some of these methods can be practiced, for example, by identifying a compound that is related to a tobacco-related disease (e.g., nicotine or a sterol), removing the compound or a precursor for the compound by modification to the tobacco or tobacco product, and analyzing the ability of the modified tobacco or modified tobacco product to contribute to a tobacco related disease by monitoring the impact of the modified tobacco or modified tobacco product on a marker for cellular homeostasis. In one example, a cellular marker for a tobacco related disease is monitored. In another example, the transcriptome and/or proteome of the cell is monitored. These methods can be used for both in vitro and in vivo testing. That is, the same cellular markers that have been identified in the in vitro studies can be analyzed in smokers that consume reduced risk cigarettes developed according to the methods above and this data can be compared to the impact on the same cellular markers in smokers that consume conventional cigarettes. By these approaches, a cigarette that minimizes the disruptions of the cellular environment of a smoker can be obtained.

Further provided herein are kits that contain the modified tobacco or modified tobacco products provided herein, and smoking cessation programs, which utilize the modified tobacco or modified tobacco products provided herein.

Provided herein are methods of making a tobacco product with a reduced potential to contribute to a tobacco related disease by providing a genetically modified tobacco configured to deliver a reduced amount of a compound that contributes to a tobacco related disease, as compared to a reference tobacco or a conventional tobacco, contacting a mammalian cell with smoke, or a smoke condensate obtained from said genetically modified tobacco, identifying a modulation of homeostasis of said cell, as compared to a control cell, which has been contacted with smoke, or a smoke condensate obtained from said reference tobacco or said conventional tobacco, wherein a decreased modulation of homeostasis in said cell compared to modulation of homeostasis in said control cell indicates a reduction in the potential to contribute to a tobacco related disease, and incorporating said identified genetically modified tobacco into a tobacco product. In some such methods, modulation of homeostasis in the cell is identified by determining the presence, absence or level of a molecular marker in the cell. In some such methods, the mammalian cell is a lung cell or a cell of the oral cavity. In some such methods, the genetically modified tobacco is identified as producing a reduced amount of a compound that contributes to a tobacco related disease, as compared to a conventional tobacco product of the same class or a reference tobacco product of the same class. In some such methods, the genetically modified tobacco is incorporated into a tobacco product that contains a filter, which retains an increased amount of a compound that contributes to a tobacco related disease, as compared to a reference filter or a conventional filter. In some such methods, the genetically modified tobacco comprises a heterologous nucleic acid that inhibits expression of an enzyme in the nicotine biosynthetic pathway. In some such methods, the heterologous nucleic acid inhibits expression of at least two enzymes in the nicotine biosynthetic pathway. In some such methods, the genetically modified tobacco comprises a heterologous nucleic acid that inhibits expression of an enzyme in the sterol biosynthetic pathway. In some such methods, the heterologous nucleic acid inhibits expression of at least two enzymes in the sterol biosynthetic pathway. In some such methods, the genetically modified tobacco comprises a heterologous nucleic acid that inhibits expression of an enzyme in the nicotine biosynthetic pathway and an enzyme in the sterol biosynthetic pathway. In some such methods, the genetically modified tobacco has a reduced amount of nornicotine and a conventional amount of nicotine. In some such methods, genetically modified tobacco comprises a nucleic acid construct selected from the group consisting of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50. Also provided herein are tobacco products made by the method provided herein.

Also provided herein are tobacco products comprising a genetically modified tobacco that comprises a reduced amount of nicotine as compared to a conventional tobacco product of the same class or a reference tobacco product of the same class and a heterologous nucleic acid that inhibits expression of at least two enzymes involved in nicotine biosynthesis. Also provided herein are tobacco products comprising a genetically modified tobacco that comprises a reduced amount of a sterol as compared to a conventional tobacco product of the same class or a reference tobacco product of the same class and a heterologous nucleic acid that inhibits expression of an enzyme involved in sterol biosynthesis. In some such tobacco products, the genetically modified tobacco comprises a nucleic acid construct as described herein. In some such tobacco products, the genetically modified tobacco comprises a nucleic acid construct selected from the group consisting of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50.

In the methods and tobacco products provided herein, the genetically modified tobacco comprises a reduced activity of a gene selected from the group consisting of arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT), S-adenosyl-methionine synthetase (SAMS), or A622 or comprises an inhibition of a gene that regulates the production of sterol biosynthesis include HMG-CoA reductase, 14alpha demethylase, squalene synthase, SMT2, SMT1, C14 sterol reductase, A8-A7-isomerase, and C4-demethylase. In the methods and tobacco products provided herein, the genetically modified tobacco has reduced production of a compound that contributes to a tobacco related disease which is stable over at least 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 40 or 50 generations. In the methods and tobacco products provided herein, the genetically modified tobacco has agronomic characteristics suitable for commercial production. In the methods and tobacco products provided herein, the agronomic characteristics are phenotypically different from conventional tobacco, and said agronomic characteristics can be compensated for by conventional agronomic methods. In the methods and tobacco products provided herein, the conventional agronomic methods are selected from the group consisting of irrigation, administration of fertilizer, and administration of nutrients.

Also provided herein are genetically modified tobaccos that produce a reduced amount of a compound that contributes to a tobacco related disease, as compared to a conventional tobacco product of the same class or a reference tobacco product of the same class, comprising a heterologous nucleic acid that inhibits expression of an enzyme in the biosynthetic pathway of a compound that contributes to a tobacco related disease. Also provided herein are reduced risk tobacco products comprising a genetically modified tobacco that produces a reduced amount of a compound that contributes to a tobacco related disease, as compared to a conventional tobacco product of the same class or a reference tobacco product of the same class. In some such tobaccos or tobacco products, the modified tobacco comprises a nucleic acid construct as described herein. In some such tobaccos or tobacco products, the modified tobacco comprises a heterologous nucleic acid that inhibits expression of at least two enzymes in the nicotine biosynthetic pathway. In some such tobaccos or tobacco products, the modified tobacco comprises a heterologous nucleic acid that inhibits expression of at least two enzymes in the sterol biosynthetic pathway.

Also provided herein are methods of making a reduced risk tobacco product by providing a modified tobacco or modified tobacco product configured to deliver to a user a reduced amount of a compound that contributes to a tobacco related disease, as compared to a reference tobacco or tobacco product or a conventional tobacco or tobacco product, contacting smoke or smoke condensate obtained from said modified tobacco or modified tobacco product with a cell, identifying a modulation of homeostasis of said cell, as compared to a control cell, which has been contacted with smoke or a smoke condensate obtained from said reference tobacco or tobacco product or said conventional tobacco or tobacco product, wherein a decreased modulation of homeostasis in said cell compared to modulation of homeostasis in said control cell indicates a reduction in the potential to contribute to a tobacco related disease, and incorporating said modified tobacco or modified tobacco product into said reduced risk tobacco product. In some such methods, modulation of homeostasis in the cell is identified by determining the presence, absence or level of a molecular marker in the cell. In some such methods, the modified tobacco is genetically modified tobacco. In some such methods, the genetically modified tobacco is modified according to the methods provided herein.

Also provided are reduced risk tobaccos as substantially described herein. Also provided are reduced risk tobacco products as substantially described herein. Also provided are uses of the tobaccos or tobacco products provided herein.

Also provided are isolated nulcleic acids substantially as described herein. Also provided are isolated inhibition cassettes substantially as described herein.

Also provided is a genetically modified tobacco having a reduced amount of nicotine as compared to conventional tobacco, further comprising a heterologous nucleic acid that encodes a gene that produces a composition selected from the group consisting of a medicinal compound, industrial oil, or dietary supplement, wherein said composition is substantially not present in conventional or wild-type tobacco. In some such tobaccos, the medicinal compound is an antibody or fragment thereof or an immunogenic preparation. In some such tobaccos, the medicinal compound is a vaccine preparation. In some such tobaccos, the medicinal compound is a veterinary product.

Also provided are genetically modified tobaccos that produce a reduced amount of a compound that contributes to a tobacco related disease, as compared to a conventional tobacco product of the same class or a reference tobacco product of the same class, comprising a heterologous nucleic acid that inhibits expression of an enzyme in the biosynthetic pathway of a compound that contributes to a tobacco related disease. Also provided are reduced risk tobacco products comprising a genetically modified tobacco that produces a reduced amount of a compound that contributes to a tobacco related disease, as compared to a conventional tobacco product of the same class or a reference tobacco product of the same class. In some such tobaccos or tobacco products, the compound is nicotine. In some such tobaccos or tobacco products, the compound is a sterol. In some such tobaccos or tobacco products, the compound is a TSNA. In some such tobaccos or tobacco products, the compound is a PAH. In some such tobaccos or tobacco products, the compound is nornicotine. In some such tobaccos or tobacco products, the genetically modified tobacco has a reduced amount of nornicotine and a conventional amount of nicotine. In some such tobaccos or tobacco products, the genetically modified tobacco comprises a nucleic acid construct as described herein. In some such tobaccos or tobacco products, the genetically modified tobacco comprises a nucleic acid construct selected from the group consisting of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50. In some such tobaccos or tobacco products, expression of two or more genes in the biosynthetic pathway of said compound is inhibited. In some such tobaccos or tobacco products, the genetically modified tobacco comprises two or more nucleic acid constructs as described herein. In some such tobaccos or tobacco products, the genetically modified tobacco comprises two or more nucleic acid constructs selected from the group consisting of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50. Some such tobaccos or tobacco products comprise reduced activity of a gene selected from the group consisting of arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT), S-adenosyl-methionine synthetase (SAMS), or A622 or comprises an inhibition of a gene that regulates the production of sterol biosynthesis include HMG-CoA reductase, 14alpha demethylase, squalene synthase, SMT2, SMT1, C14 sterol reductase, A8-A7-isomerase, and C4-demethylase. Some such tobaccos or tobacco products comprise a genetically modified tobacco for which reduced production of a compound that contributes to a tobacco related disease is stable over at least 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 40 or 50 generations. Some such tobaccos or tobacco products comprise a genetically modified tobacco having agronomic characteristics suitable for commercial production.

Also provided herein are methods of making a reduced risk tobacco product by providing a modified tobacco or modified tobacco product configured to deliver to a user a reduced amount of a compound that contributes to a tobacco related disease, as compared to a reference tobacco or tobacco product or a conventional tobacco or tobacco product, contacting smoke or smoke condensate obtained from said modified tobacco or modified tobacco product with a cell identifying a modulation of homeostasis of said cell, as compared to a control cell, which has been contacted with smoke or a smoke condensate obtained from said reference tobacco or tobacco product or said conventional tobacco or tobacco product, wherein a decreased modulation of homeostasis in said cell compared to modulation of homeostasis in said control cell indicates a reduction in the potential to contribute to a tobacco related disease, and incorporating said modified tobacco or modified tobacco product into said reduced risk tobacco product. In some such methods, modulation of homeostasis in the cell is identified by determining the presence, absence or level of a molecular marker in the cell. In some such methods, the modified tobacco is genetically modified tobacco. In some such methods, the genetically modified tobacco is modified according to any of methods provided herein. In some such methods, the genetically modified tobacco is identified as producing a reduced amount of a compound that contributes to a tobacco related disease, as compared to a conventional tobacco product of the same class or a reference tobacco product of the same class. In some such methods, the modified tobacco product contains a filter that retains an increased amount of a compound that contributes to a tobacco related disease, as compared to a reference filter or a conventional filter. Also provided herein are reduced risk tobacco products made by any of the methods provided herien. Also provided herein are methods of using a reduced risk tobacco product of any of the methods provided herein to reduce the potential of an individual that smokes to acquire a tobacco related disease comprising identifying an individual in need of a reduced risk tobacco product and providing the individual the tobacco product of the methods provided herein.

Also provided herein are plant cells resistant to norflurazone comprising providing said cell the nucleic acid of SEQ ID No 10, 11, or 12; and also provided herein are method of making the same.

Also provided herein are crops of plants comprising the nucleic acid of SEQ ID No 10, 11, or 12. Also provided herein are methods of cultivation of a crop of plants comprising obtaining plants with the nucleic acid of SEQ ID No 10, 11, or 12, cultivating said plants, and contacting said plants with norflurazone.

Also provided herein are methods of selecting positively transformed plant cells comprising providing the nucleic acid of SEQ ID No 10, 11, or 12 to said plant cells and contacting said plant cells with norflurazone, whereby the cells that survive contact with norflurazone are positively transformed plant cells.

Also provided herein are isolated nulcleic acids substantially as described herein. Also provided herein are isolated inhibition cassettes substantially as described herein. Also provided herein are isolated selection cassettes substantially described herein, wherein said selction cassette comprises the sequence of SEQ ID No 10, 11, or 12. Also provided herein are reduced risk tobaccos substantially described herein. Also provided herein are reduced risk tobacco products substantially described herein.

Also provided herein are reduced risk tobacco products comprising a transgenic tobacco that comprises a reduced expression of a plurality of genes that regulate the production of at least two different compounds in said tobacco that contribute to a tobacco related disease. In some such tobacco products, the two different compounds in said tobacco are nicotine and a sterol.

Also provided herein are kits comprising two or more different tobaccos or tobacco products in accordance with any of the methods provided herein. In some such kits, the different tobaccos or tobacco products are differently labeled.

Also provided herein are uses of a tobacco or tobacco product of any of the methods, tobaccos, tobacco products or kits provided herein. Some such uses are tobacco-use cessation methods.

FIG. 1 . An illustration of a QPTase inhibition construct comprising a QPTase inhibition cassette including full-length QPTase coding sequence and a GUS selection cassette.

FIG. 2 . An illustration of a QPTase inhibition construct comprising a QPTase inhibition cassette including a 360 bp fragment of the QPTase gene and a norflurazone resistance selection cassette including a mutant phytoene desaturase gene (PDSM-1).

FIG. 3 . An illustration of a PMTase inhibition construct comprising a PMTase inhibition cassette including a 241 bp fragment of the PMTase gene and a norflurazone resistance selection cassette including a mutant phytoene desaturase gene (PDSM-1).

FIG. 4 . An illustration of a A622 inhibition construct comprising a A622 inhibition cassette including a 628 bp fragment of the A622 gene and a norflurazone resistance selection cassette including a mutant phytoene desaturase gene (PDSM-1).

FIG. 5 . An illustration of a QPTase/A622 double inhibition construct comprising a QPTase/A622 inhibition cassette including a 360 bp fragment of the QPTase gene and a 628 bp fragment of the A622 gene and a norflurazone resistance selection cassette including a mutant phytoene desaturase gene (PDSM-1).

FIG. 6 . An illustration of a SMT2/A622 double inhibition construct comprising a A622 inhibition cassette including a 628 bp fragment of the A622 gene, an SMT2 inhibition cassette including a 779 bp fragment of the SMT2 gene and a norflurazone resistance selection cassette including a mutant phytoene desaturase gene (PDSM-1).

FIG. 7 . An illustration of a QPTase inhibition construct comprising a QPTase inhibition cassette including a 360 bp fragment of the QPTase gene and a norflurazone resistance selection cassette including a mutant phytoene desaturase gene (PDSM-1).

FIG. 8 . An illustration of a QPTase inhibition construct comprising a QPTase inhibition cassette including a 360 bp fragment of the QPTase gene and a norflurazone resistance selection cassette including a mutant phytoene desaturase gene (PDSM-1).

FIG. 9 . An illustration of a PMTase inhibition construct comprising a PMTase inhibition cassette including a 202 bp fragment of the PMTase gene and a norflurazone resistance selection cassette including a mutant phytoene desaturase gene (PDSM-1).

FIG. 10 . An illustration of a PMTase inhibition construct comprising a PMTase inhibition cassette including a 344 bp fragment of the PMTase gene and a norflurazone resistance selection cassette including a mutant phytoene desaturase gene (PDSM-1).

FIG. 11 . An illustration of a QPTase inhibition construct comprising a QPTase inhibition cassette including a 360 bp fragment of the QPTase gene and a kanamycin resistance selection cassette including a neomycin phosphotransferase gene (NPTII).

FIG. 12A-B . Fluorescence photomicrographs of NHBE cells exposed to 25 μg/ml of tobacco smoke condensate for 24 h. The cells stained with DAPI and immuno-stained with γH2AX Ab were examined under UV light-(A) or blue light-(B) fluorescence excitation (Nikon Microphot FXA, 60× Objective).

FIG. 13A-C . Bivariate (cellular DNA content vs cell immunofluorescence) distributions (scatterplots) of A549 cells, mock-treated (B) or exposed for 30 min to tobacco smoke (A, C), immuno-stained either with γH2AX Ab (B,C) or with an isotype control IgG (A). The dashed-line represents the maximal fluorescence level (for 99% cells) of the IgG control.

FIG. 14 . Plots showing the percent increase (Δ) in mean γH2AX immunofluorescence of A549 cells (per unit of DNA) exposed to smoke for different time intervals, calculated for cells in particular phases of the cell cycle, as described in Example 1. The value for mock-exposed cells was subtracted from those exposed to smoke.

FIG. 15 . Plots showing percent increase (Δ) in mean γH2AX immunofluorescence of NHBE cells treated with 10, 25 or 50 μg/ml concentrations of smoke condensate for different periods of time. As in FIG. 14 , the γH2AX value for the mock-exposed cells was subtracted from the values of the cells exposed to different concentrations of condensate.

FIG. 16 . Percent increase (Δ) in mean γH2AX immunofluorescence of NHBE cells treated with 10 μg/ml of smoke condensate for different intervals of time, in relation to cell cycle phase. As in FIG. 14 , the γH2AX value for the mock-exposed cells was subtracted from the values of the cells exposed to condensate.

FIG. 17 . Plots showing the percent increase (Δ) in mean γH2AX immunofluorescence of A549 cells (per unit of DNA) exposed to smoke of IM16 cigarettes for different time intervals, calculated for cells in particular phases of the cell cycle, as described in Example 2.

FIG. 18A-D . (A) Plots showing increase (Δ) in mean γH2AX immunofluorescence of A549 cells exposed to smoke of IM16 cigarettes for 15 minutes, relative to mock exposed cells. (B) Scatter plots showing the increase in γH2AX following 60 min of recovery of the A549 cells in particular phases of the cell cycle for mock exposed (upper plot) and for IM16 smoke exposed (lower plot) cells. (C) Plots showing increase (Δ) in mean γH2AX immunofluorescence of NHBE cells exposed to smoke of IM16 cigarettes for 20 minutes, relative to mock exposed cells. (D) Scatter plot relative increase in γH2AX following 60 min of recovery of the NHBE cells in particular phases of the cell cycle for mock exposed (upper plot) and for IM16 smoke exposed (lower plot) cells.

FIG. 19 . Plots showing the increase (Δ) in mean γH2AX immunofluorescence during different time points of the recovery of A549 cells (per unit of DNA) after exposure to smoke of IM16, Quest 3®, and Omni® cigarettes for 20 minutes, calculated for cells in particular phases of the cell cycle.

FIG. 20 . Bar plots showing the increase (Δ) in mean γH2AX immunofluorescence of A549 cells (top) and NHBE cells (bottom) exposed to smoke of IM16 cigarettes for 20 minutes, followed by a 1 hour recovery, for cells treated with phosphate-buffered saline (PBS) or N-acetyl-L-cysteine (NAC) during exposure (first value) and during recovery (second value).

FIG. 21 . Bar plot showing the increase (Δ) in mean γH2AX immunofluorescence of A549 cells exposed to smoke from IM16, Omni® and Quest 3® in the presence of PBS or NAC.

FIG. 22 . Plot of the relative amount of mean γH2AX immunofluorescence of A549 cells exposed to smoke from IM16 as a function of different concentrations of NAC, calculated for cells in particular phases of the cell cycle. Horizontal dashed line indicates 50% reduction in γH2AX immunofluorescence. Vertical dashed lines indicate the estimated NAC concentration for each cell type at 50% reduction.

FIG. 23 . Bar plots showing the increase (Δ) in mean γH2AX immunofluorescence of A549 cells (upper plot) and NHBE cells (lower plot) exposed to the vapor phase of smoke from IM16, Quest 1® and Quest 3®, and smoke from IM16 in the presence of PBS or NAC.

FIG. 24 . Bar plots showing the increase (Δ) in mean γH2AX immunofluorescence of G 1 , S and G 2 M phase A549 cells (left plots) and G 1 , S and G 2 M phase NHBE cells (right plots) exposed to the vapor phase of smoke from IM16, Quest 1® and Quest 3® in the presence of PBS or NAC.

FIG. 25 . Bar plot showing the relative percent cloning efficiency of A549 cells 5 days after exposure to smoke from IM16 or Marlboro® for 10, 15 or 20 minutes.

FIG. 26 . Bar plots showing the relative percent cloning efficiency of A549 cells 5 days after exposure to smoke from IM16, Quest 1® or Quest 3® for 10, 20 or 30 minutes (top two plots), or 6 days after (bottom plot) exposure to smoke from IM16, Marlboro® or Omni®, for 10, 15 or 20 minutes.

FIG. 27 . Bar plot showing the relative percent cloning efficiency of A549 cells 5 days after exposure to smoke from IM16 for 20 minutes in the presence of PBS or 1 mM, 5 mM, 10 mM or 25 mM NAC.

FIG. 28 . Bar plot showing the relative percent cloning efficiency of A549 cells 5 days after exposure to smoke from IM16, Omni® or Quest 3® for 20 minutes in the presence of PBS or 25 mM NAC.

FIG. 29 . Bar plot showing the relative percent cloning efficiency of A549 cells 5 days after exposure to vapor phase of smoke from IM16, Quest 1® or Quest 3®, or smoke of IM16 for 20 minutes in the presence of PBS or 25 mM NAC.

FIG. 30 . Bar plot of results from Example 2 showing the increase (Δ) in mean γH2AX immunofluorescence of A549 cells exposed to smoke from IM16, Omni® and Quest 3® in the presence of PBS or NAC, calculated for cells in particular phases of the cell cycle.

FIG. 31 . Plot depicting γH2AX associated fluorescence (γH2AX; X-axis) and the number of cells having the corresponding γH2AX fluorescence level (Y axis), for buccal cells of a subject subsequent to smoking a cigarette (smoker) or a subject who did not smoke a cigarette (non-smoker).

FIG. 32 . Bar plot of results from Example 2 showing the increase (Δ) in mean γH2AX immunofluorescence of A549 cells exposed to smoke from IM16, Marlboro®, Marlboro Light®, and Quest 3®, calculated for cells in particular phases of the cell cycle.

FIG. 33 . Bar plot of results from FIG. 32 showing the increase (Δ) in mean γH2AX immunofluorescence of A549 cells exposed to smoke from IM16, Marlboro®, Marlboro Light®, and Quest 3®, averaged for all cell cycles.

FIG. 34A is a Venn diagram comparing gene expression modulations induced by cigarette smoke condensates of two different tobacco products (e.g., cigarettes) CSC-A (3665) and CSC-B (3668). The number of genes uniquely affected by exposure to each product CSC-A (1226) and CSC-B (1229) is given in each sector. The intersections between sectors reflect the number of genes that are affected by both CSCs (2439).

FIG. 34B is a Venn diagram comparing gene expression modulations induced by CSC-A (3665), CSC-B (3668), and S9 metabolic fraction (1680). The number of unique genes affected by each treatment is given, CSC-A (992), CSC-B (1039), and S9 (383) and the intersections between sectors reflect the number of genes that are affected by more than one treatment (e.g., a common set of 873 genes is affected by CSC-A, CSC-B and S9).

FIG. 35A-C illustrate gene expression profiles between 0 and 12 hours, which are expressed a percent of highest expression value for each gene. F-cluster numbers are given at the top of each cluster of profiles. The number of member genes in each cluster (n) is shown for each cluster. FIG. 35A shows Clusters that contain 50 or more genes in CSC-A-treated cells. FIG. 35B shows Clusters containing 50 or more genes in CSC-B-treated cells. FIG. 35C shows Clusters containing 50 or more genes in S9-treated cells.

FIG. 36 illustrates a cluster analysis of genes that were hypervariable (HV) in all three treatment groups (A: CSC-A, B: CSC-B, and S9) in the form of a Dendrogram that depicts the hierarchical relationship between the three treatments based on their gene expression patterns at all time points from 0-12 hours.

FIG. 37 shows correlation mosaics of the genes listed in Table 2. Correlation coefficients were generated for each of the 40 genes in Table 2, comparing the set to itself in each of the three conditions. The same gene order runs across the x and y axes of the mosaics. Correlation mosaics for HV genes highly correlated in response to CSC-A and CSC-B, and not correlated with responses to S9. Each pixel in the plot represents a correlation coefficient of gene expression. Genes highly positively correlated are denoted in gray and those highly negatively correlated are in black. The same order of the genes along axis is used for all three mosaics. Genes highly correlated in CSC-A and CSC-B, but not in S9-treated cells are denoted as a gray cluster in the lower left hand corner of CSC-A and the CSC-B mosaic. This cluster is disrupted in the S9 mosaic demonstrating the variance in gene regulation that occurred in S9-treated cells.

FIG. 38 shows the functional associations of HV genes specific for CSC-A and CSC-B treatment. The expression patterns of this set of genes are highly correlated in CSC-treated NHBE cells and not correlated with those seen in cells treated with S9 alone. Cross-hatched ovals indicate genes from Table 2 (i.e., HV genes specific for CSC-A and CSC-B treatment). Ovals with slanted lines (indicating additional proteins not in Table 2) were added to better define the regulatory networks of the genes identified in this analysis. Ovals with dashed lines indicate classes of functional peptides. Rectangles indicate cellular processes in which these genes participate. Each line indicates a regulatory relationship (binding, regulation, etc.) based upon a literature reference. Regulatory relationships are denoted in a box on the line with positive regulation represented as a plus sign, negative regulation as a minus sign, and unknown relationships by no sign.

FIG. 39 shows the functional associations of genes, which are highly correlated in all three treatment groups (CSC-A, CSC-B, and S9). The genes, pathways, and functional interconnections among these elements for genes correlated in all three treatment groups are represented. Gene and pathway symbols are described in FIG. 38 . Cross-hatched ovals indicate genes from Table 3 (i.e., genes specific for S9 treatment). Ovals with slanted lines (indicate additional proteins not in Table 3), cross-hatched oval (cell object—DNA) and white triangle (indicating small molecule—estrogen) were added to better define the regulatory networks of the genes identified in this analysis. Ovals with dashed lines indicate classes of functional peptides. White rectangles indicate cellular processes in which these genes participate. Each line indicates a regulatory relationship (binding, regulation, etc.) based upon a literature reference. Regulatory relationships are denoted in a box on the line with positive regulation represented as a plus sign, negative regulation as a minus sign, and unknown relationships by no sign.

FIG. 40 shows the results of a discriminant function analysis (DFA), which identified genes having high discriminatory capabilities. Values of the roots obtained by DFA analysis were used to graphically depict the differences of the gene expression values obtained for the three treatments (CSC-A, CSC-B, and S9). Root values for the 2-12 h time points for each treatment are represented by filled circles (CSC-A), open circles (CSC-B), and filled triangles (S9).

FIG. 41 shows the functional associations of genes, which are provided in Table 3. The genes, pathways, and functional interconnections among these elements for genes having the highest discriminatory potential among all three treatment groups are represented. Gene and pathway symbols are described in previous figures.

FIGS. 42A and B show a comparison of expression behavior of heat shock protein family members DNAJA1 and DNAJB1 in Experiment 1 ( FIG. 42A ) and 2 ( FIG. 42B ). Each time point represents the average of 2 or 3 replicates per condition.

FIG. 43 is a hierarchical clustering of samples using 105 genes that were both over-expressed upon treatment of NHBE cells with CS in two separate experiments, and encoded protein products that modulate one of the 4 major CS-affected GO-defined cellular functions identified. Samples a-b are from Experiment 1, samples c-e are from Experiment 2. A bar indicates heat shock and heat shock-associated genes showing greatly increased expression exclusively at 4 h. Markings indicate genes whose expression is known to be regulated by transcription factor NRF2.

FIG. 44 shows a plot of γH2AX immunofluorescence in A549 cells exposed to smoke of different combinations of tobaccos and filters from IM16, Omni® and Quest 3® cigarettes, corrected according to the γH2AX immunofluorescence for mock-exposed cells. FIG. 44A depicts γH2AX immunofluorescence for the unmodified cigarettes. FIG. 44B depicts γH2AX immunofluorescence for cigarettes containing IM16 tobacco and IM16, Omni® and Quest 3® filters. FIG. 44C depicts γH2AX immunofluorescence for cigarettes containing Omni® tobacco and either an IM16 or Omni® filter. FIG. 44D depicts γH2AX immunofluorescence for cigarettes containing Quest 3® tobacco and either an IM16 or Quest 3® filter.

FIG. 45 shows bar plots showing the relative percent cloning efficiency of A549 cells 5 days after exposure to smoke of different combinations of tobaccos and filters from IM16, Omni® or Quest 3® cigarettes, relative to mock cloning efficiency.

FIG. 46 shows bar plots showing the relative percent cloning efficiency of A549 cells 5 days after exposure to smoke of different combinations of tobaccos and filters from IM16, Quest 1® or Quest 3®, relative to mock cloning efficiency.

FIG. 47 shows bar plots showing the relative percent cloning efficiency of A549 cells 5 days after exposure to smoke of different combinations of tobaccos and filters from IM16, Omni® or Quest 3® cigarettes, relative to mock cloning efficiency.

I. Introduction

The health consequences of tobacco consumption are known but many people continue to use tobacco products. The addictive properties of tobacco products are largely attributable to the presence of nicotine. In addition to being one of the most addictive substances known, nicotine is also a precursor for a large number of carcinogenic compounds present in tobacco and the body. Many other harmful compounds in addition to nicotine are present in conventional tobacco, however.

There is currently a great interest in developing approaches to decrease the levels of noxious, carcinogenic, or addictive substances including tar, TSNAs, and nicotine in tobacco. Although researchers have developed several approaches to reduce some of these harmful compounds, many conventional techniques result in a product that has poor taste, fragrance, or smoking properties. Some processes, for example, reduce the nicotine content of tobacco by microbial enzymatic degradation, chemical extraction, or high pressure extraction. (See e.g., U.S. Pat. Nos. 4,557,280; 4,561,452; 4,848,373; 4,183,364; and 4,215,706, all of which are hereby expressly incorporated by reference in their entireties). More recently, techniques in genetic engineering and chemically-induced gene suppression have been employed to make reduced nicotine and/or reduced tobacco specific nitrosamine (TSNA) tobacco. (See e.g., Conkling et al., WO98/56923; U.S. Pat. Nos. 6,586,661; 6,423,520; and U.S. patent application Ser. Nos. 09/963,340; 10/356,076; 09/941,042; 10/363,069; 10/729,121; 10/943,346; Timko et al., WO 00/67558, which designated the United States and was published in English, Nakatani et al., U.S. Pat. Nos. 5,684,241; 5,369,023; 5,260,205; and Roberts et al. 6,700,040, all of which are hereby expressly incorporated by reference in their entireties). In view of the foregoing, and notwithstanding the various efforts exemplified in the above reports, there remains a need for tobacco that has a reduced potential to contribute to a tobacco-related disease and methods of producing such tobacco.

Embodiments provided herein relate to tobacco and/or tobacco products having a reduced amount of a harmful compound, and methods of developing, screening and using such tobacco and tobacco products. Several approaches are provided to reduce the amount of one or more harmful compounds in tobacco by, for example, modifying the expression of a gene that is involved in the production of a harmful compound in tobacco. Also provided are methods of determining whether the removal of a harmful compound yields a tobacco and/or a tobacco product that has a reduced potential to contribute to a tobacco-related disease. Also provided are reduced-risk tobacco and tobacco products made in accordance with the methods provided herein. Also provided are methods of using the reduced-risk tobacco and tobacco products made in accordance with the methods provided herein.

II. Modified Tobacco

Several approaches to create a reduced risk tobacco product having a reduced amount of a harmful compound are described. At least some of the reduced risk tobacco products provided herein contain modified tobacco. As used herein, “modified tobacco” refers to a tobacco that has been subjected to one or more genetic, chemical or processing steps that is different than the conventional treatment or processing of traditional “wild-type” tobacco products. In one example, a tobacco product can be genetically modified, by, for example, administering to a tobacco plant a nucleic acid molecule that modulates expression of one or more genes in the tobacco plant that produce a compound. Genetically modified tobacco and methods of preparing same are provided elsewhere herein. In another example, a tobacco product can be chemically modified, by, for example, extracting or chemically altering one or more components of tobacco, according to methods known in the art as exemplified in U.S. Pat. Nos. 6,789,548, 4,557,280; 4,561,452; 4,848,373; 4,183,364; 4,215,706; 4,257,430; 4,248,251; 4,235,251; 4,216,784; 4,177,822; 4,055,191 (all of which are herein expressly incorporated by reference in their entireties) or by adding one or more compounds to a tobacco plant prior to harvesting the tobacco, as known in the art and exemplified in U.S. Pat. Pub. No. 20050072047, herein expressly incorporated by reference in its entirety. Additional modified tobaccos contemplated herein include reconstituted tobacco, extracted tobacco, and expanded or puffed tobacco. In some embodiments, the tobacco is modified to have a reduced amount of a compound that contributes to a tobacco-related disease, including, but not limited to, a compound associated with a tobacco-related disease or a metabolite thereof (e.g., tobacco sterols, nicotine, a TSNA, and a gene product that is involved in the production of a compound associated with a tobacco-related disease or a metabolite thereof).

The modified tobacco described herein is suitable for conventional growing and harvesting techniques (e.g. topping or no topping, bagging the flowers or not bagging the flowers, cultivation in manure rich soil or without manure) and the harvested leaves and stems are suitable for use in any traditional tobacco product including, but not limited to, pipe, cigar and cigarette tobacco and chewing tobacco in any form including leaf tobacco, shredded tobacco or cut tobacco. It is also contemplated that the modified tobacco (e.g., reduced nicotine/TSNA and/or sterol tobacco) described herein can be processed and blended with conventional tobacco so as to create a wide-range of tobacco products with varying amounts of nicotine, TSNAs, and/or sterols.

In some embodiments, the modified tobacco has reduced levels of nicotine, nornicotine, and/or sterols in tobacco. Alkaloids such as nicotine and nornicotine are precursors for a number of harmful compounds that contribute to tobacco-related disease (e.g., the tobacco specific nitrosamines (TSNAs): N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB), 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(N-nitrosomethylamino)-4-(3-pyridyl)-1-butanal (NNA)-4-N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAL), 4-N-nitrosomethylamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and/or 4-(N-nitrosomethylamino)-4-(3-pyridyl)-butanoic acid (iso-NNAC) and acrolein). Sterols are precursors for a number of harmful compounds, which are generated by pyrolysis of tobacco, that also contribute to tobacco-related disease (e.g., polycyclic aromatic hydrocarbons (PAHs), such as benz[a]pyrene (BAP), heterocyclic hydrocarbons, terpenes, paraffins, aromatic amines, and aldehydes). Because the presence of these harmful compounds in tobacco contributes to tobacco-related disease, a modified tobacco that comprises a reduced amount of any one of these compounds, as compared to a reference tobacco (e.g., the industry standard reference tobacco IM16 (Philip Morris® USA) or the low tar reference cigarette 2R4F or the ultra low tar cigarette 1R5F, which are Kentucky reference cigarettes that can be obtained from the Tobacco and Health Institute at the University of Kentucky), a conventional tobacco (e.g., a commercially available tobacco of the same class (e.g., “full-flavor” or “light” or “ultra-light”)) or a non-transgenic tobacco (e.g., a tobacco of the same variety, such as Burley, Va. Flue-cured, or Oriental, or strain, such as LA Burley 21, K326, Tn90, Djebel174, as the transgenic tobacco prior to genetic modification) has a reduced potential to contribute to a tobacco-related disease. Tobacco products comprising the modified tobacco can also be analyzed by various approaches to confirm that the tobacco is “reduced risk,” as compared to a parental strain or a reference tobacco using one or more of the assays described herein or otherwise known in the art. This “reduced risk” modified tobacco can then be processed, optionally, sterilized or otherwise made substantially-free of microbes, and said tobacco can be incorporated into tobacco products, preferably, cigarettes, optionally, by an aseptic approach so as to not introduce microbes (e.g., bacteria, mold, yeast, and fungi) into the products. Tobacco products comprising the modified tobacco can then be packaged, optionally, by an aseptic approach in air-tight or microbe-free packaging so as to not introduce microbes into the products.

In this manner, the conversion of alkaloid to TSNA, which results from microbial growth on the tobacco when microbes are introduced during processing, packaging, and storage, is significantly reduced. By using the embodied tobacco preparative methods, which may include several aseptic processing, manufacturing, and packaging procedures, one can maintain an amount of total TSNA (e.g., the collective content of NNN, NAT, NAB, and NNK) in or delivered by (e.g., as measured by FTC or ISO methodologies) a commercially available tobacco product of less than or equal to 0.5 μg/g (e.g., 0.05 μg/g, 0.1 μg, 0.2 μg/g, 0.3 μg/g, 0.4 μg/g, or 0.5 μg/g) for a period of at least 1 week, 1 month, or 1-5 years after packaging or incorporation of the tobacco into a tobacco product (e.g., at least 1-30 days, 30-90 days, 90-180 days, 180-270 days, 270 days-365 days, 1 year-1.5 years, 1.5-2.0 years, 2.0 years-2.5 years, 2.5 years-3.0 years, 3.0 years-4 years, and 4.0 years-5.0 years).

In some embodiments, a modified tobacco comprising a reduced amount of alkaloid (e.g., a reduced amount of nicotine, nornicotine, and/or TSNAs) is contacted with an exogenous nicotine so as to raise the level of nicotine in the contacted transgenic tobacco in a controlled fashion. By this approach, nicotine levels in transgenic tobacco that comprises a reduced amount of endogenous nicotine (i.e., nicotine that is produced by the transgenic plant from which the transgenic tobacco is obtained) can be selectively raised to levels that are commensurate with conventional full-flavor cigarettes, light cigarettes, or ultra-light cigarettes. (See e.g., WO 2005/018307, which designates the United States and was published in English, herein expressly incorporated by reference in its entirety). For example, modified tobacco comprising a reduced amount of endogenous nicotine and/or TSNAs can be contacted with an amount of exogenous nicotine that is at least, equal to, or more than 0.3 mg/g-20.0 mg/g (nicotine/gram of tobacco). That is, modified tobacco comprising a reduced amount of endogenous nicotine and/or TSNAs can be contacted with an amount of exogenous nicotine that is or delivers (e.g., as measured by FTC or ISO methodologies) at least, equal to, or more than 0.3 mg/g, 0.4 mg/g, 0.5 mg/g, 0.6 mg/g, 0.7 mg/g, 0.8 mg/g, 0.9 mg/g, 1.0 mg/g, 1.1 mg/g, 1.2 mg/g, 1.3 mg/g, 1.4 mg/g, 1.5 mg/g, 1.6 mg/g, 1.7 mg/g, 1.8 mg/g, 1.9 mg/g, 2.0 mg/g, 2.1 mg/g, 2.2 mg/g, 2.3 mg/g, 2.4 mg/g, 2.5 mg/g, 2.6 mg/g, 2.7 mg/g, 2.8 mg/g, 2.9 mg/g, 3.0 mg/g, 3.1 mg/g, 3.2 mg/g, 3.3 mg/g, 3.4 mg/g, 3.5 mg/g, 3.6 mg/g, 3.7 mg/g, 3.8 mg/g, 3.9 mg/g, 4.0 mg/g, 4.1 mg/g, 4.2 mg/g, 4.3 mg/g, 4.4 mg/g, 4.5 mg/g, 4.6 mg/g, 4.7 mg/g, 4.8 mg/g, 4.9 mg/g, 5.0 mg/g, 5.1 mg/g, 5.2 mg/g, 5.3 mg/g, 5.4 mg/g, 5.5 mg/g, 5.6 mg/g, 5.7 mg/g, 5.8 mg/g, 5.9 mg/g, 6.0 mg/g, 6.1 mg/g, 6.2 mg/g, 6.3 mg/g, 6.4 mg/g, 6.5 mg/g, 6.6 mg/g, 6.7 mg/g, 6.8 mg/g, 6.9 mg/g, 7.0 mg/g, 7.1 mg/g, 7.2 mg/g, 7.3 mg/g, 7.4 mg/g, 7.5 mg/g, 7.6 mg/g, 7.7 mg/g, 7.8 mg/g, 7.9 mg/g, 8.0 mg/g, 8.1 mg/g, 8.2 mg/g, 8.3 mg/g, 8.4 mg/g, 8.5 mg/g, 8.6 mg/g, 8.7 mg/g, 8.8 mg/g, 8.9 mg/g, 9.0 mg/g, 9.1 mg/g, 9.2 mg/g, 9.3 mg/g, 9.4 mg/g, 9.5 mg/g, 9.6 mg/g, 9.7 mg/g, 9.8 mg/g, 9.9 mg/g, 10.0 mg/g, 10.1 mg/g, 10.2 mg/g, 10.3 mg/g, 10.4 mg/g, 10.5 mg/g, 10.6 mg/g, 10.7 mg/g, 10.8 mg/g, 10.9 mg/g, 11.0 mg/g, 11.1 mg/g, 11.2 mg/g, 11.3 mg/g, 11.4 mg/g, 11.5 mg/g, 11.6 mg/g, 11.7 mg/g, 11.8 mg/g, 11.9 mg/g, 12.0 mg/g, 12.1 mg/g, 12.2 mg/g, 12.3 mg/g, 12.4 mg/g, 12.5 mg/g, 12.6 mg/g, 12.7 mg/g, 12.8 mg/g, 12.9 mg/g, 13.0 mg/g, 13.1 mg/g, 13.2 mg/g, 13.3 mg/g, 13.4 mg/g, 13.5 mg/g, 13.6 mg/g, 13.7 mg/g, 13.8 mg/g, 13.9 mg/g, 14.0 mg/g, 14.1 mg/g, 14.2 mg/g, 14.3 mg/g, 14.4 mg/g, 14.5 mg/g, 14.6 mg/g, 14.7 mg/g, 14.8 mg/g, 14.9 mg/g, 15.0 mg/g, 15.1 mg/g, 15.2 mg/g, 15.3 mg/g, 15.4 mg/g, 15.5 mg/g, 15.6 mg/g, 15.7 mg/g, 15.8 mg/g, 15.9 mg/g, 16.0 mg/g, 16.1 mg/g, 16.2 mg/g, 16.3 mg/g, 16.4 mg/g, 16.5 mg/g, 16.6 mg/g, 16.7 mg/g, 16.8 mg/g, 16.9 mg/g, 17.0 mg/g, 17.1 mg/g, 17.2 mg/g, 17.3 mg/g, 17.4 mg/g, 17.5 mg/g, 17.6 mg/g, 17.7 mg/g, 17.8 mg/g, 17.9 mg/g, 18.0 mg/g, 18.1 mg/g, 18.2 mg/g, 18.3 mg/g, 18.4 mg/g, 18.5 mg/g, 18.6 mg/g, 18.7 mg/g, 18.8 mg/g, 18.9 mg/g, 19.0 mg/g, 19.1 mg/g, 19.2 mg/g, 19.3 mg/g, 19.4 mg/g, 19.5 mg/g, 19.6 mg/g, 19.7 mg/g, 19.8 mg/g, 19.9 mg/g, and 20.0 mg/g (nicotine/gram tobacco). In some of the aforementioned embodiments, the modified tobacco contacted with the exogenous nicotine is a transgenic tobacco comprising, for example, one or more of the isolated nucleic acids, isolated nucleic acid cassettes, or isolated nucleic acid constructs described herein.

Nicotine-containing fractions, nicotine, or nicotine salts of organic acids are added to the reduced-nicotine transgenic tobacco by contacting said tobacco (e.g., spraying or additive application), with or without propylene glycol, solvent, flavoring, or water at any stage of the harvesting, curing, fermenting, aging, reconstituting, expanding, or otherwise processing of the tobacco, preferably at a stage that is post-cure, when flavorings and additives are provided. By “exogenous nicotine” is meant nicotine, nicotine derivatives, nicotine analogs, nicotine-containing fractions (e.g., extracts of Nicotiana ), and nicotine salts of organic acids obtained from a source outside of the transgenic tobacco to which the exogenous nicotine is applied. In this manner, a modified tobacco that provides virtually any amount of nicotine can be obtained.

In some embodiments, the exogenous nicotine (e.g., commercially available nicotine salts, liquid, or a nicotine-containing extract prepared from a Nicotiana plant or portion thereof) is contacted with a reduced-alkaloid modified tobacco (e.g., a transgenic tobacco comprising a reduced amount of nicotine and/or TSNA as prepared as described herein) after the modified tobacco has been made substantially free of microbes (e.g., bacteria, yeast, mold, or fungi). The reduced alkaloid modified tobacco can be made substantially-free of microbes (e.g., an aseptic preparation) by employing sterilization, heat treatment, pasteurization, steam treatment, gas treatment, and radiation (e.g., gamma, microwave, and ultraviolet). The term “substantially-free of microbes” in some contexts can mean an amount of bacteria, mold, fungi, or yeast that is reduced to the point that the conversion of nicotine or total alkaloid to TSNA is negligible (e.g., the resultant concentration of or the amount of delivered or provided total TSNA (e.g., NNN, NNK, NAT, and NAB) in or delivered by a tobacco or tobacco product is equal to or below 0.5 μg/g (e.g., 0.05 μg/g, 0.1 μg, 0.2 μg/g, 0.3 μg/g, 0.4 μg/g, or 0.5 μg/g) after prolonged storage (e.g., at least 1-30 days, 30-90 days, 90-180 days, 180-270 days, 270 days-365 days, 1 year-1.5 years, 1.5-2.0 years, 2.0 years-2.5 years, 2.5 years-3.0 years, 3.0 years-4 years, and 4.0 years-5.0 years)). The term “substantially-free of microbes” also includes the term “substantially-free of bacteria,” which means in some contexts that the tobacco or tobacco product is substantially-free of Arthrobacter, Proteus , nicotine oxidizing bacteria, such as P-34 , Psuedomonas, Xantomonas , or Zoogloea strains of bacteria. For example, a tobacco or tobacco product is substantially-free of bacteria or a particular strain of bacteria when said tobacco or tobacco product has less than or equal to 20% of the bacteria or a specific strain of bacteria normally present on the tobacco or tobacco product in the absence of application of a technique to rid the tobacco or tobacco product of bacteria (e.g., less than or equal to 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%). With respect to modified tobacco described herein, the term “substantially-free of bacteria” can refer to tobacco or a tobacco product containing the modified tobacco that has less than or equal to 20% of the bacteria normally present on the strain of tobacco prior to modification and/or application of a technique to rid the tobacco or tobacco product of bacteria (e.g., less than or equal to 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%).

Once the exogenous nicotine has been contacted with the microbe-free modified tobacco, it is preferably processed and packaged aseptically and the tobacco product is maintained in an airtight container so as to not re-introduce microbes that convert the exogenous nicotine to TSNAs. By using the aseptic processing, manufacturing, and packaging procedures, described herein, one can maintain an amount of total TSNA (e.g., the collective content of NNN, NAT, NAB, and NNK) in a commercially available tobacco product or delivered by a commercially available tobacco product, which comprises exogenous nicotine, of less than or equal to 0.5 μg/g (e.g., 0.05 μg/g, 0.1 μg, 0.2 μg/g, 0.3 μg/g, 0.4 μg/g, or 0.5 μg/g) for at least 1 week, 1 month, or 1-5 years after packaging (e.g., at least 1-30 days, 30-90 days, 90-180 days, 180-270 days, 270 days-365 days, 1 year-1.5 years, 1.5-2.0 years, 2.0 years-2.5 years, 2.5 years-3.0 years, 3.0 years-4 years, and 4.0 years-5.0 years). In some embodiments, the exogenous nicotine is contacted with a modified tobacco and a collective content of NNN, NAT, NAB, and NNN that is present or delivered by the tobacco is less than or equal to 0.5 μg/g (e.g., 0.05 μg/g, 0.1 μg, 0.2 μg/g, 0.3 μg/g, 0.4 μg/g, or 0.5 μg/g). In some embodiments, a collective content of NNN, NAT, NAB, and NNN of less than or equal to 0.5 μg/g (e.g., 0.05 μg/g, 0.1 μg, 0.2 μg/g, 0.3 μg/g, 0.4 μg/g, or 0.5 μg/g) in or delivered by a tobacco product containing said transgenic tobacco can be maintained for at least at least 1 week, 1 month, or 1-5 years after packaging (e.g., at least 1-30 days, 30-90 days, 90-180 days, 180-270 days, 270 days-365 days, 1 year-1.5 years, 1.5-2.0 years, 2.0 years-2.5 years, 2.5 years-3.0 years, 3.0 years-4 years, and 4.0 years-5.0 years). An exemplary modified tobacco is transgenic tobacco comprising, for example, one of the nucleic acid constructs described herein. Accordingly, several embodiments address the problem of gradually increasing TSNA levels in alkaloid-containing tobacco products by employing processing, storage, and packaging methods that reduce the amount of microbial flora on the tobacco, limit the re-introduction of microbes during processing and maintain a reduced amount of microbes (e.g., bacteria) once the product is packaged, stored, and sold. Tobacco and tobacco products comprising modified tobacco having a reduced amount of endogenous nicotine and an amount of exogenous nicotine can be analyzed by various methods to confirm that said tobacco and said tobacco products are “reduced risk” or have less of a potential to contribute to a tobacco-related disease, as compared to the parent strain of tobacco having conventional amounts of endogenous nicotine or a reference tobacco.

Tobacco products that comprise a modified tobacco described herein include “full-flavor,” “lights,” and “ultra light” cigarettes typically having both reduced levels of alkaloids and levels of alkaloids commensurate with a level of alkaloid common to the particular class of cigarette (i.e., a conventional amount of nicotine). The term “tobacco products” includes, but is not limited to, smoking materials (e.g., cigarettes, cigars, pipe tobacco), snuff, chewing tobacco, gum, and lozenges.

The term “reduced risk tobacco product” or “reduced risk tobacco” includes, but is not limited to, a tobacco product or tobacco comprising a modified tobacco that has a reduced amount of a compound that contributes to a tobacco-related disease, or increased amounts of a compound that reduces the harmful effects of a compound that contributes to a tobacco-related disease such as nicotine, nornicotine, a sterol, or the metabolites thereof including, but not limited to, a TSNA, an acrolein, an aldehyde, or harmful compounds generated upon pyrolysis of tobacco, including but not limited to, PAH, BAP, a heterocyclic hydrocarbon, or an aromatic amine, as compared to the amount of these compounds in or generated by a reference tobacco or reference tobacco product (e.g., IM16, 2R4F or 1R5F), a commercially available tobacco product of the same class (e.g., full-flavor, lights, and ultra-lights), or, preferably, a tobacco of the same variety (e.g., Burley, Va. Flue-cured, or Oriental) or strain (e.g., LA Burley 21, K326, Tn90, Djebel174) as the transgenic tobacco prior to genetic modification). For example, a reduced risk tobacco or a reduced risk tobacco product can include a transgenic tobacco or a tobacco product comprising transgenic tobacco that up-regulates fewer genes associated with a tobacco-related disease as compared to a reference tobacco or reference tobacco product (e.g., IM16, 2R4F or 1R5F), a commercially available tobacco product of the same class (e.g., full-flavor, lights, and ultra-lights), or, preferably, a tobacco of the same variety (e.g., Burley, Va. Flue-cured, or Oriental) or strain (e.g., LA Burley 21, K326, Tn90, Djebel174) as the transgenic obacco prior to genetic modification).

Nitrosamines and Tobacco-Specific Nitrosamines

The term nitrosamine generally refers to any of a class of organic compounds with the general formula R 2 NNO or RNHNO (where R denotes an amine-containing group). Nitrosamines are present in numerous foods and have been found to be carcinogenic in laboratory animals. These compounds are formed by nitrosation reactions of amines such as amino acids and alkaloids with nitrites and/or nitrous oxides. By themselves, nitrosamines are not carcinogenic substances, but in mammals nitrosamines undergo decomposition by enzymatic activation to form alkylating metabolites which appear to react with biopolymers to initiate their tumorogenic effect. Thus, by reducing the amount of nitrosamine intake, one has effectively reduced the carcinogenic potential in humans.

Nitrosamines have been identified in tobacco, tobacco products, and tobacco smoke by the use of techniques such as gas chromatography-thermal energy analysis (GC-TEA). Some of these nitrosamines have been identified as tobacco-specific nitrosamines (TSNAs). TSNAs are primarily formed by reactions between the two most abundant alkaloids, nicotine and nornicotine, with nitrous oxides (NOx), and they account proportionately for the highest concentration of nitrosamines in both tobacco products and in mainstream smoke. Of the TSNAs identified, and the subset that have been found to be present in cigarette smoke, the most characterized is N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (N-nitrosamine-ketone), or NNK. When injected at relatively high doses, NNK is carcinogenic in rodents. Minimal amounts of TSNAs are found in green tobacco, indicating that TSNA formation may occur during processing steps such as curing, drying, fermentation, burning or storage of tobacco.

TSNA formation is attributed to chemical, enzymatic and bacterial influences during tobacco processing, particularly during curing, fermentation and aging. Nitrosation of nornicotine, anatabine, and anabasine gives the corresponding nitrosamines: N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT) and N′-nitrosoanabasine (NAB). Nitrosation of nicotine in aqueous solution affords a mixture of 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), NNN, and 4-(N-nitrosomethylamino)-4-(3-pyridyl)-1-butanal (NNA). Less commonly encountered TSNAs include NNAL (4-N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol), iso-NNAL (4-N-nitrosomethylamino)-4-(3-pyridyl)-1-butanol, 11) and iso-NNAC (4-(N-nitrosomethylamino)-4-(3-pyridyl)-butanoic acid, 12). See, U.S. Pat. No. 6,135,121, the entire disclosure of which is hereby expressly incorporated by reference in its entirety.

TSNA levels are particularly high in chewing tobaccos and snuff. The partially anaerobic processes that occur during fermentation promote the formation of TSNAs from tobacco alkaloids by promoting increased nitrite levels; in particular, over-fermentation can increase TSNA levels in snuff by its effects on nitrate levels and microbial enzymatic activity. The reduction of the TSNA level in snuff in recent years has been achieved by maintaining a better control over the bacterial content in these products.

Since the nitrate level of tobacco is important for TSNA formation in cigarette smoke, a significant reduction of TSNAs in smoke can be achieved by low-nitrate leaf and stem blends. However, these methods may negatively impact the smokability or the taste of the tobacco. The TSNA content of mainstream smoke can be reduced by as much as 80% by cellulose acetate filters, and it can be reduced still further by filter ventilation.

Air-cured tobaccos such as Burley and dark-fired may have higher levels of TSNAs than certain types of Flue-cured bright, Burley, or dark tobaccos apparently because the high temperatures associated with flue-curing can kill the micro-organisms that transform the alkaloids into TSNAs. In air-cured types, nitrate (N—NO 3 ) is more abundant in the leaf (particularly in the leaf and stems) than in Flue-cured tobacco and the alkaloid content is also much higher. This N—NO 3 is reduced to nitrite (NO 2 − ) by microbes during curing and the NO 2 − can be further reduced to NOx or react directly with alkaloids to form TSNAs.

It is contemplated that, in addition to the techniques described above, nitrate levels in tobacco (especially in the leaf) can be reduced by limiting exposure to nitrosating agents or conditions. Air-curing experiments at a higher temperature have shown that considerably higher levels of N-nitrosamines are formed at a curing temperature of 32° C. than at 16° C., which is associated with a rise of the nitrite level in the tobacco, and may also be associated with a rise in microbial enzymatic activity. Modified curing that involves faster drying from wider spacing or from more open curing structures has been shown to reduce TSNA levels in Burley tobacco. The climatic conditions prevailing during curing exert a major influence on N-nitrosamine formation, and the relative humidity during air-curing can be of importance. Stalk curing results in higher TSNA levels in the smoke than primed-leaf curing. Sun-cured Oriental tobaccos have lower TSNA levels than flue- and air-cured dark tobaccos. Accelerated curing of crude tobaccos such as homogenized leaf curing limits the ability of bacteria to carry out the nitrosation reactions. However, many of the methods described above for reducing TSNAs in Burley tobacco can have undesirable effects on tobacco taste.

TSNA formation in Flue-cured tobacco also results from exposure of the tobacco to combustion gases during curing, where nearly all of the TSNAs in Flue-cured tobacco (e.g., Virginia Flue-cured) result from a reaction involving NOx and nicotine. The predominant source of NOx is the mixture of combustion gases in direct-fired barns. At present, Flue-cured tobacco is predominantly cured in commercial bulk barns. As a result of energy pressures in the U.S. during the 1960's, farmer-built “stick barns” with heat-exchanged flue systems were gradually replaced with more energy efficient bulk barns using direct-fired liquid propane gas (LPG) burners. These LPG direct-fired burner systems exhaust combustion gases and combustion by-products directly into the barn where contact is made with the curing tobacco. Studies indicate that LPG combustion by-products react with naturally occurring tobacco alkaloids to form TSNA.

In contrast to direct-fired curing, heat-exchange burner configurations completely vent combustion gases and combustion by-products to the external atmosphere rather than into the barn. The heat-exchange process precludes exposure of the tobacco to LPG combustion by-products, thereby eliminating an important source of nitrosating agent for TSNA formation, without degrading leaf quality or smoking quality. The use of heat exchangers reduces TSNA levels by about 90%. Steps are being taken to reduce TSNA levels in US tobacco by converting barns to indirect heat through the use of a heat exchanger, but these methods are very expensive. Although many of the approaches described in this section have significant drawbacks, it should be understood that any or all of these techniques can be used with other techniques, as described herein, to make tobacco and tobacco products having reduced TSNAs. The section below provides more detail on nicotine and approaches to reduce nicotine in tobacco.

Nicotine

Nicotine is formed primarily in the roots of the tobacco plant and is subsequently transported to the leaves, where it is stored (Tso, Physiology and Biochemistry of Tobacco Plants, pp. 233-34, Dowden, Hutchinson & Ross, Stroudsburg, Pa. (1972)). Classical crop breeding techniques have produced tobacco with lower levels of nicotine, including varieties with as low as 8% of the amount of nicotine found in wild-type tobacco. The many methods described herein can be used with virtually any tobacco variety but are preferably used with Burley, Oriental or Flue-cured (e.g., Virginia Flue-cured) varieties.

Nicotine is produced in tobacco plants by the condensation of nicotinic acid and 4-methylaminobutanal. Two regulatory loci (Nic1 and Nic2) act as co-dominant regulators of nicotine production. Enzyme analyses of root tissue from single and double Nic mutants show that the activities of two enzymes, quinolate phosphoribosyl transferase (“QPTase”) and putrescene methyl transferase (PMTase), are directly proportional to levels of nicotine biosynthesis. An obligatory step in nicotine biosynthesis is the formation of nicotinic acid from quinolinic acid, a step that is catalyzed by QPTase. QPTase appears to be a rate-limiting enzyme in the pathway supplying nicotinic acid for nicotine synthesis in tobacco. (See, eg., Feth et al., Planta, 168, pp. 402-07 (1986) and Wagner et al., Physiol. Plant., 68, pp. 667-72 (1986), herein expressly incorporated by reference in its entirety). A comparison of enzyme activity in tobacco tissues (root and callus) with different capacities for nicotine synthesis shows that QPTase activity is strictly correlated with nicotine content (Wagner and Wagner, Planta 165:532 (1985), herein expressly incorporated by reference in its entirety). In fact, Saunders and Bush (Plant Physiol 64:236 (1979), herein expressly incorporated by reference in its entirety), showed that the level of QPTase in the roots of low nicotine mutants is proportional to the level of nicotine in the leaves.

The modification of nicotine levels in tobacco plants by antisense regulation of putrescene methyl transferase expression has been proposed in U.S. Pat. Nos. 5,369,023 and 5,260,205, to Nakatani and Malik, and in PCT application WO 94/28142 and U.S. Pat. No. 5,668,295 to Wahad and Malik, which describe DNA encoding PMT and the use of sense and antisense PMT constructs, the entire disclosures of each of which are hereby expressly incorporated by reference in their entireties. Other genetic modifications proposed to reduce nicotine levels are described in PCT application WO 00/67558, to Timko, and WO 93/05646, to Davis and Marcum; the entire contents of each are hereby expressly incorporated by reference in their entireties. Although these investigators made significant contributions, there were significant drawbacks to their experimental design.

Provided herein are tobacco and tobacco products in which a plurality of genes involved in nicotine biosynthesis are inhibited. Most notably, it is presently revealed that there are several different PMT genes and each may play a role in nicotine biosynthesis. Knocking-out only one PMT gene may create a leaky system allowing the other genes to compensate for the reduction in nicotine biosynthesis. Accordingly, the PMT constructs described herein were designed to inhibit a plurality of different PMT genes. That is, in some embodiments, the PMT constructs described herein are designed to complement common regions to all five of the PMT genes so that inhibition of each of the PMT genes can be accomplished with a single construct. Although many of the approaches described in this section have significant drawbacks, it should be understood that any or all of these techniques can be used with other techniques, as described herein, to make tobacco and tobacco products having reduced nicotine. The section below explains several approaches to reduce the amount of nicotine and sterols in tobacco and tobacco products.

Reducing the Amount of Nicotine and Sterols in Tobacco

As discussed above, TSNAs, nicotine, nornicotine, and sterols contribute significantly to tobacco-related disease, most notably the carcinogenic potential of tobacco and tobacco products. Thus, tobacco and tobacco products that have or produce reduced amounts of these compounds are reduced risk compositions (e.g., products that have a reduced potential to contribute to a tobacco-related disease). Without wishing to be bound by any particular theory, it is contemplated that the creation of tobacco plants, tobacco and tobacco products that have a reduced amount of nicotine will also have reduced amounts of TSNAs. That is, by removing nicotine from tobacco plants, tobacco and tobacco products, one effectively removes the most significant alkaloid substrate for TSNA formation. It was found that the reduction of nicotine in tobacco was directly related to the reduction of TSNAs. Similarly, it is contemplated that by removing sterols from tobacco, one can reduce the amount of PAHs generated from pyrolysis of the tobacco. Unexpectedly, the methods described herein not only produce tobacco with a reduced addictive potential but, concomitantly, produce a tobacco that has a reduced potential to contribute to a tobacco related disease.

It should be emphasized that the phrase “a reduced amount” as applied to nicotine and/or TSNAs is intended to refer to an amount of nicotine and/or TSNAs in a treated or transgenic tobacco plant, tobacco or a tobacco product that is less than what would be found in a tobacco plant, tobacco or a tobacco product from the same variety of tobacco, processed in the same manner, which has not been treated or was not made transgenic for reduced nicotine and/or TSNAs. Thus, in some contexts, wild-type tobacco of the same variety that has been processed in the same manner is used as a control by which to measure whether a reduction in nicotine, nornicotine, a sterol and/or TSNAs or PAHs has been obtained by the inventive methods described herein.

The amount of TSNAs (e.g., collective content of NNN, NAT, NAB, and NNK) and nicotine in wild-type tobacco varies significantly depending on the variety and the manner it is grown, harvested and cured. For example, a cured Burley tobacco leaf can have approximately 30,000 parts per million (ppm) nicotine and 8,000 parts per billion (ppb) TSNA (e.g., collective content of NNN, NAT, NAB, and NNK); a Flue-cured leaf can have approximately 20,000 ppm nicotine and 300 ppb TSNA (e.g., collective content of NNN, NAT, NAB, and NNK); and an Oriental cured leaf can have approximately 10,000 ppm nicotine and 100 ppb TSNA (e.g., collective content of NNN, NAT, NAB, and NNK). Tobacco having a reduced amount of nicotine and/or TSNA, can have no detectable nicotine and/or TSNA (e.g., collective content of NNN, NAT, NAB, and NNK), or may contain some detectable amounts of one or more of the TSNAs and/or nicotine, so long as the amount of nicotine and/or TSNA is less than that found in tobacco of the same variety, grown under similar conditions, and cured and/or processed in the same manner. That is, cured Burley tobacco, as described herein, having a reduced amount of nicotine can have between 0 and 30,000 ppm nicotine and 0 and 8,000 ppb TSNA, desirably between 0 and 20,000 ppm nicotine and 0 and 6,000 ppb TSNA, more desirably between 0 and 10,000 ppm nicotine and 0 and 5,000 ppb TSNA, preferably between 0 and 5,000 ppm nicotine and 0 and 4,000 ppb TSNA, more preferably between 0 and 2,500 ppm nicotine and 0 and 2,000 ppb TSNA and most preferably between 0 and 1,000 ppm nicotine and 0 and 1,000 ppb TSNA. Embodiments of cured Burley leaf prepared by the methods described herein can also have between 0 and 1000 ppm nicotine and 0 and 500 ppb TSNA, 0 and 500 ppm nicotine and 0 and 250 ppb TSNA, 0 and 250 ppm nicotine and 0 and 100 ppb TSNA, 0 and 100 ppm nicotine and 0 and 50 ppb TSNA, 0 and 50 ppm nicotine and 0 and 5 ppb TSNA and some embodiments of cured Burley leaf described herein have virtually no detectable amount of nicotine or TSNA. In some embodiments above, the amount of TSNA refers to the collective content of NNN, NAT, NAB, and NNK.

Similarly, a Flue-cured tobacco embodiment having a reduced amount of nicotine can have between 0 and 20,000 ppm nicotine and 0 and 300 ppb TSNA, desirably between 0 and 15,000 ppm nicotine and 0 and 250 ppb TSNA, more desirably between 0 and 10,000 ppm nicotine and 0 and 200 ppb TSNA, preferably between 0 and 5,000 ppm nicotine and 0 and 150 ppb TSNA, more preferably between 0 and 2,500 ppm nicotine and 0 and 100 ppb TSNA and most preferably between 0 and 1,000 ppm nicotine and 0 and 50 ppb TSNA. Embodiments of Flue-cured tobacco, as described herein, can also have between 0 and 500 ppm nicotine and 0 and 25 ppb TSNA, 0 and 200 ppm nicotine and 0 and 10 ppb TSNA, 0 and 100 ppm nicotine and 0 and 5 ppb TSNA and some embodiments of Flue-cured tobacco have virtually no detectable amount of nicotine or TSNA. In some embodiments above, the amount of TSNA refers to the collective content of NNN, NAT, NAB, and NNK.

Further, a cured Oriental tobacco embodiment having a reduced amount of nicotine can have between 0 and 10,000 ppm nicotine and 0 and 100 ppb TSNA, desirably between 0 and 7,000 ppm nicotine and 0 and 75 ppb TSNA, more desirably between 0 and 5,000 ppm nicotine and 0 and 50 ppb TSNA, preferably between 0 and 3,000 ppm nicotine and 0 and 25 ppb TSNA, more preferably between 0 and 1,500 ppm nicotine and 0 and 10 ppb TSNA and most preferably between 0 and 500 ppm nicotine and no detectable TSNA. Embodiments of cured Oriental tobacco can also have between 0 and 250 ppm nicotine and no detectable TSNA and some embodiments of cured Oriental tobacco have virtually no detectable amount of nicotine or TSNA. In some embodiments above, the amount of TSNA refers to the collective content of NNN, NAT, NAB, and NNK.

Some embodiments comprise cured tobaccos (e.g., Burley, Flue-cured, or Oriental) with reduced amounts of nicotine as compared to control varieties, wherein the amount of nicotine in or delivered by the product (e.g., as measured by FTC or ISO methodologies) is less than about 2 mg/g, 1 mg/g, 0.75 mg/g, 0.5 mg/g or desirably less than about 0.1 mg/g, and preferably less than 0.08 mg/g, 0.07 mg/g, 0.06 mg/g, 0.05 mg/g, 0.04 mg/g, 0.03 mg/g, 0.02 mg/g, 0.01 mg/g. Tobacco products made from these reduced nicotine and TSNA tobaccos are also embodiments. The term “tobacco products” include, but are not limited to, smoking materials (e.g., cigarettes, cigars, pipe tobacco), snuff, chewing tobacco, gum, and lozenges. As mentioned above, these reduced nicotine and TSNA tobaccos can be treated with exogenous nicotine so as to incrementally increase the amount of nicotine in the product and by employing aseptic processing and packaging techniques, the amounts of total TSNAs in the product can be kept at or below 0.5 μg/g for prolonged periods of time.

In some contexts, the phrase “reduced amount of nicotine and/or TSNAs” refers to the tobacco plants, cured tobacco, and tobacco products, as described herein, which have less nicotine and/or TSNAs (e.g., the collective content of NNN, NAT, NAB, and NNK) by weight than the same variety of tobacco grown, processed, and cured in the same way. For example, wild type cured tobacco can have has approximately 1-4% dry weight nicotine and approximately 0.2%-0.8% dry weight TSNA depending on the manner it was grown, harvested and cured. A typical cigarette has between 2-11 mg of nicotine and approximately 5.0 μg of TSNAs. Thus, the tobacco plants, tobacco and tobacco products provided herein can have or deliver, in dry weight for example, less than 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1%, 0.15%, 0.175%, 0.2%, 0.225%, 0.25%, 0.275%, 0.3%, 0.325%, 0.35%, 0.375%, 0.4%, 0.425%, 0.45%, 0.475%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, and 1.0% nicotine and less than 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, and 0.08% TSNA (e.g., collective content of NNN, NAT, NAB, and NNK).

Alternatively, a cigarette provided herein can have or deliver, for example, less than 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1.0 mg, 1.1 mg, 1.15 mg, 1.2 mg, 1.25 mg, 1.3 mg, 1.35 mg, 1.4 mg, 1.45 mg, 1.5 mg, 1.55 mg, 1.6 mg, 1.65 mg, 1.7 mg, 1.75 mg, 1.8 mg, 1.85 mg, 1.9 mg, 1.95 mg, 2.0 mg, 2.1 mg, 2.15 mg, 2.2 mg, 2.25 mg, 2.3 mg, 2.35 mg, 2.4 mg, 2.45 mg, 2.5 mg, 2.55 mg, 2.6 mg, 2.65 mg, 2.7 mg, 2.75 mg, 2.8 mg, 2.85 mg, 2.9 mg, 2.95 mg, 3.0 mg, 3.1 mg, 3.15 mg, 3.2 mg, 3.25 mg, 3.3 mg, 3.35 mg, 3.4 mg, 3.45 mg, 3.5 mg, 3.55 mg, 3.6 mg, 3.65 mg, 3.7 mg, 3.75 mg, 3.8 mg, 3.85 mg, 3.9 mg, 3.95 mg, 4.0 mg, 4.1 mg, 4.15 mg, 4.2 mg, 4.25 mg, 4.3 mg, 4.35 mg, 4.4 mg, 4.45 mg, 4.4 mg, 4.45 mg, 4.5 mg, 4.55 mg, 4.6 mg, 4.65 mg, 4.7 mg, 4.75 mg, 4.8 mg, 4.85 mg, 4.9 mg, 4.95 mg, 5.0 mg, 5.5 mg, 5.7 mg, 6.0 mg, 6.5 mgmg, 6.7 mg, 7.0 mg, 7.5 mg, 7.7 mg, 8.0 mg, 8.5 mg, 8.7 mg, 9.0 mg, 9.5 mg, 9.7 mg, 10.0 mg, 10.5 mg, 10.7 mg, and 11.0 mg nicotine and less than 0.001 μg, 0.002 μg, 0.003 μg, 0.004 μg, 0.005 μg, 0.006 μg, 0.007 μg, 0.008 μg, 0.009 μg, 0.001 μg, 0.02 μg, 0.03 μg, 0.04 μg, 0.05 μg, 0.06 μg, 0.07 μg, 0.08 μg, 0.09 μg, 0.1 μg, 0.15 μg, 0.4 μg, 0.25 μg, 0.3 μg, 0.336 μg, 0.339 μg, 0.345 μg, 0.35 μg, 0.375 μg, 0.4 μg, 0.414 μg, 0.45 μg, 0.5 μg, 0.515 μg, 0.55 μg, 0.555 μg, 0.56 μg, 0.578 μg, 0.58 μg, 0.6 μg, 0.611 μg, 0.624 μg, 0.65 μg, 0.7 μg, 0.75 μg, 0.8 μg, 0.85 μg, 0.9 μg, 0.95 μg, 1.0 μg, 1.1 μg, 1.114 μg, 1.15 μg, 1.2 μg, 1.25 μg, 1.3 μg, 1.35 μg, 1.4 μg, 1.45 μg, 1.5 μg, 1.55 μg, 1.6 μg, 1.65 μg, 1.7 μg, 1.75 μg, 1.8 μg, 1.85 μg, 1.9 μg, 1.95 μg, 2.0 μg, 2.1 μg, 2.15 μg, 2.2 μg TSNA (e.g., collective content of NNN, NAT, NAB, and NNK).

Unexpectedly, it was discovered that several methods for reducing endogenous levels of nicotine in a plant are suitable for producing tobacco that is substantially free of nitrosamines, especially TSNAs. Any method that reduces levels of other alkaloids, including norniticotine, is likewise suitable for producing tobacco substantially free of nitrosamines, especially TSNAs. As described, embodiments comprise methods of reducing the carcinogenic potential of a tobacco product comprising providing a cured tobacco as described herein and preparing a tobacco product from said cured tobacco, whereby the carcinogenic potential of said tobacco product is thereby reduced.

In some embodiments that employed the A622 inhibition construct, it was found that transgenic tobacco that had conventional levels of nicotine but significantly reduced levels of nornicotine were produced. This particular line of tobacco is particularly useful because nornicotine may be the most significant precursor for NNN in tobacco. Accordingly, reduced risk conventional cigarettes and other tobacco products (e.g., snuff) comprising the A622 inhibition construct are embodiments.

Other embodiments include the use of the cured tobacco described herein for the preparation of a tobacco product that contains reduced amounts of carcinogens as compared to control varieties and/or that reduces the amount of a TSNA or TSNA metobolite in a human that uses tobacco. In some embodiments, for example, the tobacco smoking products described herein reduce the carcinogenic potential of side stream or main stream tobacco smoke in humans exposed to said side stream or main stream tobacco smoke. By providing the modified cured tobacco described herein in a product that undergoes pyrolysis, for example, the side stream and/or main stream smoke produced by said product comprises a reduced amount of TSNAs and/or nicotine. Thus, the cured tobacco described herein can be used to prepare a tobacco smoking product that comprises a reduced amount of TSNAs in side stream and/or mainstream smoke.

In the United States, tar, nicotine, and carbon monoxide yields are obtained using the Federal Trade Commission (FTC) smoking-machine test method, which defines the measurement of tar as that material captured by a Cambridge pad when a cigarette is machine smoked, minus nicotine and water (Pillsbury, et al., 1969, “Tar and nicotine in cigarette smoke”. J. Assoc. Off. Analytical Chem., 52, 458-62). Specifically, the FTC cigarette-testing method collects smoke samples by simulating puffing volumes of 35 ml of cigarette smoke for two seconds every 58 seconds, with none of the filter ventilation holes blocked (if any), until the burn line reaches the tipping paper plus 2 mm, or a line drawn 23 mm from the end of a non-filter cigarette. This FTC smoking-machine test method has been used in the United States since 1967 to determine smoke cigarette yields for tar and nicotine. The determination of carbon monoxide yields in cigarette smoke was added to this method in 1980.

In 1967, when the FTC introduced its testing method, it issued a news release and explained that the purpose of the testing “is not to determine the amount of tar and nicotine inhaled by any human smoker, but rather to determine the amount of tar and nicotine generated when a cigarette is smoked by a machine in accordance with the prescribed method.” Nevertheless, the method serves an important role in providing an accurate way to rank and compare cigarettes according to tar, nicotine and carbon monoxide yields.

The International Standards Organization (ISO) developed a very similar smoking-machine test method for tar, nicotine, and carbon monoxide yields of cigarettes (ISO, 1991 “Cigarettes—determination of total and nicotine-free dry particulate matter using a routine analytical smoking machine” ISO: 4387:1991).

The FTC and ISO smoking methods differ in the following eight areas.

The FTC method specifies laboratory environmental conditions of 75° F.±1° F. (23.8° C.±1° C.) and a relative humidity of 60%±2% for both the equilibration and testing. The time of equilibration is a minimum of 24 hours and a maximum of 14 days. This is compared to the ISO specifications of 22° C.±1° C. and 60%±2% relative humidity for equilibration, 22° C.±2° C. and 60% relative humidity±5% for testing. The equilibration time is a minimum of 48 hours and a maximum of 10 days. The FTC defines the cigarette butt length as a minimum of 23 millimeters or the tipping paper plus three millimeters whichever is longer. ISO defines butt length as the longest of 23 millimeters or tipping paper plus three millimeters or the filter plus eight millimeters. Both methods specify a 23-millimeter butt length for non-filter cigarettes. ISO defines the position of the ashtray at 20-60 millimeters below the cigarettes in the smoking machine. FTC does not specify a position. ISO specifies a two-piece snap together reusable filter holder. This filter holder contains the Cambridge pad and uses a synthetic rubber perforated washer to partly obstruct the butt end of the cigarette. The FTC method defines the use of a Cambridge filter pad but does not specify a filter pad holder assembly. The ISO method specifies airflow across the cigarettes at the cigarette level. FTC specifies the use of a monitor cigarette to adjust airflow. The ISO procedure defines the process of wiping the excess total particulate matter (TPM) out of the used filter holder. The inner surfaces of the filter holder are wiped with two separate quarters of an unused conditioned filter pad. The FTC method uses the backside (the side opposite of the trapped TPM) to wipe the inner surface of the filter holder. ISO specifies using 20 ml per Cambridge pad of extraction solution to analyze nicotine and water in TPM. The FTC procedure defines 10 ml per Cambridge pad. ISO defines the internal standards for the gas chromatographic determination of nicotine and water. The FTC procedure does not specify the internal standards.

These differences typically result in slightly lower measured deliveries for the ISO Method versus the FTC Method. The measured values between FTC and ISO methods are within the detection limits of the test or about no greater than 0.4 mg tar and about 0.04 mg nicotine for cigarettes that yield over about 10 mg.

In some embodiments, for example, the collective content of NNN, NAT, NAB, and NNK in the mainstream or side stream smoke from a tobacco product comprising the modified tobacco, including genetically modified tobacco, described herein is between about 0-5.0 μg/g, 0-4.0 μg/g, 0-3.0 μg/g, 0-2.0 μg/g, 0-1.5 μg/g, 0-1.0 μg/g, 0-0.75 μg/g, 0-0.5 μg/g, 0-0.25 μg/g, 0-0.15 μg/g, 0-0.1 μg/g, 0-0.05 μg/g, 0-0.02 μg/g, 0-0.015 μg/g, 0-0.01 μg/g, 0-0.005 μg/g, 0-0.002 μg/g, or 0-0.001 μg/g. That is, some embodiments are genetically modified Burley tobacco, wherein the side stream or mainstream smoke produced from a tobacco product comprising said Burley tobacco has a collective content of NNN, NAT, NAB, and NNK in the mainstream or side stream smoke between about 0-5.0 μg/g, 0-4.0 μg/g, 0-3.0 μg/g, 0-2.0 μg/g, 0-1.5 μg/g, 0-1.0 μg/g, 0-0.75 μg/g, 0-0.5 μg/g, 0-0.25 μg/g, 0-0.15 μg/g, 0-0.1 μg/g, 0-0.05 μg/g, 0-0.02 μg/g, 0-0.015 μg/g, 0-0.01 μg/g, 0-0.005 μg/g, 0-0.002 μg/g, or 0-0.001 μg/g.

Other embodiments concern modified Flue-cured tobacco, such as genetically modified Flue-cured tobacco, wherein the sidestream or mainstream smoke produced from a tobacco product comprising said Flue-cured tobacco has a collective content of NNN, NAT, NAB, and NNK in the mainstream or side stream smoke between about 0-5.0 μg/g, 0-4.0 μg/g, 0-3.0 μg/g, 0-2.0 μg/g, 0-1.5 μg/g, 0-1.0 μg/g, 0-0.75 μg/g, 0-0.5 μg/g, 0-0.25 μg/g, 0-0.15 μg/g, 0-0.1 μg/g, 0-0.05 μg/g, 0-0.02 μg/g, 0-0.015 μg/g, 0-0.01 μg/g, 0-0.005 μg/g, 0-0.002 μg/g, or 0-0.001 μg/g.

More embodiments concern modified Oriental tobacco, wherein the sidestream or mainstream smoke produced from a tobacco product comprising said Oriental tobacco has a collective content of NNN, NAT, NAB, and NNK in the mainstream or side stream smoke between about 0-5.0 μg/g, 0-4.0 μg/g, 0-3.0 μg/g, 0-2.0 μg/g, 0-1.5 μg/g, 0-1.0 μg/g, 0-0.75 μg/g, 0-0.5 μg/g, 0-0.25 μg/g, 0-0.15 μg/g, 0-0.1 μg/g, 0-0.05 μg/g, 0-0.02 μg/g, 0-0.015 μg/g, 0-0.01 μg/g, 0-0.005 μg/g, 0-0.002 μg/g, or 0-0.001 μg/g.

Additional Tobacco Modifications

Additional modified tobaccos that can be used in the methods and tobacco products provided herein include, but are not limited to, chemically modified tobacco, expanded, extracted, or puffed tobacco, and reconstituted tobacco.

Any of a variety of chemically modified tobaccos can be included in the methods and tobacco products provided herein. For example, the chemical modification can include palladium, or can include an auxin, auxin analog, or jasmonate antagonist (see e.g., U.S. Pat. No. 6,789,548 and U.S. Pat. App. Pub. No. 20050072047, both of which are hereby expressly incorporated by reference in their entirety).

By one approach, a chemically modified tobacco is made as follows. A tobacco is provided and a casing solution is applied thereto. Thereafter, a plurality of metallic or carbonaceous catalytic particles having a mean average or a mode average particle size of less than about 20 microns is applied to the tobacco in a form separate from the casing solution. Next, a nitrate or nitrite source in a form separate from the casing solution and in a form separate from the plurality of metallic or carbonaceous catalytic particles is applied to the tobacco, before, after or simultaneously with applying the plurality of particles but after applying the casing solution, whereby a smoking composition is obtained. In some embodiments of this modified tobacco, a polyaromatic hydrocarbon, azaarene, carbazole, or a phenolic compound is reduced. Using this approach, the Omni® tobacco product was developed.

By another approach, a chemically modified tobacco is made by identifying a tobacco plant in a field for nicotine reduction; and contacting said tobacco plant with a composition selected from the group consisting of an auxin, auxin analog, and jasmonate antagonist from between about 21 days before topping to about 21 days after topping said tobacco plant, whereby the amount of nicotine in said topped tobacco plant contacted with said composition is below that of a topped tobacco plant of the same variety, grown under the same conditions, which has not been contacted with said composition.

In another example, the chemically modified tobacco can be extracted tobacco. By some approaches the chemically modified tobacco is extracted with an organic solvent and other processes use super-critical fluid extraction or carbon dioxide. In another example, the chemical modification can be a biotic modification. Microbes that ingest nitrates and alkaloids can be applied to tobacco so as to obtain a reduced nicotine tobacco; for example such a biotic modification can include bacteria. In another example, the tobacco is processed to remove the presence of a microbe. In another example the chemically modified tobacco can be sterilized, pasteurized, or radiated.

In another example, the chemically modified tobacco can have added thereto an exogenous component of tobacco or analog thereof. Tobacco can be modified to increase or decrease one or more compounds such as proteins, metabolites, nicotine-related compounds and sterols. In some methods provided herein, a tobacco which has been modified to produce lower levels of one or more compounds such as nicotine or a nicotine metabolite, or a sterol, can have exogenously added thereto, one of these lower-level compounds, one or more but not all lower-level compounds, or all lower-level compounds or an analog of the compound(s).

Such tobaccos with one or more exogenously added compounds can be compared in accordance with the methods provided herein to the same tobacco to which no exogenous compound has been added, to which a different exogenous compound has been added, or to which a different level of the same exogenous compound has been added. For example, the methods provided herein can be used to compare a tobacco that has been genetically modified to produce reduced nicotine levels with the same tobacco to which exogenous nicotine or a nicotine analog has been added thereto. By performing such methods, the role of the exogenously added compound on cell damage or other response determined according to the methods provided herein (e.g., apoptosis or cell proliferation), can be determined.

In another example, the chemically modified tobacco has had added thereto a compound or composition containing antioxidants. Tobacco at any stage of its processing can have added thereto an antioxidant compound or a composition with antioxidant properties. Any of a variety of known antioxidant compounds can be added to the tobacco, including, but not limited to, lycopene, tocopherol, tocopherol metabolites, ascorbic acid, unsaturated fatty acids, N-acetyl cysteine, and other antioxidants known in the art. A composition with antioxidant properties can include a biological composition or extract that can neutralize oxidants, such as milk or milk proteins, tumeric or tumeric extracts, barley or barley extracts, alfalfa or alfalfa extracts. Other compounds that can be added to the tobacco include thiol-containing proteins, plant extracts, aromatic compounds (e.g., caffeine or pentoxyfyllen, which are contemplated to scavenge carcinogens).

Another form of modified tobacco is expanded or puffed tobacco. Included herein are methods to produce reduced-exposure tobacco products by utilizing the tobacco provided herein, deproteinized tobacco fiber, and freeze dried tobacco in any combination and in conjunction with expanded or puffed tobacco. More than 150 patents have been issued related to tobacco expansion (e.g., U.S. Pat. No. 3,991,772, herein expressly incorporated by reference in its entirety). “Expanded tobacco” is an important part of tobacco filler which is processed through expansion of suitable gases so that the tobacco is “puffed” resulting in reduced density and greater filling capacity. It reduces the weight of tobacco used in cigarettes. Advantageously, expanded tobacco reduces tar, nicotine and carbon monoxide deliveries and finds use, for example, in making low tar, low nicotine, and low carbon monoxide delivery cigarettes. Expanded tobacco is particularly useful in making low-tar delivery cigarettes. Carlton® cigarettes, which have had claims of being the lowest tar and nicotine delivery cigarette, are reportedly made with a very large percentage of expanded tobacco. However, use of expanded tobacco also results in reduced nicotine delivery, which can result in compensation.

Any method for expansion of tobacco known in the art can be used in the methods provided herein. The most common method used today incorporates liquid carbon dioxide (U.S. Pat. Nos. 4,340,073 and 4,336,814, herein expressly incorporated by reference in its entirety). Liquid propane has also been used for making commercial cigarettes, predominantly in Europe (U.S. Pat. No. 4,531,529, herein expressly incorporated by reference in its entirety). Liquid propane offers advantages over carbon dioxide since higher 3Q degrees of expansion are possible, in the range of 200%. Under pressure, the liquid carbon dioxide (or liquid propane) permeates the tobacco cell structure. When the tobacco is rapidly heated the carbon dioxide (or liquid propane) expands the cell back to its pre-cured size.

Another form of modified tobacco is reconstituted tobacco. Included herein are methods to produce reduced-exposure tobacco products by utilizing the tobacco provided herein, deproteinized tobacco fiber, and freeze dried tobacco in any combination and in conjunction with reconstituted tobacco. “Reconstituted tobacco” (“Recon”) is an important part of tobacco filler made from tobacco dust and other tobacco scrap material, processed into sheet form and cut into strips to resemble tobacco. In addition to the cost savings, reconstituted tobacco is very important for its contribution to cigarette taste from processing flavor development using reactions between ammonia and sugars.

The process to produce sheets of Recon began during the 1950s. U.S. patents that describe such processes include: U.S. Pat. Nos. 3,499,454, 4,182,349, 4,962,774, and 6,761,175, herein expressly incorporated by reference in their entirety. Recon is traditionally produced from tobacco stems and/or smaller leaf particles in a process that closely resembles a typical paper making process. The tar and nicotine yields of reconstituted tobacco are lower than those from equivalent quantities of whole tobacco leaf. This process entails processing the various tobacco portions that are to be made into Recon. After the Recon sheets are produced they are cut into a size and shape that resembles cut rag tobacco made from whole leaf tobacco. This cut Recon then gets mixed with cut-rag tobacco and is ready for cigarette making. Cigarettes can be manufactured with all Recon, no Recon, or any combination thereof. Most major brands have at least 10% of Recon in the Filler.

In another embodiment nicotine can be added, or nicotine salts, to produce Recon, which is made from reduced-nicotine transgenic tobacco or any non-tobacco plant material including but not limited to herbal blends so that when the Recon is burned it yields substantially less tobacco-specific nitrosamines and other carcinogens produced from conventional cigarettes, yet satisfactory amounts are nicotine are present.

Processes of removing proteins from tobacco, thereby creating “deproteinized tobacco fiber” are known in the art, as exemplified in U.S. Pat. Nos. 4,289,147 and 4,347,324, herein expressly incorporated by reference in its entirety. Tobacco fiber is a major byproduct after removing protein. The fibrous remains from deproteinized tobacco can be included in any percentage as an ingredient of Recon. Cigarettes made from deproteinized tobacco have a different taste than conventional cigarettes. However, appropriate amounts of additives, including flavorings and nicotine, can be added to help alleviate this taste deficiency.

Cigarettes containing deproteinized tobacco have a significant advantage over conventional cigarettes since they produce reduced levels of carcinogens and harmful combustion products. “A 71% reduction in protein content of a Flue-cured tobacco sheet resulted in an 81% reduction in the TA98 Ames mutagenicity” of the pyrolytic condensate (Clapp, W. L., et al., “Reduction in Ames Salmonella mutagenicity of cigarette mainstream smoke condensate by tobacco protein removal”, Mutation Research, 446, pg 167-174, 1999). Previous research in this area had determined that tobacco leaf protein might be the principal precursor of mutagens in TSC (Matsumoto, et al., “Mutagenicities of the pyrolysis of peptides and proteins”, Mutation Research, 56, pg 281-288, 1978).

Extracting tobacco fiber from genetically modified reduced-nicotine tobacco effectively eliminates virtually all carcinogenic TSNAs in such tobacco, since nitrosamines require relatively high concentrations of nicotine and other alkaloids to form at detectable levels. Therefore, it can be advantageous to utilize reduced-nicotine tobacco in reduced-exposure cigarettes or other tobacco products to further reduce TSNAs. Nicotine can be either left out or introduced later in the process, which can also be in the form of nicotine salts.

PAHs are formed from high temperature pyrolysis of amino acids, sugars, paraffins, terpenes, phytosterols, celluloses and other components of tobacco. Most of these components are greatly reduced in tobacco fiber, effectively reducing formation of PAHs. Catechols and phenols, recognized carcinogenic co-factors in CS, would also be reduced since low levels of soluble sugar are present in tobacco fiber.

Harmful gas phase compounds such as hydrogen cyanide, nitrogen oxides, and carbon monoxide are also reduced when cigarette containing only tobacco fiber is smoked compared to cigarettes made with whole-leaf tobacco. Hydrogen cyanide is formed from burning proteins and chlorophyll. Nitrogen oxides are formed from burning soluble protein, chlorophyll, nitrates, and alkaloids. These components would not be present in significant amounts in deproteinized tobacco. Tobacco fiber has approximately 85 percent less starches and cellulosic material thus reducing the major pyrolytic precursors of carbon monoxide.

In another embodiment, methods are provided to produce reconstituted tobacco that includes extracted tobacco fiber derived from conventional tobacco, reduced-nicotine transgenic tobacco, or increased-nicotine transgenic tobacco.

If the tobacco curing process is circumvented, virtually no TSNAs will be present in traditional tobacco products such as cigarettes, cigar filler or wrapper, roll-your-own tobacco for cigarettes, pipe tobacco, chewing tobacco, snuff, reconstituted tobacco and other preparations made with freeze-dried tobacco would contain virtually no TSNAs since traditional curing processes are eliminated.

In another embodiment TSNAs can be virtually eliminated through processing freshly harvested tobacco using lyophilization. This is accomplished by processing freshly harvested tobacco through freeze-drying units located near tobacco farms. Tobacco processed in this manner can be grown in a traditional fashion with spacing of plants or in a biomass setting. In addition to the economic advantages of eliminating the costs associated with the curing process, the tobacco can now be grown in a biomass fashion that can create hundreds of thousands of pounds of fresh tobacco per acre.

By growing tobacco in a biomass setting and immediately freeze drying the fresh tobacco for cigarettes, roll-your-own-tobacco, pipe tobacco, cigar filler or wrapper, chewing tobacco, snuff, and other versions of smokeless tobacco, labor is reduced not only by eliminating the transplant of each plant from greenhouse to the field but also by eliminating traditional harvesting and curing of the tobacco. Also, farmland needed for this purpose is greatly reduced. The yield of tobacco from one acre of tobacco grown in biomass is equivalent to approximately 100 acres of tobacco grown in a traditional manner.

“Tobacco biomass” is achieved by directly sowing an acre of land with copious quantities of tobacco seed within a few inches of each other in the field. Unlike tobacco planted with traditional spacing, individual plants can no longer be differentiated when tobacco is planted in a biomass fashion. An acre of tobacco biomass has the appearance of a continuous, dense, green carpet. U.S. Pub. Pat. App. No. 20020197688, herein expressly incorporated by reference in its entirety, describes such methods.

Lyophilization removes most of the water (−80%) from the weight of fresh harvested tobacco biomass. The result is Freeze Dried Tobacco (“FDT”). FDT is easily pulverized into fine particles suitable for processing into Recon. This Recon can be cut and made into any type of tobacco product such as filler for cigarettes, roll-your-own-tobacco, pipe tobacco, cigar filler or wrapper, chewing tobacco, snuff, and other forms of smokeless tobacco. Flavorings and additives, including sugars, can be incorporated into the recon process.

Such Recon can be made from 100 percent FDT or in any proportion that consumers prefer. The lyophilization process can have adverse affects on the taste of such tobacco products. Therefore, FDT can even be mixed in any percentage with traditional pulverized, cured tobacco so that the mixture can be made into Recon. Alternatively, FDT can be mixed in any percentages with any forms of traditional tobacco conducive for manufacturing cigarettes, roll-your-own tobacco, pipe tobacco, and cigar filler or wrapper, chewing tobacco, snuff and other versions of smokeless tobacco in order to satisfy the tastes of the mass market.

In another embodiment, genetically modified reduced-nicotine tobacco can be used for reducing TSNAs as described elsewhere herein, thereby creating an additional benefit of such cigarettes, roll-your-own-tobacco, pipe tobacco, cigar filler or wrapper, chewing tobacco, snuff and other versions of smokeless tobacco being non-addictive and without any TSNAs.

In another embodiment, nicotine can be added, in amounts that deliver the desired physiological response, back to the FDT for uses in cigarettes, cigar filler or wrapper, roll-your-own tobacco for cigarettes, pipe tobacco, chewing tobacco, snuff, and other versions of smokeless tobacco so that they will contain virtually no TSNAs. Cigarettes produced from tobacco fiber obtained from green leaf cured tobacco.

In another embodiment, Nicotiana rustica and/or increased-nicotine transgenic Nicotiana tabacum are freeze dried after harvest and are incorporated into recon. The benefits are that the high alkaloid content is preserved for low TNR cigarettes and that the tobacco curing step is saved. Also, the associated increase in TSNAs with high alkaloid tobaccos will not materialize. Preferred tobaccos for use with the methods described herein include genetically modified tobaccos as described in the following sections.

Curing

The curing process, which typically lasts about 1 week, brings out the flavor and aroma of tobacco. Several methods for curing tobacco may be used, and indeed many methods have been previously disclosed. For example, U.S. Pat. No. 4,499,911 to Johnson; U.S. Pat. No. 5,685,710 to Martinez Sagrera; U.S. Pat. No. 3,905,123 to Fowler; U.S. Pat. No. 3,840,025 to Fowler; and U.S. Pat. No. 4,192,323 to Home describe aspects of the tobacco curing process which may be used for some embodiments provided herein. Conventionally, “sticks” that are loaded with tobacco are placed into bulk containers and placed into closed buildings having a heat source known as a curing barn. A flue is often used to control the smoke (thus earning the term “Flue-cured”). The method of curing will depend, in some cases, on the type of tobacco-use cessation product desired, (i.e., snuff, cigarettes, or pipe tobacco may preferably utilize different curing methods) and preferred methods may vary from region to region and in different countries. In some approaches, the stems and midveins of the leaf are removed from the leaves prior to curing to yield a high quality, low TSNA tobacco product.

“Flue-curing” is a popular method for curing tobacco in Virginia, North Carolina, and the Coastal Plains regions of the United States. This method is used mainly in the manufacture of cigarettes. Flue-curing requires a closed building equipped with a system of ventilation and a source of heat. The heating can be direct or indirect (e.g., radiant heat). When heat and humidity are controlled, leaf color changes, moisture is quickly removed, and the leaf and stems dry. Careful monitoring of the heating and humidity can reduce the accumulation of TSNAs.

Another curing method is termed “air-curing”. In this method, an open framework is prepared in which sticks of leaves (or whole plants) are hung so as to be protected from both wind and sun. Leaf color changes from green to yellow, as leaves and stems dry slowly.

“Fire-curing” employs an enclosed barn similar to that used for flue-curing. The tobacco is hung over low temperature fire so that the leaves cure in a smoke-laden atmosphere. This process uses lower temperatures, so the process may take up to a month, in contrast to flue-curing, which takes about 6 to 8 days.

A further curing method, termed “sun-curing” is the drying of uncovered sticks or strings of tobacco leaves in the sun. The best known sun-cured tobaccos are the so-called Oriental tobaccos of Turkey, Greece, Yugoslavia, and nearby countries.

The curing process, and most particularly the flue-curing process, is generally divided into the following four stages:

A) Firing Up: During this step, the tobacco leaves turn bright lemon-orange in color. This is achieved by a gradual increase in temperature. B) Leaf Yellowing: In this step any moisture is removed. This creates the “yellowing” of the tobacco. It also prepares the tobacco for drying in the next step. C) Leaf Drying: Leaf drying, an important step in the curing process, requires much time for the tobacco to dry properly. Additionally, air flow is increased in this step to facilitate the drying process. D) Stem Drying: The drying process continues, as the stem of the tobacco leaf becomes dried.

The cured tobacco may then be blended with other tobaccos or other materials to create the product to be used for the tobacco-use cessation method. The section below describes typical methods of blending and preparing a tobacco product provided herein.

Tobacco Blending

It may be desirable to blend tobacco of varying nicotine levels to create the cessation product having the desired level of nicotine. This blending process is typically performed after the curing process, and may be performed by conventional methods. Preferred tobacco blending approaches are provided below. In some embodiments, blending of the transgenic tobacco is conducted to prepare the tobacco so that it will contain specific amounts of nicotine, nornicotine, sterol and/or TSNA in specific products. Preferably, the blending is conducted so that tobacco products of varying amounts of nicotine are made in specific products.

A mixture that contains different types of tobacco is desirably substantially homogeneous throughout in order to avoid undesirable fluctuations in taste or nicotine levels. Typically, tobacco to be blended may have a moisture content between 30 and 75%. As an example, the tobacco is first cut or shredded to a suitable size, then mixed in a mixing device, such as a rotating drum or a blending box. One such known mixing device is a tumbling apparatus that typically comprises a rotating housing enclosing mixing paddles which are attached to and, therefore, rotate with the housing to stir the tobacco components together in a tumbling action as the drum turns.

After the desired tobaccos are thoroughly mixed, the resulting tobacco blend is removed from the mixing apparatus and bulked to provide a continuous, generally uniform quantity of the tobacco blend. The tobacco is then allowed to remain relatively undisturbed (termed the “bulking step”) for the required period of time before subsequent operations are performed. The bulking step typically takes 30 minutes or less, and may be carried out on a conveyor belt. The conveyor belt allows the blended tobacco to remain in bulk form in an undisturbed condition while it is continuously moving the tobacco blend through the process from the mixing stage to the expansion stage.

The tobacco blend is typically expanded by the application of steam. The tobacco mixture is typically subjected to at least 0.25 pounds of saturated steam at atmospheric conditions per pound of blended tobacco for at least 10 seconds to provide an increase in moisture of at least 2 weight percent to the tobacco blend. After the tobacco blend has been expanded, it is dried. A typical drying apparatus uses heated air or superheated steam to dry the tobacco as the tobacco is conveyed by the heated air or steam stream through a drying chamber or series of drying chambers. Generally, the wet bulb temperature of the drying air may be from about 150 degrees F. to about 211 degrees F. The tobacco blend is typically dried to a moisture content of from about 60% to about 5%. The dried, expanded tobacco blend is then in a suitable mode to be processed into the tobacco-use cessation product as described below.

Some blending approaches begin with tobacco prepared from varieties that have extremely low amounts of nicotine, nornicotine, sterols and/or TSNAs. By blending prepared tobacco from a low nicotine/TSNA variety (e.g., undetectable levels of nicotine and/or TSNAs) with a conventional tobacco (e.g., Burley, which has 30,000 parts per million (ppm) nicotine and 8,000 parts per billion (ppb) TSNA; Flue-cured, which has 20,000 ppm nicotine and 300 ppb TSNA; and Oriental, which has 10,000 ppm nicotine and 100 ppb TSNA), tobacco products having virtually any desired amount of nicotine and/or TSNAs can be manufactured. Other approaches blend only low nicotine/TSNA tobaccos (e.g., genetically modified Burley, genetically modified Virginia Flue-cured, and genetically modified Oriental tobaccos that contain reduced amounts of nicotine and/or TSNAs) and/or low sterol tobaccos (e.g., Burley, Flue-cured, and Oriental). Tobacco products having various amounts of nicotine and/or TSNAs can be incorporated into tobacco-use cessation kits and programs to help tobacco users reduce or eliminate their dependence on nicotine and reduce the carcinogenic potential.

By one approach, a step 1 tobacco product is comprised of approximately 25% low nicotine/TSNA tobacco and 75% conventional tobacco; a step 2 tobacco product can be comprised of approximately 50% low nicotine/TSNA tobacco and 50% conventional tobacco; a step 3 tobacco product can be comprised of approximately 75% low nicotine/TSNA tobacco and 25% conventional tobacco; and a step 4 tobacco product can be comprised of approximately 100% low nicotine/TSNA tobacco and 0% conventional tobacco. By another approach, a step 1 tobacco product is comprised of approximately 25% low sterol/PAH tobacco and 75% conventional tobacco; a step 2 tobacco product can be comprised of approximately 50% low sterol/PAH tobacco and 50% conventional tobacco; a step 3 tobacco product can be comprised of approximately 75% low sterol/PAH tobacco and 25% conventional tobacco; and a step 4 tobacco product can be comprised of approximately 100% low sterol/PAH tobacco and 0% conventional tobacco. By another approach, a step 1 tobacco product is comprised of approximately 25% low sterol/PAH and low nicotine/TSNA tobacco and 75% conventional tobacco; a step 2 tobacco product can be comprised of approximately 50% low sterol/PAH and low nicotine/TSNA tobacco and 50% conventional tobacco; a step 3 tobacco product can be comprised of approximately 75% low sterol/PAH and low nicotine/TSNA tobacco and 25% conventional tobacco; and a step 4 tobacco product can be comprised of approximately 100% low sterol/PAH and low nicotine/TSNA tobacco and 0% conventional tobacco. A tobacco-use cessation kit can comprise an amount of tobacco product from any combination of the aforementioned blends to satisfy a consumer for a single month program. That is, if the consumer is a one pack per day smoker, for example, a single month kit would provide 7 packs from each step, a total of 28 packs of cigarettes. Each tobacco-use cessation kit would include a set of instructions that specifically guide the consumer through the step-by-step process. Of course, tobacco products having specific amounts of nicotine, TSNA, sterol and/or PAH would be made available in conveniently sized amounts (e.g., boxes of cigars, packs of cigarettes, tins of snuff, and pouches or twists of chew) so that consumers could select the amount of nicotine, TSNA, sterol and/or PAH they individually desire. There are many ways to obtain various low nicotine/low TSNA and/or low sterol/low PAH tobacco blends using the tobaccos and teachings described herein and the following is intended merely to guide one of skill in the art to one possible approach.

To obtain a step 1 tobacco product, which is a 25% low nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm nicotine/TSNA tobacco can be mixed with conventional Burley, Flue-cured, or Oriental in a 25%/75% ratio respectively to obtain a Burly tobacco product having 22,500 ppm nicotine and 6,000 ppb TSNA, a Flue-cured product having 15,000 ppm nicotine and 225 ppb TSNA, and an Oriental product having 7,500 ppm nicotine and 75 ppb TSNA. Similarly, to obtain a step 2 product, which is 50% low nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm nicotine/TSNA tobacco can be mixed with conventional Burley, Flue-cured, or Oriental in a 50%/50% ratio respectively to obtain a Burly tobacco product having 15,000 ppm nicotine and 4,000 ppb TSNA, a Flue-cured product having 10,000 ppm nicotine and 150 ppb TSNA, and an Oriental product having 5000 ppm nicotine and 50 ppb TSNA. Further, a step 3 product, which is a 75%/25% low nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm nicotine/TSNA tobacco can be mixed with conventional Burley, Flue-cured, or Oriental in a 75%/25% ratio respectively to obtain a Burly tobacco product having 7,500 ppm nicotine and 2,000 ppb TSNA, a Flue-cured product having 5,000 ppm nicotine and 75 ppb TSNA, and an Oriental product having 2,500 ppm nicotine and 25 ppb TSNA.

By a preferred method, conventional Virginia Flue-cured tobacco was blended with genetically modified Burley (i.e., Burley containing a significantly reduced amount of nicotine and TSNA) to yield a blended tobacco that was incorporated into three levels of reduced nicotine cigarettes: a step 1 cigarette containing 0.6 mg nicotine, a step 2 cigarette containing 0.3 mg nicotine, and a step 3 cigarette containing less than 0.05 mg nicotine. The amount of total TSNA was found to range between approximately 0.17 μg/g-0.6 μg/g

In some cigarettes, approximately, 28% of the blend was Virginia Flue-cured tobacco, approximately 29% of the blend was genetically modified (i.e., reduced nicotine Burley), approximately 14% of the blend was Oriental, approximately 17% of the blend was expanded Flue-cured stem, and approximately 12% was standard commercial Recon. The amount of total TSNAs in cigarettes containing this blend was approximately 1.5 μg/g.

It should be appreciated that tobacco products are often a blend of many different types of tobaccos, which were grown in many different parts of the world under various growing conditions. As a result, the amount of nicotine, TSNAs, sterols and PAHs will differ from crop to crop. Nevertheless, by using conventional techniques one can easily determine an average amount of nicotine, TSNA, sterol and PAH per crop used to create a desired blend. It should also be appreciated that reconstituted, expanded, chemically treated, or microbial treated tobacco can be blended with the modified tobacco described herein, such as, for example the transgenic tobacco described herein. By adjusting the amount of each type of tobacco that makes up the blend one of skill can balance the amount of nicotine, TSNA, sterol and/or PAH with other considerations such as appearance, flavor, and smokability. In this manner, a variety of types of tobacco products having varying level of nicotine, TSNA, sterol and/or PAH, as well as, appearance, flavor and smokability can be created.

A. Genetically Modified Tobacco

In some embodiments, the modified tobacco is a genetically modified tobacco. Several approaches to create genetically modified tobacco having a reduced amount of a harmful compound are described. Many embodiments concern nucleic acid constructs that inhibit the expression of a gene, which regulates production of a compound that is associated with a tobacco-related disease. Since these nucleic acid constructs efficiently reduce the presence of a compound that contributes to a tobacco-related disease, the genetically modified tobacco, prepared as described herein, can be used to create a tobacco product, such as a cigarette, snuff or pipe tobacco, which has a reduced potential to contribute to a tobacco-related disease. That is, embodiments provided herein concern reduced risk tobacco products made from reduced risk transgenic tobacco created using the nucleic acid constructs described herein.

More specifically, embodiments provided herein concern nucleic acid constructs that inhibit the expression of a number of genes involved in the synthesis and regulation of the production of nicotine, nornicotine, and/or sterols in tobacco. Alkaloids such as nicotine and nornicotine are precursors for a number of harmful compounds that contribute to tobacco-related disease (e.g., the tobacco specific nitrosamines (TSNAs): N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB), 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(N-nitrosomethylamino)-4-(3-pyridyl)-1-butanal (NNA)-4-N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAL), 4-N-nitrosomethylamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and/or 4-(N-nitrosomethylamino)-4-(3-pyridyl)-butanoic acid (iso-NNAC) and acrolein). Sterols are precursors for a number of harmful compounds, which are generated by pyrolysis of tobacco, that also contribute to tobacco-related disease (e.g., polyaromatic hydrocarbons (PAHs), such as benz[a]pyrene (BAP), heterocyclic hydrocarbons, terpenes, paraffins and aromatic amines). Because the presence of these harmful compounds in tobacco contributes to tobacco-related disease, a transgenic or genetically modified tobacco that comprises a reduced amount of any one of these compounds, as compared to a reference tobacco has a reduced potential to contribute to a tobacco-related disease.

Other embodiments concern nucleic acid constructs for heterologous expression of a gene that reduces, or is related to production of a compound that reduces, the harmful effect of one or more compounds associated with a tobacco-related disease. Since these nucleic acid constructs introduce or increase the presence of a compound that results in reduction of the harmful effect of a compound associated with a tobacco-related disease, the genetically modified tobacco, prepared as described herein, can be used to create a tobacco product, such as a cigarette, snuff or pipe tobacco, which has a reduced potential to contribute to a tobacco-related disease. That is, embodiments provided herein concern reduced risk tobacco products made from reduced risk transgenic tobacco created using the nucleic acid constructs described herein.

Other embodiments are directed to genetically modified tobacco in which expression of two or more genes in the biosynthetic pathway of a compound associated with a tobacco-related disease is inhibited. Inhibition of two or more genes in the biosynthetic pathway of a compound associated with a tobacco-related disease can be attained by inhibition of two or more genes that act on a substrate at the same step in the biosynthetic pathway (e.g., inhibition of two or more isoforms of a biosynthetic gene) or inhibition of two or more genes that act on a substrate at different steps in the biosynthetic pathway. In such embodiments, the genetically modified tobacco can contain one or more heterologous nucleic acids such as the nucleic acids and constructs provided herein, where the heterologous nucleic acids can contain one or more sequences that can inhibit expression of two or more genes in the biosynthetic pathway of a compound associated with a tobacco-related disease.

Other embodiments are directed to genetically modified tobacco in which the active form of a gene in the biosynthetic pathway of a compound associated with a tobacco-related disease is inhibited. The active form of a gene in the biosynthetic pathway of a compound associated with a tobacco-related disease can be inhibited by any of a variety of methods for inhibiting protein activity, including, but not limited to: knocking out part or all of a gene encoding the endogenous protein using, for example, homologous recombination; and heterologous expression of a dominant negative protein that inhibits the activity of the endogenous protein.

By using the constructs described herein, the amount of harmful compounds in tobacco or the harmful effects thereof, such as alkaloids and sterols, can be reduced or removed and a tobacco product comprising this genetically modified tobacco, with or without exogenous nicotine, will have a reduced potential to contribute to a tobacco-related disease. That is, genetically modified tobacco comprising the constructs described herein can be used to manufacture “reduced risk” tobacco products (e.g., a tobacco product comprising a reduced endogenous nicotine, reduced endogenous nornicotine, and/or reduced sterol tobacco), such as a cigarette, snuff or pipe tobacco, which may have exogenous nicotine incorporated therein.

Accordingly, embodiments provided herein concern genetically modified tobacco and tobacco products containing a tobacco that comprises a genetic modification, which have a reduced amount or are substantially free of a harmful compound including, but not limited to, nicotine, nornicotine, a sterol, an acrolein, an aldehyde, a TSNA selected from the group consisting of N′-nitrosonornicotine (NNN), 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosoanatabine (NAT), and/or N′-nitrosoanabasine (NAB) or generate a reduced amount of a PAH, a BAP, a heterocyclic hydrocarbon, an aromatic amine upon pyrolysis, wherein this reduced risk genetically modified tobacco is made by lowering the expression of a gene in said tobaccos with one of the constructs described herein. Preferred embodiments include a transgenic tobacco and a tobacco product (e.g., cigarette) that comprises a cured tobacco comprising a genetic modification and comprising or delivering by FTC method a reduced amount of nicotine or total alkaloid (e.g., below a conventional level of nicotine or total alkaloid typical for the strain of plant, preferably, less than or equal to 3,000 ppm, 2000 ppm, 1000 ppm, or 500 ppm), wherein said genetic modification comprises an inhibition of a gene that regulates the production of nicotine and/or nornicotine, such as arginine decarboxylase (ADC), methylputrescine oxidase (WO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT), S-adenosyl-methionine synthetase (SAMS), or A622 or comprises an inhibition of a gene that regulates the production of sterol biosynthesis include HMG-CoA reductase, 14alpha demethylase, squalene synthase, SMT2, SMT1, C14 sterol reductase, A8-A7-isomerase, or C4-demethylase, using one or more of the constructs described herein.

Preferred embodiments also include a transgenic tobacco and a tobacco product (e.g., cigarette, snuff or pipe tobacco) that comprises a cured tobacco comprising a genetic modification and a reduced amount of a sterol (e.g., comprises an amount of sterol or delivers and amount of sterol that is below a conventional level of said sterol typical for the strain of plant) wherein said genetic modification comprises an inhibition of a gene that regulates the production of a sterol in tobacco using one or more of the constructs described herein. Related embodiments include a transgenic tobacco and tobacco product made therefrom (e.g., a cigarette, snuff or pipe tobacco) that upon pyrolysis generates a reduced amount of a PAH, BAP, a heterocyclic hydrocarbon, or an aromatic amine, as compared to that generated by a reference tobacco or reference tobacco product (e.g., 1M16, 2R4F or 1R5F), a commercially available tobacco product of the same class (e.g., full-flavor, lights, and ultra-lights), or, preferably, a tobacco of the same variety (e.g., Burley, Va. Flue-cured, or Oriental) or strain (e.g., LA Burley 21, K326, Tn90, Djebel174) as the transgenic tobacco prior to genetic modification).

Preferred embodiments also include a transgenic tobacco and a tobacco product (e.g., cigarette, snuff or pipe tobacco) that comprises a cured tobacco comprising a genetic modification and a reduced amount of nicotine or total alkaloid and a sterol (e.g., comprise or provides an amount of nicotine or total alkaloid an/or sterols that is below a conventional level of nicotine, total alkaloid, or sterol typical for the strain of plant) wherein said genetic modification comprises an inhibition of a gene that regulates the production of both nicotine and sterols in tobacco. That is, embodiments provided herein concern isolated nucleic acids, isolated nucleic acid cassettes, and isolated nucleic acid constructs that inhibit the expression of a plurality of genes that regulate the production of nicotine and TSNAs, isolated nucleic acids, isolated nucleic acid cassettes, and isolated nucleic acid constructs that inhibit the expression of a plurality of genes that regulate the production of sterols and, thus PAHs, and isolated nucleic acids, isolated nucleic acid cassettes, and isolated nucleic acid constructs that inhibit the expression of a plurality of genes that regulate the production of nicotine and TSNAs and sterols and, thus, PAHs (e.g., a double knock-out of at least two different genes that regulate the production of at least two different harmful compounds in tobacco).

In some embodiments, the tobacco that is substantially free or comprises a reduced amount of nicotine, nornicotine, TSNAs, sterols, and/or produces a reduced amount of PAHs upon pyrolysis is made by exposing at least one tobacco cell of a selected variety (e.g., Burley, Va. Flue-cured, or Oriental) to an exogenous nucleic acid construct encoding an interfering RNA comprising an RNA duplex that comprises a first strand having a sequence that is substantially similar or identical to at least a portion of the coding sequence of a target gene and/or target gene product involved in nicotine biosynthesis or sterol biosynthesis, and a second strand that is complementary or substantially complementary to the first strand. In some embodiments, the nucleic acid construct further comprises a nucleotide sequence encoding the interfering RNA operably linked to a promoter operable in a plant cell. The tobacco cell is transformed with the nucleic acid construct, transformed cells are selected and at least one transgenic tobacco plant is regenerated from the transformed cells. The transgenic tobacco plants described herein can contain a reduced amount of anyone of nicotine, nornicotine, TSNAs and/or a sterol as compared to a control tobacco plant of the same variety. In some embodiments, nucleic acid constructs encoding interfering RNAs (RNAi) comprising a first strand having a sequence substantially similar or identical to the entire coding sequence of a target gene and/or target gene product involved in nicotine or sterol biosynthesis, and a second strand that is complementary or substantially complementary to the first strand, are contemplated.

In some embodiments, the genetically modified tobacco provided herein will be genetically stable for at least 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 40 or 50, or more, generations. For example, the genetically modified tobacco produces a reduced amount of a compound associated with a tobacco related disease for at least 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 40 or 50, or more, generations. It is contemplated, for example, that crossings of multiple tobaccos each having different genetic modifications that are stable over many generations can be performed so as to obtain a genetically modified tobacco having a reduced level of expression of a plurality of genes that encode precursors for various tobacco related diseases.

In some embodiments, the genetically modified tobacco provided herein will have agronomic characteristics suitable for commercial production. Although in some instances genetically modified tobacco can have agronomic characteristics that are different from conventional tobacco, such a tobacco can be suitable for commercial production because these different agronomic characteristics can be compensated for by employing techniques common to those of skill in the art. That is, although the agronomic characteristics for a genetically modified tobacco created as described herein may differ from those of conventional tobacco, such alterations may not necessarily yield a plant that is no longer suitable for commercial production. For example, a genetically modified tobacco may have a reduced root mass, but tobacco plants having reduced root mass can nevertheless be suitable for commercial production when such tobaccos are raised under conditions in which the plants are thoroughly irrigated and/or not subjected to drought conditions. Additional nutritional requirements (e.g., nitrogen) may be required. Any of a variety of conventional agronomic methods can be used to produce commercial quantities of a genetically modified tobacco, where such methods include, but are not limited to, irrigation, fertilization, providing nutrients for plant growth, and use of pesticides. As referred to herein, a genetically modified tobacco that is suitable for commercial production is a genetically modified tobacco that, under appropriate agronomic conditions will produce at least 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, tobacco useful for creation of a tobacco product relative to an unmodified or conventional tobacco, grown under its standard growing conditions.

1. Genes to Modify

In some embodiments, the gene product is one that is involved in nicotine biosynthesis. Such enzymes include, but are not necessarily limited to, putrescene N-methyltransferase (PMTase), N-methylputrescene oxidase, ornithine decarboxylase, S-adenosylmethionine synthetase, NADH dehydrogenase, phosphoribosylanthranilate isomerase and quinolate phosphoribosyl transferase (QPTase). In preferred embodiments, the gene product that is inhibited using a construct described herein is QPTase, PMTase, and A622. In some embodiments, the tobacco that is made substantially free of nicotine and/or TSNAs (e.g., comprises or delivers less than or equal to 0.5 mg/g nicotine and/or less than or equal to 0.5 μg/g collective content of NNN, NAT, NAB, and NNK) is prepared from a variety of Burley tobacco (e.g., Burley 21 or Tn90), Oriental tobacco (Djebal 174), or Virginia Flue-cured (K326) tobacco. It should be understood, however, that most tobacco varieties can be made to have reduced amounts of nicotine and/or TSNAs or can be made substantially free of nicotine and/or TSNAs by using the embodiments described herein. For example, plant cells of the variety Burley 21 are used as the host for the genetic engineering that results in the reduction of nicotine and/or TSNAs so that the resultant transgenic plants are a Burley 21 variety that has a reduced amount of nicotine and/or TSNAs.

Accordingly, some embodiments concern a tobacco that comprises a genetic modification comprising a reduced amount or a reduced level of expression of QPTase, PMTase, or A622, comprising or delivering a reduced amount of nicotine or total alkaloid and/or a collective content of TSNA (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 0.5 μg/g (e.g., 0.05 μg/g, 0.1 μg, 0.2 μg/g, 0.3 μg/g, 0.4 μg/g, or 0.5 μg/g). More embodiments concern a tobacco that comprises or delivers a reduced amount or a reduced level of expression of A622, a normal or conventional amount of nicotine (e.g., comprising or delivering by FTC methodology an amount of nicotine equal to, less than, or greater than 0.9 mg/g, 1.0 mg/g, 1.1 mg/g, 1.2 mg/g, 1.3 mg/g, 1.4 mg/g, 1.5 mg/g, 1.6 mg/g, 1.7 mg/g, 1.8 mg/g, 1.9 mg/g, and 2.0 mg/g), and a reduced amount of nornicotine (e.g., comprising or delivering by FTC methodology an amount of nornicotine less than or equal to 0.5 μg/g), and/or a reduced amount of NNN (e.g., comprising or delivering by FTC methodology an amount of totoal TSNAs equal to or less than 0.05 μg/g, 0.1 μg, 0.2 μg/g, 0.3 μg/g, 0.4 μg/g, or 0.5 μg/g). That is, particular lines of transgenic tobacco containing the A622 inhibition cassette described herein were unexpectedly found to have a reduced level of nornicotine but conventional levels of nicotine. This finding is particularly important since nornicotine may be a more important precursor for NNN than nicotine. (See Carmella et al., Carcinogenesis, Vol. 21, No. 4, 839-843, (April 2000), herein expressly incorporated by reference in its entirety). In other transgenic lines, wherein the A622 gene was inhibited using one of the constructs described herein, it was found that both nicotine and nornicotine were effectively reduced (e.g., total alkaloids were less than or equal to 7,000 ppm, 5000 ppm, 3000 ppm, 1000 ppm, or 500 ppm).

Some of the nucleic acid constructs provided herein employ interfering RNAs (e.g., siRNAs or dsRNAs) that comprise an RNA duplex wherein each RNA portion of the duplex is at least, greater than, or equal to 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 750, 1000, 1500, 2000, 2500, or 5000 consecutive nucleotides complementary or substantially complementary to an mRNA that encodes a gene product or the entire coding sequence of the enzyme or complement thereof of an enzyme that regulates nicotine or sterol biosynthesis. In some embodiments, the RNA duplex comprises a first RNA strand that is complementary to an mRNA that encodes a gene product involved in nicotine or sterol biosynthesis and a second RNA stand that is complementary to said first strand. Some interfering RNAs provided herein can comprise two separate RNA strands hybridized to each other by hydrogen bonding. Other interfering RNAs comprise a single RNA strand comprising a first and second regions of nucleotide sequence that are complementary to each other. In such embodiments, the first and second regions of nucleotide sequence are separated by a nucleotide sequence (e.g., a “linker”) that permits or, in the case of the FAD2 intron described herein, facilitates formation of a hairpin structure upon hybridization of the first and second regions. This “linker” that permits formation of a hairpin structure is preferably at least, greater than, or equal to 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 800, 900, 1000 or more nucleotides in length.

A preferred method of producing tobacco having a reduced amount of nicotine and TSNAs, involves genetic engineering directed at reducing the levels of nicotine and/or nornicotine or other alkaloids. Any enzyme involved in the nicotine synthesis pathway can be a suitable target for genetic engineering to reduce levels of nicotine and, optionally, levels of other alkaloids including nornicotine. Suitable targets for genetic engineering to produce tobacco having a reduced amount of nicotine and/or nitrosamines, especially TSNAs, include but are not limited to putrescene N-methyltransferase, N-methylputrescene oxidase, ornithine decarboxylase, S-adenosylmethionine synthetase, NADH dehydrogenase, phosphoribosylanthranilate isomerase, quinolate phosphoribosyl transferase (QPTase) or a combination of any of the above targets. Additionally, enzymes that regulate the flow of precursors into the nicotine or sterol synthesis pathway are suitable targets for genetic engineering to produce tobacco with a reduced amount of nicotine and nitrosamines, especially TSNAs, and tobaccos with reduced amounts of sterols, which produce a reduced amount of PAHs upon pyrolysis. Suitable methods of genetic engineering are known in the art and include, for example, the use of antisense and sense suppression technology to reduce or eliminate the production of enzymes, the use of interfering RNA molecules (gene silencing) as described herein to reduce or eliminate the expression of gene products, and the use of random or targeted mutagenesis to disrupt gene function, for example, using T-DNA insertion or EMS mutagenesis. The next section provides more description of these techniques.

2. Modification Methods

a) Knockouts

Inhibition of Gene Expression Using Nucleic Acids

Inhibition of gene expression refers to the absence or observable reduction in the level of polypeptide and/or mRNA gene product. Some embodiments provided herein relate to inhibiting the expression of one or more genes involved in the biosynthesis of nicotine, nornicotine, and/or sterols by genetically modifying a plant cell, such as a tobacco cell, by providing the cell with an inhibitory nucleic acid that reduces or eliminates the production of a gene product involved in nicotine or sterol biosynthesis. Inhibitory nucleic acids include, but are not limited to, interfering RNAs, antisense nucleic acids and catalytic RNAs. Some preferred embodiments provided herein relate to interfering RNAs (RNAi).

RNA interference and gene silencing are terms that are used to describe a phenomenon by which the expression of a gene product is inhibited by an interfering RNA molecule. Interfering RNA molecules are double-stranded RNAs (dsRNA) that are expressed in or otherwise introduced into a cell. The dsRNA molecules may be of any length, however, short dsRNA constructs are commonly used. Such constructs are known as small interfering RNAs (siRNA), and are typically 21-23 bp in length.

RNA interference is exhibited by nearly every eukaryote and is thought to function by a highly conserved mechanism (Dillin, A. PNAS, 100:6289-91). As with antisense inhibition of gene expression, inhibition mediated by RNA interference is gene specific. However, in contrast to antisense-mediated inhibition, inhibition mediated by interfering RNA appears to be inherited (Dillin, A. PNAS, 100:6289-91). Without being bound by theory, it is believed that specificity is achieved through nucleotide sequence interaction between complementary portions of a target mRNA and the interfering RNA. The target mRNA is selected based on the specific gene to be silenced. In particular, the target mRNA, corresponds to the sense strand of the gene to be silenced. An interfering RNA, such as a dsRNA or an siRNA, comprises an RNA duplex, which includes a first strand that is substantially similar or identical to at least a portion of the nucleotide sequence of the target mRNA, and a second strand having a nucleotide sequence that is complementary or substantially complementary to the first strand.

When used herein with reference to an RNA duplex of the interfering RNA, it will be appreciated that the terms “first strand” and “second strand” are used in a relative sense. For example, the first strand of an RNA duplex can be selected to comprise either a nucleotide sequence substantially similar or identical to at least a portion of the nucleotide sequence of the target mRNA or a nucleotide sequence that is complementary or substantially complementary to at least a portion of the nucleotide sequence of the target mRNA. If the first strand is selected to be substantially similar or identical to at least a portion of the nucleotide sequence of the target mRNA, then the second strand will be complementary to at least a portion of the target mRNA because it is complementary to the first strand. If the first strand is selected to be complementary or substantially complementary to at least a portion of the target mRNA, then the second strand will be substantially similar or identical to at least a portion of the nucleotide sequence of the target mRNA because it is complementary to the first strand.

As used herein with reference to nucleic acids, “portion” means at least 5 consecutive nucleotides, at least 6 consecutive nucleotides, at least 7 consecutive nucleotides, at least 8 consecutive nucleotides, at least 9 consecutive nucleotides, at least 10 consecutive nucleotides, at least 11 consecutive nucleotides, at least 12 consecutive nucleotides, at least 13 consecutive nucleotides, at least 14 consecutive nucleotides, at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, at least 20 consecutive nucleotides, at least 21 consecutive nucleotides, at least 22 consecutive nucleotides, at least 23 consecutive nucleotides, at least 24 consecutive nucleotides, at least 25 consecutive nucleotides, at least 30 consecutive nucleotides, at least 35 consecutive nucleotides, at least 40 consecutive nucleotides, at least 45 consecutive nucleotides, at least 50 consecutive nucleotides, at least 60 consecutive nucleotides, at least 70 consecutive nucleotides, at least 80 consecutive nucleotides, at least 90 consecutive nucleotides, at least 100 consecutive nucleotides, at least 125 consecutive nucleotides, at least 150 consecutive nucleotides, at least 175 consecutive nucleotides, at least 200 consecutive nucleotides, at least 250 consecutive nucleotides, at least 300 consecutive nucleotides, at least 350 consecutive nucleotides, at least 400 consecutive nucleotides, at least 450 consecutive nucleotides, at least 500 consecutive nucleotides, at least 600 consecutive nucleotides, at least 700 consecutive nucleotides, at least 800 consecutive nucleotides, at least 900 consecutive nucleotides, at least 1000 consecutive nucleotides, at least 1200 consecutive nucleotides, at least 1400 consecutive nucleotides, at least 1600 consecutive nucleotides, at least 1800 consecutive nucleotides, at least 2000 consecutive nucleotides, at least 2500 consecutive nucleotides, at least 3000 consecutive nucleotides, at least 4000 consecutive nucleotides, at least 5000 consecutive nucleotides or greater than at least 5000 consecutive nucleotides. In some preferred embodiments, a portion of a nucleotide sequence is between 20 and 25 consecutive nucleotides. In other preferred embodiments, a portion of a nucleotide sequence is between 21 and 23 consecutive nucleotides. In some embodiments provided herein, a portion of a nucleotide sequence includes the full-length coding sequence of the gene or the target mRNA.

Some preferred interfering RNAs that are described herein comprise an RNA duplex, which comprises a nucleotide sequence that is substantially similar or identical to at least a portion of the coding strand of a gene involved in nicotine or sterol biosynthesis. Although nucleic acid sequences that are substantially similar or identical to at least a portion of the coding strand of the target gene involved in nicotine biosynthesis are preferred, it will be appreciated that nucleotide sequences with insertions, deletions, and single point mutations relative to the target sequence are also effective for inhibition of gene expression. Sequence identity may be determined by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the interfering RNA and a portion of the target gene is preferred. In especially preferred embodiments, at least about 21 to about 23 contiguous nucleotides in the target gene are greater than 90% identical to a sequence present in the interfering RNA.

In other embodiments provided herein, the duplex region of the RNA may be defined functionally as including a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript. Exemplary hybridization conditions are 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed by washing.

The modification of nicotine levels in tobacco plants by antisense regulation of putrescene methyl transferase (PMTase) expression has been proposed in U.S. Pat. Nos. 5,369,023 and 5,260,205, to Nakatani and Malik, and in PCT application WO 94/28142 and U.S. Pat. No. 5,668,295 to Wahad and Malik, which describe DNA encoding PMT and the use of sense and antisense PMT constructs, the entire disclosures of each of which are hereby expressly incorporated by reference in their entireties. Other genetic modifications proposed to reduce nicotine levels are described in PCT application WO 00/67558, to Timko, and WO 93/05646, to Davis and Marcum; the entire contents of each are hereby expressly incorporated by reference in their entireties. Although these investigators made significant contributions, there were significant drawbacks to their experimental design.

Provided herein are tobacco and tobacco products in which a plurality of genes involved in nicotine biosynthesis are inhibited. Most notably, it is presently revealed that there are several different PMT genes and each may play a role in nicotine biosynthesis. Knocking-out only one PMT gene can create a leaky system allowing the other PMT genes to compensate for the reduction. Accordingly, each of the PMT constructs described herein were designed to inhibit a plurality of different PMT genes with a single construct. That is, the PMT constructs described herein are designed to complement common regions to all five of the PMT genes so that inhibition of each of the PMT genes can be accomplished with one inhibitory fragment. Although many of the approaches described in this section have significant drawbacks, it should be understood that any or all of these techniques can be used with other techniques, as described herein, to make tobacco and tobacco products having reduced nicotine.

In some embodiments that employed the A622 inhibition construct, it was found that transgenic tobacco that had conventional levels of nicotine but significantly reduced levels of nornicotine were produced. This particular line of tobacco is particularly useful because nornicotine may be the most significant precursor for NNN in tobacco. Accordingly, reduced risk conventional cigarettes and other tobacco products (e.g., snuff) comprising the A622 inhibition construct are embodiments.

As described above, interfering RNAs disclosed herein comprise a sequence that is complementary to at least a portion of the sense strand of a gene encoding a target mRNA, which produces a polypeptide that is involved in nicotine biosynthesis. Preferred targets are the products of the quinolate phosphoribosyltransferase (QTPase) gene, the putrescene N-methyltransferase (PMTase) gene, and the A622 gene. However, it will be appreciated that interfering RNAs specific for other gene products or combinations of gene products involved in nicotine and nornicotine biosynthesis and/or sterol biosynthesis are contemplated. For example, additional gene products involved in nicotine biosynthesis include, but are not limited to, N-methylputrescene oxidase, ornithine decarboxylase, S-adenosylmethionine synthetase, NADH dehydrogenase, and phosphoribosylanthranilate isomerase. Additionally, it will be appreciated that interfering RNAs specific for other gene products or combinations of gene products involved in in sterol biosynthesis include HMG-CoA reductase, 14alpha demethylase, squalene synthase, SMT2, SMT1, C14 sterol reductase, A8-A7-isomerase, and C4-demethylase.

Additionally, the interfering RNAs described herein can comprise a plurality nucleotide sequences that are each complementary to different portions of the sense strand of a gene involved in nicotine and/or sterol biosynthesis. Alternatively, the interfering RNAs described herein can comprise a plurality nucleotide sequences that are each complementary to at least a portion of the sense strands of different genes involved in nicotine and/or sterol biosynthesis. Still further, a single RNAi construct or inhibition cassette can be used to inhibit a plurality of genes involved in the regulation of the production of nicotine, nornicotine, or sterols. For example, as described below, it was found that the A622 inhibitory fragment and inhibition cassette (SEQ. ID. Nos. 5 and 26) efficiently reduced production of nicotine and nornicotine in some lines of tobacco and in other lines of tobacco conventional levels of nicotine were maintained but the amount of nornicotine in said tobacco was 0.00 mg/g. Still further, the PMTase inhibitory sequence and PMTase inhibition cassette (SEQ. ID. Nos. 4 and 25) were designed to complement common regions of a plurality of PMTase genes so that the production of multiple gene products can be inhibited or reduced with a single construct.

In still more embodiments, it is contemplated that a single T-DNA containing construct be used to overexpress one gene and, in the same construct, inhibiting expression of a second gene. That is, some embodiments concern constructs, tobacco containing said constructs, and tobacco products containing said tobacco, wherein said constructs comprise an overexpression cassette that comprises a gene that regulates the production of a compound that improves the composition of the tobacco (e.g, overexpression of a gene encoding an antioxidant) and, on the same construct, an inhibition cassette that comprises an inhibitory sequence that reduces the production of a compound that contributes to a tobacco related disease (e.g., nicotine, nornicotine, or a sterol).

In preferred embodiments, the interfering RNAs described herein comprise at least one region of double-stranded RNA (duplex RNA). This duplex RNA can range from about 10 bp in length to about 10,000 bp in length. In some embodiments, the duplex RNA ranges from about 15 bp in length to about 1500 bp in length. In other embodiments, the duplex RNA ranges from about 20 bp in length to about 1200 bp in length. In still other embodiments, the duplex RNA ranges from about 21 bp in length to about 23 bp in length. In a preferred embodiment, the duplex RNA has a length of 22 bps. Short regions of duplex RNA are often designated siRNA, whereas longer regions of RNA duplex are often termed dsRNA. In some embodiments provided herein, the interfering RNA duplex region is a dsRNA. In other embodiments, the interfering RNA duplex region is an siRNA. In a preferred embodiment, the duplex region about the length of the coding sequence of a target mRNA encoding a polypeptide involved in nicotine biosynthesis.

Interfering RNAs described herein can be generated using a variety of techniques. For example, an interfering RNA can be generated in a host cell in vivo by providing the cell with one or more a nucleic acid constructs that comprise the nucleic acids necessary to encode the strands of a double-stranded RNA. Such constructs can be included in various types of vectors. Exemplary vectors contemplated herein include, but are not limited to, plasmids, viral vectors, viroids, replicable and nonreplicable linear DNA molecules, replicable and nonreplicable linear RNA molecules, replicable and nonreplicable circular DNA molecules and replicable and nonreplicable circular RNA molecules. Preferred vectors include plasmid vectors, especially vector systems derived from the Agrobacterium Ti plasmid, such as pCambia vectors and derivatives thereof.

In some embodiments, both strands of the double-stranded region of the interfering RNA can be encoded by a single vector. In such cases, the vector comprises a first promoter operably linked to a first nucleic acid which is substantially similar or identical to at least a portion of the target mRNA. The vector also comprises a second promoter operably linked to a second nucleic acid, which is complementary or substantially to the first nucleic acid.

Another type of single vector construct, which can be used to generate interfering RNA, encodes a double-stranded RNA hairpin. In such embodiments, the vector comprises a promoter operably linked to a nucleic acid that encodes both strands of the duplex RNA. The first nucleotide sequence, which encodes the strand that is substantially similar or identical to at least a portion of the target mRNA, is separated from the second nucleotide sequence, which encodes a strand complementary or substantially complementary to the first strand, by a region of nucleotide sequence that does not substantially hybridize with either of the strands. This nonhybridizing region permits the RNA sequence transcribed from the vector promoter to fold back on itself, thereby permitting the complementary RNA sequences to hybridize so as to produce an RNA hairpin. Vectors comprising a plurality of nucleic acids, each of which encode both strands of the duplex RNA are also contemplated.

Other embodiments provided herein relate to multiple vector systems for the production of interfering RNA. In one example, a multiple vector system is used to produce a single interfering RNA that is specific for a single gene product involved in nicotine biosynthesis. In such embodiments, at least two vectors are used. The first vector comprises a promoter operably linked to a first nucleic acid that encodes a first strand of the RNA duplex that is present in the interfering RNA. The second vector comprises a promoter operably linked to a second nucleic acid that encodes the second strand of the RNA duplex, which is complementary to the first strand.

Other multiple vector systems are combinations of vectors, wherein each vector in the system encodes a different interfering RNA. Each of the interfering RNAs is specific for different gene products involved in nicotine biosynthesis. In some embodiments, the vectors in a multiple vector system can encode different interfering RNAs that are specific to different portions of a single gene product involved in nicotine biosynthesis.

It will be appreciated that the promoters used in the above-described vectors can either be constitutive or regulated. Constitutive promoters are promoters that are always expressed. The constitutive promoters selected for use in the above-described vectors can range from weak promoters to strong promoters depending on the desired amount of interfering RNA to be produced. Regulated promoters are promoters for which the desired level of expression can be controlled. An example of a regulated promoter is an inducible promoter. Using an inducible promoter in the above-described vector constructs permits expression of a wide range of concentrations of interfering RNA inside a cell.

It will also be appreciated that there is no requirement that the same or same types of promoters be used in vectors or multiple vector systems that comprise a plurality of promoters. For example, in some vectors or vector systems, a first promoter, which controls the expression of the first interfering RNA strand, can be an inducible promoter, whereas the second promoter, which controls the expression of the second RNA strand, can be a constitutive promoter. This same principal can also be illustrated in a multiple vector system. For example, a multiple vector system may have three vectors each of which includes one or more different types of promoters. Such a system can include, for example, a first vector having repressible promoter that controls the expression of an interfering RNA specific for a first gene product involved in nicotine biosynthesis, a second vector having a constitutive promoter that controls the expression of an interfering RNA specific for a second gene product involved in nicotine biosynthesis and a third vector having an inducible promoter that controls the expression of an interfering RNA specific for a third gene product involved in nicotine biosynthesis.

In other embodiments provided herein, interfering RNAs can be produced synthetically and introduced into a cell by methods known in the art. Synthetic interfering RNAs can include a variety of RNA molecules, which include, but are not limited to, nucleic acids having at least one region of duplex RNA. The duplex RNA in such molecules can comprise, for example, two antiparallel RNA strands that form a double-stranded RNA having flush ends, two antiparallel RNA strands that form a double-stranded RNA having at least one end that forms a hairpin structure, or two antiparallel RNA strands that form a double-stranded RNA, wherein both ends form a hairpin structure. In some embodiments, synthetic interfering RNAs comprise a plurality of RNA duplexes.

The regions of RNA duplex in synthetic interfering RNAs can range from about 10 bp in length to about 10,000 bp in length. In some embodiments, the duplex RNA ranges from about 15 bp in length to about 1500 bp in length. In other embodiments, the duplex RNA ranges from about 20 bp in length to about 1200 bp in length. In still other embodiments, the duplex RNA ranges from about 21 bp in length to about 23 bp in length. In a preferred embodiment, the duplex RNA has a length of 22 bps. In preferred embodiments, synthetic interfering RNAs are siRNAs. In another preferred embodiment, the synthetic interfering RNA is an siRNA specific for the coding sequence of a target mRNA encoding a polypeptide involved in nicotine biosynthesis. In another preferred embodiment, the synthetic interfering RNA is an siRNA specific for the coding sequence of a target mRNA encoding a polypeptide involved in sterol biosynthesis.

Some embodiments provided herein relate to interfering nucleic acids that are not comprised entirely of RNA. Still other aspects relate to interfering nucleic acids that do not comprise any RNA. Such interfering nucleic acids are synthetic interfering RNA analogs. These analogs substantially mimic the specificity and activity of interfering RNA from which they are modeled; however, they typically include additional properties which make their use desirable. For example, one or both strands of the interfering nucleic acid may contain one or more normatural nucleotide bases that improve the stability of the molecule, enhance that affinity of the molecule for the target mRNA and/or enhance cellular uptake of the molecule. Other modifications are also contemplated. For example, an interfering nucleic acid can include one or more nucleic acid strands composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as non-naturally-occurring nucleobases, sugars and covalent internucleoside linkages.

As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or the 5′ hydroxyl moiety of the sugar. In forming nucleic acids, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure. Within the nucleic acid structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

Specific examples of interfering nucleic acids useful in certain embodiments of provided herein include one or more nucleic acid strands containing modified backbones or non-natural internucleoside linkages. As used herein, nucleic acids having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

In some embodiments, modified nucleic acid backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Certain nucleic acids having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

In some embodiments, modified nucleic acid backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.

In other embodiments, the interfering nucleic acid can comprise one or more mimetic regions, wherein both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. In such embodiments, the base units are maintained for hybridization with an appropriate nucleic acid target compound. One such compound, a mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference in its entirety. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

In still other embodiments provided herein, interfering nucleic acids may include nucleic acid strands having phosphorothioate backbones and/or heteroatom backbones. Modified interfering nucleic acids may also contain one or more substituted sugar moieties. In some embodiments, the interfering nucleic acids comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl. Particularly preferred are O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 and O(CH 2 ) n ON[(CH 2 ) n CH 3 ] 2 , where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C 1 to C 10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Another modification includes 2′-methoxyethoxy (2′ OCH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Hely. Chim. Acta, 1995, 78, 486-504).

An embodiment provided herein includes the use of Locked Nucleic Acids (LNAs) to generate interfering nucleic acids having enhanced affinity and specificity for the target polynucleotide. LNAs are nucleic acid in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH 2 —) n group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226, the disclosures of which are incorporated herein by reference in their entireties.

Other modifications include 2′-methoxy (2′-O—CH 3 ), 2′-aminopropoxy (2′-OCH 2 CH 2 CH 2 NH 2 ), 2′-allyl (2′-CH 2 —CH═CH 2 ), 2′-O-allyl (2′-O—CH 2 —CH═CH 2 ) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Interfering nucleic acids may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

The interfering nucleic acids contemplated herein may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazi-n-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrimido [3′,2′: 4,5] pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993, the disclosures of which are incorporated herein by reference in their entireties. Certain of these nucleobases are particularly useful for increasing the binding affinity of the interfering nucleic acids described herein. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcyto sine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Another modification of the interfering nucleic acids described herein involves chemically linking to at least one of the nucleic acid strands one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the of the interfering nucleic acid. The interfering nucleic acids can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of nucleic acids, and groups that enhance the pharmacokinetic properties of such molecules. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve interfering nucleic acid uptake, enhance its resistance to degradation, and/or strengthen sequence-specific hybridization with target molecules. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve the uptake, distribution, metabolism or excretion of the interfering nucleic acid. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylaminocarbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

As described above, it is not necessary for all positions in a given compound to be uniformly modified, and in fact, more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within a nucleic acid. The methods described herein also contemplate the use of interfering nucleic acids which are chimeric compounds. “Chimeric” interfering nucleic acid compounds or “chimeras,” as used herein, are interfering nucleic acid compounds, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a nucleic acid compound. These interfering nucleic acids typically contain at least one region wherein the nucleic acid is modified so as to confer upon the interfering nucleic acid increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the nucleic acid may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby contributes further to the inhibition of gene expression by the interfering nucleic acid.

The above-described interfering nucleic acids may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare nucleic acids such as the phosphorothioates and alkylated derivatives.

The interfering nucleic acid compounds for use with the methods described herein encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound.

Although terms, such as interfering RNA, dsRNA and siRNA, are used throughout the remainder of the specification, it will be appreciated that in the context of synthetically produced interfering nucleic acids, that such terms are meant to include interfering nucleic acids of all types, including those which incorporate modifications, such as those described above.

Some embodiments provided herein relate to methods of reducing or eliminating the expression of one or more target genes involved in nicotine, nornicotine, and/or sterol biosynthesis. Target genes that are involved in nicotine, nornicotine, and/or sterol biosynthesis are expressed through the transcription a first gene product, the target mRNA, which is then translated to produce a second gene product, the target polypeptide. Thus, reduction or elimination of the expression of one or more target genes results in the reduction or elimination of one or more target mRNAs and/or target polpypeptides. Target polypeptides involved in nicotine and nornicotine biosynthesis include, for example, putrescene N-methyltransferase, N-methylputrescene oxidase, ornithine decarboxylase, S-adenosylmethionine synthetase, NADH dehydrogenase, phosphoribosylanthranilate isomerase, and quinolate phosphoribosyl transferase (QPTase). In a preferred embodiment, the expression of the QPTase, PMTase, and A622 product is inhibited. Target polypeptides involved in sterol biosynthesis include, for example, HMG-CoA reductase, 14alpha demethylase, squalene synthase, SMT2, SMT1, C14 sterol reductase, A8-A7-isomerase, and C4-demethylase.

Reduction of the expression of one or more target genes and/or target gene products that are involved in nicotine, nornicotine, and/or sterol biosynthesis leads to a reduction in the amount of nicotine, sterols, and TSNAs produced in tobacco and PAHs upon pyrolysis of the tobacco. In certain embodiments, the expression of one or more target gene products involved in nicotine, nornicotine, and/or sterol biosynthesis is eliminated. Elimination of such target gene products can result in the elimination of nicotine, nornicotine, and/or sterol biosynthesis, thereby reducing the amount of nicotine, nornicotine, and/or sterol present in tobacco to levels below the detection limit of methods commonly used. Reduction of the amount of nicotine and nornicotine present in tobacco can lead to a reduction in the amount of TSNAs produced in the tobacco. In some embodiments, the amount of TSNA present in tobacco is reduced to levels below the detection limit of methods commonly used to detect TSNAs. Similarly, the reduction in the amount of sterol present in tobacco can lead to a reduction in the amount of PAH generated from the tobacco upon pyrolysis. In some embodiments, the amount of PAH present in tobacco is reduced to levels below the detection limit of methods commonly used to detect PAH.

The reduction in or elimination of the expression of target genes or target gene products involved in nicotine, nornicotine, and/or sterol biosynthesis is achieved by providing an interfering RNA specific to one or more such target genes to a tobacco cell, thereby producing a genetically modified tobacco cell. The interfering RNA can be provided as a synthetic double-stranded RNA, or alternatively, as a nucleic acid construct capable of encoding the interfering RNA. Synthetic double-stranded interfering RNAs are taken up by the cell directly whereas interfering RNAs encoded by a nucleic acid construct are expressed from the construct subsequent to the entry of the construct inside the cell. The reduction in or elimination of the expression of the target genes and/or the target gene products is mediated by the presence of the interfering RNA inside the cell.

In general, the interfering RNAs that are produced inside the cell, whether expressed from a nucleic acid construct or provided as synthetic double-stranded RNA molecules, include an RNA duplex having a first and second strand. At least a portion the first strand of the duplex is substantially similar or identical to at least a portion of a target mRNA or a target gene involved in nicotine biosynthesis. Correspondingly, at least a portion of the second strand of the duplex is complementary or substantially complementary to the first strand, and thus, at least a portion of the second strand is complementary or substantially complementary to at least a portion of the mRNA encoded by the target gene. In some embodiments provided herein, the interfering RNA can comprise a first strand that is substantially similar or identical to the entire coding sequence of the target gene or target mRNA involved in nicotine biosynthesis and a second strand complementary or substantially complementary to the first strand. In some embodiments provided herein, the interfering RNA can comprise a first strand that is substantially similar or identical to the entire coding sequence of the target gene or target mRNA involved in sterol biosynthesis and a second strand complementary or substantially complementary to the first strand.

The reduction in or elimination of the expression of genes and/or gene products involved in nicotine, nornicotine, and/or sterol biosynthesis can be characterized by comparing the amount of nicotine, nornicotine, and/or sterol produced in genetically modified cells, with the amount of nicotine, nornicotine, and/or sterol produced in cells that have not been genetically modified. Alternatively, such reduction in or elimination of gene expression can be characterized by genetically analyzing plant cells so as to determine the level of mRNA present in the genetically modified plant cell as compared to a non-modified plant cell. Depending on the assay, quantitation of the amount of gene expression allows one to determine a degree of reduction in gene expression, which can be greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to an untreated cell. As with nicotine and nornicotine, the reduction in or elimination of TSNA production in tobacco can be characterized by comparing the amount of TSNAs produced in genetically modified cells, with the amount of TSNAs produced in cells that have not been genetically modified. The section below provides more description of the transgenic plants and cells provided herein.

b) Transgenics

Transgenic Plant Cells and Plants

Embodiments provided herein concern transgenic plant cells comprising one or more interfering RNAs that are capable of reducing or eliminating the expression of one or more target genes and/or target gene products involved in nicotine, nornicotine, and/or sterol biosynthesis. As described above, an appropriate interfering RNA comprises a duplex RNA that comprises a first strand that is substantially similar or identical to at least a portion of a target gene or target mRNA, which encodes a gene product involved in nicotine, nornicotine, and/or sterol biosynthesis. The RNA duplex also comprises a second strand that is complementary or substantially complementary to the first strand.

The interfering RNA or nucleic acid construct comprising the interfering RNA can be introduced into the plant cell in any suitable manner. Plant cells possessing stable interfering RNA activity, for example, by having a nucleic acid construct stably integrated into a chromosome, can be used to regenerate whole plants using methods known in the art. As such, some embodiments provided herein relate to plants, such as tobacco plants, transformed with one or more nucleic acid constructs and/or vectors which encode at least one interfering RNA that is capable of reducing or eliminating the expression of a gene product involved in nicotine biosynthesis. Transgenic tobacco cells and the plants described herein are characterized in that they have a reduced amount of nicotine, nornicotine, sterol and/or TSNA and/or generate a reduced amount of PAHs upon pyrolysis, as compared to unmodified or control tobacco cells and plants.

The tobacco plants described herein are suitable for conventional growing and harvesting techniques (e.g. topping or no topping, bagging the flowers or not bagging the flowers, cultivation in manure rich soil or without manure) and the harvested leaves and stems are suitable for use in any traditional tobacco product including, but not limited to, pipe, cigar and cigarette tobacco and chewing tobacco in any form including leaf tobacco, shredded tobacco or cut tobacco. It is also contemplated that the low nicotine and/or TSNA tobacco described herein can be processed and blended with conventional tobacco so as to create a wide-range of tobacco products with varying amounts of nicotine and/or TSNAs. These blended tobacco products can be used in tobacco product cessation programs so as to slowly move a consumer from a high nicotine and/or sterol product to a low nicotine and/or sterol product. Some embodiments provided herein comprise a tobacco use cessation kit, comprising two or more tobacco products with different levels of nicotine. For example, a smoker can begin the program smoking blended cigarettes having or delivering 0.6 mg of nicotine, gradually move to smoking cigarettes having or delivering 0.3 mg of nicotine, followed by cigarettes having or delivering less than 0.1 mg nicotine until the consumer decides to quit smoking altogether. Accordingly, the blended cigarettes described herein provide the basis for an approach to reduce the exposure of a tobacco consumer to a tobacco related disease in a step-wise fashion. The components of the tobacco use cessation kit described herein may include other tobacco products, including but not limited to, smoking materials (e.g., cigarettes, cigars, pipe tobacco), snuff, chewing tobacco, gum, and lozenges.

Gene silencing has been employed in several laboratories to create transgenic plants characterized by lower than normal amounts of specific gene products. As used herein, “exogenous” or “heterologous” nucleic acids, including DNAs and/or RNAs, refer to nucleic acids that have been introduced into a cell (or the cell's ancestor) through the efforts of humans. The nucleic acid constructs that are used with the transgenic plants and the methods for producing the transgenic plants described herein encode one or more interfering RNA constructs comprising regulatory sequences, which include, but are not limited to, a transcription initiation sequence (“promoter”) operable in the plant being transformed, and a polyadenylation/transcription termination sequence. Typically, the promoter is located upstream of the 5′-end of the nucleotide sequence to be expressed. The transcription termination sequence is generally located just downstream of the 3′-end of the nucleotide sequence to be transcribed.

In some preferred embodiments, the nucleic acid encoding the exogenous interfering RNA, which is transformed into a tobacco cell, comprises a first RNA strand that is identical to the an endogenous coding sequence of a gene encoding a gene product involved in nicotine biosynthesis. However, minor variations between the exogenous and endogenous sequences can be tolerated. It is preferred, but not necessarily required, that the exogenously-produced interfering RNA sequence, which is substantially similar to the endogenous gene coding sequence, be of sufficient similarity to the endogenous gene coding sequence, such that the complementary interfering RNA strand is capable of binding to the endogenous sequence in the cell to be regulated under stringent conditions as described below.

In some embodiments, the heterologous sequence utilized in the methods provided herein may be selected so as to produce an interfering RNA product comprising a first strand that is substantially similar or identical to the entire QTPase mRNA sequence, or to a portion thereof, and a second strand that is complementary to the entire QPTase mRNA sequence, or to a portion thereof. The interfering RNA may be complementary to any contiguous sequence of the natural messenger RNA. For example, it may be complementary to the endogenous mRNA sequence proximal to the 5′-terminus or capping site, downstream from the capping site, between the capping site and the initiation codon and may cover all or only a portion of the non-coding region, may bridge the non-coding and coding region, be complementary to all or part of the coding region, complementary to the C-terminus of the coding region, or complementary to the 3′-untranslated region of the mRNA.

As used herein, the term “gene” refers to a DNA sequence that incorporates (1) upstream (5′) regulatory signals including the promoter, (2) a coding region specifying the product, protein or RNA of the gene, (3) downstream regions including transcription termination and polyadenylation signals and (4) associated sequences required for efficient and specific expression. The DNA sequence provided herein may consist essentially of the sequence provided herein, or equivalent nucleotide sequences representing alleles or polymorphic variants of these genes, or coding regions thereof. Use of the phrase “substantial sequence similarity” or “substantially similar” in the present specification and claims means that DNA, RNA or amino acid sequences which have slight and non-consequential sequence variations from the actual sequences disclosed and claimed herein are considered to be equivalent to the sequences provided herein. In this regard, “slight and non-consequential sequence variations” mean that “similar” sequences (i.e., the sequences that have substantial sequence similarity with the DNA, RNA or proteins disclosed and claimed herein) will be functionally equivalent to the sequences disclosed and claimed in the present invention. Functionally equivalent sequences will function in substantially the same manner to produce substantially the same compositions as the nucleic acid and amino acid compositions disclosed and claimed herein.

As used herein, a “native nucleotide sequence” or “natural nucleotide sequence” means a nucleotide sequence that can be isolated from non-transgenic cells or tissue. Native nucleotide sequences are those which have not been artificially altered, such as by site-directed mutagenesis. Once native nucleotide sequences are identified, nucleic acid molecules having native nucleotide sequences may be chemically synthesized or produced using recombinant nucleic acid procedures as are known in the art. As used herein, a “native plant nucleotide sequence” is that which can be isolated from non-transgenic plant cells or tissue. As used herein, a “native tobacco nucleotide sequence” is that which can be isolated from non-transgenic tobacco cells or tissue. Use of the phrase “isolated” or “substantially pure” in the present specification and claims as a modifier of nucleic acids, polypeptides or proteins means that the nucleic acids, polypeptides or proteins so designated have been separated from their in vivo cellular environments through the efforts of human beings.

The nucleotide sequences provided herein, such as interfering RNAs or nucleic acids encoding interfering RNAs, can be transformed into a variety of host cells. As used herein, “transformation” refers to the introduction of exogenous nucleic acid into cells so as to produce transgenic cells stably transformed with the exogenous nucleic acid. A variety of suitable host cells, having desirable growth and handling properties, are readily available in the art.

Standard techniques, such as restriction mapping, Southern blot hybridization, polymerase chain reaction (PCR) and/or nucleotide sequence analysis can be employed to identify clones expressing the desired interfering RNA construct. Following the introduction and verification of the desired interfering RNA or nucleic acid construct encoding the desired interfering RNA, whole plants can be regenerated from successfully transformed cells using conventional techniques.

Nucleic acid constructs, or “transcription cassettes,” encoding the interfering RNAs that are used to produce the transgenic cells and plants provided herein include, 5′ to 3′ in the direction of transcription, a promoter as described herein, a nucleotide sequence as described herein operatively associated with the promoter, and, optionally, a termination sequence including stop signal for RNA polymerase and a polyadenylation signal. All of these regulatory regions should be capable of operating in the cells of the tissue to be transformed. Any suitable termination signal may be employed in carrying out the present invention, examples thereof including, but not limited to, the nopaline synthase (nos) terminator, the octapine synthase (ocs) terminator, the CaMV terminator or native termination signals, derived from the same gene as the transcriptional initiation region or derived from a different gene. (See, e.g., Rezian et al. (1988) supra, and Rodermel et al. (1988), supra).

The term “operatively associated,” as used herein, refers to nucleotide sequences on a single nucleic acid molecule that are associated so that the function of one sequence is affected by the other. Thus, a promoter is operatively associated with a nucleotide sequence when it is capable of affecting the transcription of that sequence (i.e., the nucleic acid is under the transcriptional control of the promoter). The promoter is said to be “upstream” from the transcribed nucleotide sequence, which is in turn said to be “downstream” from the promoter.

In some embodiments, the transcription cassette may be provided in a DNA construct that also has at least one replication system. For convenience, it is common to have a replication system functional in Escherichia coli , such as ColE1, pSC101, pACYC184, or the like. In this manner, at each stage after each manipulation, the resulting construct may be cloned, sequenced, and the correctness of the manipulation determined. In addition, or in place of the E. coli replication system, a broad host range replication system may be employed, such as the replication systems of the P-1 incompatibility plasmids, e.g., pRK290. In addition to the replication system, there will frequently be at least one marker present, which may be useful in one or more hosts, or different markers for individual hosts. That is, one marker may be employed for selection in a prokaryotic host, while another marker may be employed for selection in a eukaryotic host, particularly the plant host. The markers may be protection against a biocide (such as antibiotics, toxins, heavy metals or the like), provide complementation by imparting prototrophy to an auxotrophic host and/or provide a visible phenotype through the production of a novel compound in the plant.

The various fragments comprising the various constructs, transcription cassettes, markers and the like may be introduced consecutively by restriction enzyme cleavage of an appropriate replication system and insertion of the particular construct or fragment into the available site. After ligation and cloning, the DNA construct may be isolated for further manipulation. All of these techniques are amply exemplified in the literature as demonstrated by J. Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989)(Cold Spring Harbor Laboratory).

Vectors that may be used to transform plant tissue with nucleic acid constructs provided herein include Agrobacterium and Transbacter vectors and ballistic vectors, as well as vectors suitable for DNA-mediated transformation. In this particular embodiment, the promoter is a region of a DNA sequence that incorporates the necessary signals for the efficient expression of the coding sequence. This region may include sequences to which an RNA polymerase binds, but is not limited to such sequences, and may include sequences to which other regulatory proteins bind along with sequences involved in the control of protein translation. Such regions may also include coding sequences.

Promoters employed in carrying out the invention may be constitutively active promoters. Numerous constitutively active promoters that are operable in plants are available. A preferred example is the Cauliflower Mosaic Virus (CaMV) 35S promoter, which is expressed constitutively in most plant tissues. As an alternative, the promoter may be a root-specific promoter or root cortex specific promoter, as explained in greater detail below.

Nucleic acid sequences have been expressed in transgenic tobacco plants utilizing the Cauliflower Mosaic Virus (CaMV) 35S promoter. (See, e.g., Cornelissen et al., “Both RNA Level and Translation Efficiency are Reduced by Anti-Sense RNA in Transgenic Tobacco”, Nucleic Acids Res. 17, pp. 833-43 (1989); Rezaian et al., “Anti-Sense RNAs of Cucumber Mosaic Virus in Transgenic Plants Assessed for Control of the Virus”, Plant Molecular Biology 11, pp. 463-71 (1988); Rodermel et al., “Nuclear-Organelle Interactions: Nuclear Antisense Gene Inhibits Ribulose Bisphosphate Carboxylase Enzyme Levels in Transformed Tobacco Plants”, Cell 55, pp. 673-81 (1988); Smith et al., “Antisense RNA Inhibition of Polygalacturonase Gene Expression in Transgenic Tomatoes”, Nature 334, pp. 724-26 (1988); Van der Krol et al., “An Anti-Sense Chalcone Synthase Gene in Transgenic Plants Inhibits Flower Pigmentation”, Nature 333, pp. 866-69 (1988)).

Use of the CaMV 35S promoter for expression of interfering RNAs in the transformed tobacco cells and plants provided herein is preferred. Use of the CaMV promoter for expression of other recombinant genes in tobacco roots has been well described (Lam et al., “Site-Specific Mutations Alter In Vitro Factor Binding and Change Promoter Expression Pattern in Transgenic Plants”, Proc. Nat. Acad. Sci. USA 86, pp. 7890-94 (1989); Poulsen et al. “Dissection of 5′ Upstream Sequences for Selective Expression of the Nicotiana plumbaginifolia rbcS-8B Gene”, Mol. Gen. Genet. 214, pp. 16-23 (1988)). Other promoters that are active only in root tissues (root specific promoters) are also particularly suited to the methods provided herein. See, e.g., U.S. Pat. No. 5,459,252 to Conkling et al.; Yamamoto et al., The Plant Cell, 3:371 (1991). The TobRD2 root-cortex specific promoter may also be utilized. All patents cited herein are intended to be incorporated herein by reference in their entirety.

The recombinant interfering nucleic acid molecules and vectors used to produce the transformed tobacco cells and plants provided herein may further comprise a dominant selectable marker gene. Suitable dominant selectable markers for use in tobacco include, inter alia, antibiotic resistance genes encoding neomycin phosphotransferase (NPTII) and hygromycin phosphotransferase (HPT). Preferred selectable markers include the norflurazone resistance genes described in this disclosure. Other well-known selectable markers that are suitable for use in tobacco include a mutant dihydrofolate reductase gene that encodes methotrexate-resistant dihydrofolate reductase. DNA vectors containing suitable antibiotic resistance genes, and the corresponding antibiotics, are commercially available.

Transformed tobacco cells are selected out of the surrounding population of non-transformed cells by placing the mixed population of cells into a culture medium containing an appropriate concentration of the antibiotic (or other compound normally toxic to tobacco cells) against which the chosen dominant selectable marker gene product confers resistance. Thus, only those tobacco cells that have been transformed will survive and multiply. Additionally, the positive selection techniques described by Jefferson (e.g., WO 00055333; WO 09913085; U.S. Pat. Nos. 5,599,670; 5,432,081; and 5,268,463, hereby expressly incorporated by reference in their entireties) can be used.

Methods of making recombinant plants provided herein, in general, involve first providing a plant cell capable of regeneration (the plant cell typically residing in a tissue capable of regeneration). The plant cell is then transformed with an interfering RNA or a nucleic acid construct encoding an interfering RNA comprising a transcription cassette provided herein (as described above) and a recombinant plant is regenerated from the transformed plant cell. As explained below, the transforming step is carried out by techniques as are known in the art, including but not limited to bombarding the plant cell with microparticles carrying the transcription cassette, infecting the cell with an Agrobacterium tumefaciens containing a Ti plasmid carrying the transcription cassette or any other technique suitable for the production of a transgenic plant.

Numerous Agrobacterium vector systems useful in carrying out the present invention are known. For example, U.S. Pat. No. 4,459,355 discloses a method for transforming susceptible plants, including dicots, with an Agrobacterium strain containing the Ti plasmid. The transformation of woody plants with an Agrobacterium vector is disclosed in U.S. Pat. No. 4,795,855. Further, U.S. Pat. No. 4,940,838 to Schilperoort et al. discloses a binary Agrobacterium vector (i.e., one in which the Agrobacterium contains one plasmid having the vir region of a Ti plasmid but no T region, and a second plasmid having a T region but no vir region) useful in carrying out the present invention, all references are hereby expressly incorporated by reference in their entireties.

Microparticles suitable for the ballistic transformation of a plant cell, carrying a nucleic acid construct provided herein, are also useful for making the transformed plants described herein. The microparticle is propelled into a plant cell to produce a transformed plant cell and a plant is regenerated from the transformed plant cell. Any suitable ballistic cell transformation methodology and apparatus can be used in practicing the present invention. Exemplary apparatus and procedures are disclosed in Sanford and Wolf, U.S. Pat. No. 4,945,050, and in Christou et al., U.S. Pat. No. 5,015,580. When using ballistic transformation procedures, the transcription cassette may be incorporated into a plasmid capable of replicating in or integrating into the cell to be transformed. Examples of microparticles suitable for use in such systems include 1 to 5 μm gold spheres. The nucleic acid construct may be deposited on the microparticle by any suitable technique, such as by precipitation.

Plant species may be transformed with the interfering RNA or nucleic acid construct encoding an interfering RNA provided herein by the nucleic acid-mediated transformation of plant cell protoplasts. Plants may be subsequently regenerated from the transformed protoplasts in accordance with procedures well known in the art. Fusion of tobacco protoplasts with nucleic acid-containing liposomes or with nucleic acid constructs via electroporation is known in the art. (Shillito et al., “Direct Gene Transfer to Protoplasts of Dicotyledonous and Monocotyledonous Plants by a Number of Methods, Including Electroporation”, Methods in Enzymology 153, pp. 313-36 (1987)).

These inhibition constructs or RNAi constructs can be transferred to plant cells by any known method in the art. Preferably, Agrobacterium -mediated or Biolistic-mediated transformation are used, according to well-established protocols. It is also contemplated that Transbacter-mediated transformation can be used, as described below. (See Broothaerts et al., Nature 433, 629 (2005), herein expressly incorporated by reference in its entirety).

By this approach, first bacteria are prepared as follows. YM plus antibiotic plates (see below) are streaked with bacteria and the plates are incubated for 2-3 days at 28° C. Transformation is accomplished by measuring about 20 mL Minimal A medium for each bacterial strain. Scrapping or washing the Scrape or wash bacteria from plate with sterile loop and then suspending said bacteria in 20 mL of Minimal A medium. The cell density is adjusted to an OD600 0.9-1.0.

Next, the first healthy fully expanded leaves from 4-5 week old tissue culture grown tobacco plants are cut into 0.5 cm squares (or can use a cork borer, which is about 1.0 cm diameter) in deep petri dish, under sterile RMOP liquid medium. The tissue pieces are stored in RMOP in a deep petri dish. The leaf pieces (about 20 per transformation) are then transferred to a deep petri dish containing bacterial suspension. To ensure that the bacteria have contacted a cut edge of the leaf, the suspension with leaf cutting is swirled and is left standing for 5 minutes. The leaf pieces are then removed from the suspension and blotted dry on filter paper or on the edge of the container. The leaf pieces are then placed with adaxial side (upper leaf surface) on solid RMOP at about 10 pieces per plate.

The plates are then incubated in the dark at 28° C. for: 2-3 days, if A. tumefaciens is used, 5 days if S. melilotiis used, 5 days M. loti is used, and 5-11 days if Rhizobium sp. NGR234 is used. Over the next week, selection is performed. For the purposes of this example, hygromycin selction is performed. Accordingly, the leaf pieces are transferred onto solid RMOP-TCH, with abaxial surface (lower surface of leaf) in contact with media.

The plates are incubated for 2-3 weeks in the light at 28° C., with 16 hours daylight per day. Subculture occurs every 2 weeks.

Plantlet formation is accomplished as follows. Once shoots appear, the plantlet is transferred to MST-TCH pots. The plantlets are grown with 16 hours daylight for 1-2 weeks. Once roots form the plants appear, the plants can be transferred to soil in the greenhouse.

Media and Solutions for Tobacco Transformation:

YM Media (IL)

Keep poured plates for 2 days at room temperature to visualize any contamination, then store at 4° C.

RMOP+RMOP-TCH Media

(Svab, Z., et al., 1975. Transgenic tobacco plants by cocultivation of leaf disks with pPZP Agrobacterium binary vectors. In “Methods in Plant Molecular Biology-A Laboratory Manual”, P. Maliga, D. Klessig, A. Cashmore, W. Gruissem and J. Varner, eds. Cold Spring Harbor Press: 55-77), herein expressly incorporated by reference in its entirety).

IL Final conc.

Sucrose 30 g   (3%) Myo-inositol 100 mg (0.1%) MS Macro 10x 100 mL (1x) MS Micro 1000x 1 mL (1x) Fe2EDTA Iron 100x 10 mL (1x) Thiamine-HCl (10 mg/mL stock) 100 μL (1mg) NAA (1 mg/mL stock) 100 μL 0.1 mg) BAP (1 mg/mL stock) 1 mL  (1 mg) pH 5.8 Phytagel 2.5 g/L for solid autoclave *for RMOP-TCH, when ready to pour add: Timentin (200 mg/mL stock) 1 mL, Claforan (250 mg/mL stock) 1 mL, and Hygromycin (50 mg/mL stock) 1 mL

BAP (1 mg/ml) (6-B enzylaminopurine)

Add 1N KOH drop wise to 100 mg BAP until dissolved. Make up to 100M1 with Milli-Q H2O and store at 4° C.

NAA (1 mg/ml) (Naphthalene Acetic Acid)

Dissolve 100 mg NAA in 1 mL absolute ethanol. Add 3 mL 1N KOH. Make up to 80 mL with Milli-Q H2O. Adjust pH to 6.0 with 1N HCl, make up to 100 mL with Milli-Q H2O, and store at 4° C.

Cefotaxamine (250 mg/ml)

Add 8 ml sterile Milli-Q H2O to 2 g Claforan and store at 4° C. in dark

Timentin (200 mg/ml)

Add 15 ml sterile Milli-Q H2O to 3 g Timentin and store at 4° C.

MST+MST-TCH media

(Svab, Z., et al., 1975. Transgenic tobacco plants by cocultivation of leaf disks with pPZP Agrobacterium binary vectors. In “Methods in Plant Molecular Biology-A Laboratory Manual”, P. Maliga, D. Klessig, A., Cashmore, W. Gruissem and J. Varner, eds. Cold Spring Harbor Press: 55-77), herein expressly incorporated by reference in its entirety).

IL Final Concentration

Sucrose 30 g (3%) MS Macro 10x 100 mL (1x) MS Micro 1000x 1 mL (1x) Fe2EDTA Iron 100x 10 mL (1x) pH 5.8 Phytagel 2.5 g/L Autoclave For MST-TCH, when ready to pour add: Timentin (200 mg/mL stock) (1 mL) Cefotaxamine (250 mg/mL stock) (1 mL) Hygromycin (50 mg/mL stock) (1 mL) MS Macro 10x ((Murashige and Skoog., Phys. Plant. 15: 473-497 (1962), herein expressly incorporated by reference in its entirety)).

Final Concentration

(g/L) 10x KNO3 19.0 NH4 N03 16.5 CaCl2•2H2O 4.4 MgS04•7H2O 3.7 KH2PO4 1.7 Store 4° C. Substituting chemicals: CaCl2 3.3 g/L MgS04 1.8 g/L MS Micro 1000x (Murashige and Skoog., Phys. Plant. 15: 473-497 (1962), herein expressly incorporated by reference in its entirety).

Final Concentration

Once the transformed cells are selected, by any of the approaches described above, they are induced to regenerate intact tobacco plants through application of tobacco cell and tissue culture techniques that are well known in the art. The method of plant regeneration is chosen so as to be compatible with the method of transformation. The stable presence of an interfering RNA or a nucleic acid encoding an interfering RNA in transgenic tobacco plants can be verified by Mendelian inheritance of the interfering RNA or a nucleic acid encoding an interfering RNA sequence, as revealed by standard methods of nucleic acid analysis applied to progeny resulting from controlled crosses. After regeneration of transgenic tobacco plants from transformed cells, the introduced nucleic acid sequence can be readily transferred to other tobacco varieties through conventional plant breeding practices and without undue experimentation.

For example, to analyze the segregation of the transgene, regenerated transformed plants (TO) may be grown to maturity, tested for nicotine and/or TSNA levels, and selfed to produce T 1 plants. A percentage of T 1 plants carrying the transgene are homozygous for the transgene. To identify homozygous T 1 plants, transgenic T 1 plants are grown to maturity and selfed. Homozygous T 1 plants will produce T 2 progeny where each progeny plant carries the transgene; progeny of heterozygous T 1 , plants will segregate 3:1.

Any plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a nucleic acid embodiment provided herein. Preferred plants for introduction of a nucleic acid embodiment, described herein, include Nicotiana . Preferred varieties of Nicotiana for introduction of a nucleic acid embodiment as described herein include the Nicotiana tabacum varieties provided in Table 1.

The term “organogenesis,” as used herein, means a process by which shoots and roots are developed sequentially from meristematic centers; the term “embryogenesis,” as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, callus tissue, existing meristematic tissue (e.g., apical meristems, axillary buds, and root meristems) and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).

Plants provided herein may take a variety of forms. The plants may be chimeras of transformed cells and non-transformed cells; the plants may be clonal transformants (e.g., all cells transformed to contain the transcription cassette); the plants may comprise grafts of transformed and untransformed tissues (e.g., a transformed root stock grafted to an untransformed scion in citrus species). The transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, first generation (or T 1 ) transformed plants may be selfed to give homozygous second generation (or T 2 ) transformed plants and the T 2 plants further propagated through classical breeding techniques. A dominant selectable marker (such as nptII) can be associated with the transcription cassette to assist in breeding.

As used herein, a crop comprises a plurality of plants provided herein, and of the same genus, planted together in an agricultural field. By “agricultural field” is meant a common plot of soil or a greenhouse. Thus, the present invention provides a method of producing a crop of plants having reduced amounts of nicotine, nornicotine, and/or sterol, as compared to a similar crop of non-transformed plants of the same species and variety.

The modified tobacco plants described herein are suitable for conventional growing and harvesting techniques (e.g. topping or no topping, bagging the flowers or not bagging the flowers, cultivation in manure rich soil or without manure). The harvested tobacco leaves and stems are suitable for conventional methods of processing such as curing and blending. The modified tobacco is suitable for use in any traditional tobacco product including, but not limited to, pipe, cigar and cigarette tobacco, and chewing tobacco in any form including leaf tobacco, shredded tobacco, or cut tobacco.

Some embodiments concern the production and identification of particular lines of a transgenic Burley variety (Vector 21-41), which have very low levels of nicotine and TSNAs. The constructs used to create these particular lines of transgenic Burley tobacco are provided in Conkling et al., WO98/56923; U.S. Pat. Nos. 6,586,661; 6,423,520; and U.S. patent application Ser. Nos. 09/963,340; 10/356,076; 09/941,042; 10/363,069; 10/729,121; 10/943,346, all of which are hereby expressly incorporated by reference in their entireties. After the creation and analysis of nearly 2,000 lines of transgenic Burley tobacco, these particular lines of reduced nicotine and TSNA transgenic tobacco were identified. Tobacco harvested from these lines were incorporated into tobacco products (Quest 1®, Quest 2®, and Quest 3®) and were analyzed for their ability to reduce the potential to contribute to a tobacco-related disease, as described in the sections above. It was found that tobacco products comprising these lines of transgenic Burley tobacco, had a reduced potential to contribute to a tobacco-related disease (i.e., that these tobacco products are reduced risk tobacco products).

3. Exemplary Constructs

Several embodiments concern isolated nucleic acids that comprise, consist, or consist essentially of the nucleic acids described in the sequence listing (SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50) and fragments thereof at least 30 consecutive nucleotides in length. That is, embodiments provided herein include an isolated nucleic acid comprising, consisting of, consisting essentially of, any one or more of the sequences of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, or a fragment thereof (e.g., a fragment that is at least, less than or equal to or greater than 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, or 9000 consecutive nucleotides of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50.

In preferred embodiments, the target gene or target mRNA encodes QTPase, PMTase, or the A622 gene product. In preferred embodiments, an interfering RNA comprises, consists, or consists essentially of an RNA strand that is complementary to each least a portion (e.g., less than, greater than or equal to 30, 35, 40, 45, 50, 60, 75, 100, 150, 250, 500, 750, or 1000 consecutive nucleotides) of SEQ ID NOS: 2, 3, 4, 5, 39 or 40, and inhibits the production of QTPase, PMTase, A622, nicotine, nornicotine, NNN, NNK, NAT, or NAB in a tobacco. In related embodiments, the interfering RNA comprises, consists, or consists essentially of an RNA strand that is complementary to each least a portion (e.g., less than, greater than or equal to 30, 35, 40, 45, 50, 60, 75, 100, 150, 250, 500, 750, or 1000 consecutive nucleotides) of SEQ ID NO: 5, and inhibits production of nornicotine but not nicotine in a tobacco. In still more embodiments, the interfering RNA comprises, consists, or consists essentially of an RNA strand that is complementary to each least a portion (e.g., less than, greater than or equal to 30, 35, 40, 45, 50, 60, 75, 100, 150, 250, 500, 750, or 1000 consecutive nucleotides) of SEQ ID NO: 6, 7, 8, or 9, and inhibits production of at least one sterol (e.g., squalene synthase, HMG-CoA reductase, SMT2, or 14alpha demethylase) in a tobacco and at least one PAH upon pyrolysis of said tobacco.

Some of these nucleic acid embodiments comprise, consist, or consist essentially of fragments of the QPTase, PMTase, and A622 genes that were found to inhibit gene expression unexpectedly well in the RNAi constructs described herein, producing reduced alkaloid tobacco (below 7,000 ppm, 1,000 ppm, or 500 ppm). Some of these nucleic acids concern fragments of genes involved in sterol biosynthesis (e.g., squalene synthase, HMG-CoA reductase, SMT2, or 14alpha demethylase) and these fragments are particularly useful for inhibiting production of sterols in tobacco and PAHs when said tobacco undergoes pyrolysis.

Still more of the nucleic acid embodiments concern several phytoene desaturase (PDS) mutants (e.g., PDSM-1, PDSM-2, and PDSM-3, SEQ. ID. NOs.: 10, 11, or 12) that were developed to confer resistance to norflurazone, which allows both tissue-culture selection of cells transformed with the construct, as well as, field-based selection, wherein weeds and tobacco, which do not contain an herbicide resistance gene, are removed from the field or crop by spraying the herbicide norflurazone or an herbicide of the same class or activity (e.g., herbicides that contain C 12 H 9 ClF 3 N 3 O (see U.S. Pat. No. 3,644,355, herein expressly incorporated by reference in its entirety), but plants expressing PDSM-1, PDSM-2, or PDSM-3 survive the herbicide contact). That is, some embodiments include isolated nucleic acids that comprise, consist, or consist essentially of the PDS mutant sequences provided by SEQ. ID. NOs.:10, 11, or 12 and fragments thereof at least 30 nucleotides in length (e.g., less than, greater than or equal to 30, 35, 40, 45, 50, 60, 75, 100, 150, 250, 500, 750, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1729 consecutive nucleotides) that include a mutation (e.g., T1478G, which encodes Val493Gly; G863C, which encodes Arg288Pro; and T1226C, which encodes Leu409Pro) that confers resistance to norflurazone). Preferably, the fragments of the PDS mutants described herein confer resistance to norflurazone, although fragments that do not confer resistance to the herbicide are also useful in the field in assays designed to follow the retention of constructs described herein in successive generations of transgenic plants. Approaches to develop more norflurazone-resistance genes are also provided herein.

Additional embodiments include isolated nucleic acids that comprise, consist, or consist essentially of root-specific promoters, constitutive promoters, and developmentally regulated promoters, which can be used interchangeably with the nucleic acid sequences described herein. Some embodiments, for example, include a root-specific promoter such as the RD2 promotor (SEQ. ID NO. 37 or SEQ. ID NO. 50), truncated RD2 promoter (SEQ. ID NO. 13) or the Putrescene methyl transferase promoter (PMT-1) (SEQ. ID NO. 14). Constitutive promoters that can be used with embodiments described herein include the GapC promoter (SEQ. ID. NO.: 15), Actin 2 promoter (Act2P) (SEQ. ID NO. 16), the tobacco alcohol dehydrogenase promoter (ADP) (SEQ. ID NO. 17), the Arabidopsis ribosomal protein L2 promoter (RPL2P) (SEQ. ID NO. 18), and the nopaline synthase promoter (NOS P) (SEQ ID NO. 46). Developmentally regulated promoters that can be used with the nucleic acid sequences described herein include the cinnamyl alcohol dehydrogenase promoter (SEQ. ID NO. 19) and the metallothionein I promoter (SEQ. ID NO. 20). Additional embodiments also include isolated nucleic acids that comprise, consist, or consist essentially of the GAD2 terminator (SEQ. ID NO. 21), nopaline synthase terminator (NOS T) (SEQ ID NO 38), a FAD2 intron (provided by (SEQ. ID NO. 22), ACT 11 intron 3 (SEQ ID NO 41), which was used as a spacer in several of the RNAi constructs, and the PAP1 intron (provided by nucleotides 6446-7625 of (SEQ. ID NO. 33). Because of the unique properties of the FAD2 intron, in particular the hair-pin secondary structure afforded by the interaction of splice sites in the sequence, it was found, unexpectedly, that transgenic tobacco could be made with various inhibitory sequences with nearly equivalent success (e.g., approximately 50% of the reduced nicotine lines created by multiple constructs were found to have less than 1,000 ppm total alkaloid). Accordingly, significantly improved RNAi constructs were generated using this spacer. That is, embodiments provided herein concern the use of an intronic sequence comprising splicing recognition sequences (preferably FAD2 or PAP1 intron) to link or join a first RNA sequence to a second RNA sequence that is complementary to said first RNA sequence, wherein said first or second RNA sequence is complementary to a target RNA, which, preferably, regulates the production of a harmful compound in tobacco (e.g., nicotine, nornicotine, or a sterol).

Embodiments provided herein also concern isolated nucleic acids that comprise, consist, or consist essentially of the inhibition and selection cassettes identified as SEQ. ID. Nos. 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 and fragments thereof (e.g., a fragment that is at least, less than or equal to or greater than 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, or 9000 consecutive nucleotides) of SEQ. ID. Nos. 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50).

Embodiments provided herein also concern isolated nucleic acids that comprise, consist, or consist essentially of a plurality of the nucleic acid sequences described herein. For example, a double knock-out construct comprising a portion of the A622 gene and a portion of the QPTase gene has been made and it is expected that this construct will efficiently reduce expression of at least two genes involved in the synthesis or regulation of the production of nicotine (SEQ. ID. No. 27). Another double knock-out construct comprises, consists of or consists essentially of a first isolated nucleic acid that inhibits nicotine biosynthesis (e.g., A622) and a second isolated nucleic acid that inhibits synthesis of at least one sterol (e.g., SMT2). (See (SEQ. ID. No. 33)). Accordingly, embodiments provided herein concern an isolated nucleic acid construct that inhibits the expression of a plurality of genes that regulate the production of more than one harmful compound in tobacco. In some aspects of these embodiments, said isolated nucleic acid construct inhibits the expression of at least two nicotine biosynthesis genes, a nicotine biosynthesis gene and a sterol biosynthesis gene, or two sterol biosynthesis genes. It should also be understood that embodiments provided herein concern tobacco generated by crossing the transgenic tobaccos described herein. For example, some embodiments concern progeny of a cross between a transgenic tobacco having a reduced amount of nicotine and a transgenic tobacco having a reduced amount of a sterol. Crossings of the transgenic tobacco described herein and wild-type tobacco are also embodiments provided herein.

The interfering RNAs used with the embodied nucleic acids can be expressed from nucleic acid construct that encodes one or more strands of the RNA duplex of the interfering RNA. In some embodiments, the nucleic acid construct is present on a vector. The vectors may be viral vectors, plasmids, or any other vehicles for nucleic acid delivery. In other embodiments, the interfering RNAs described herein can be generated synthetically by methods, such as direct synthesis or in vitro transcription. In some embodiments, synthetic interfering nucleic acids comprising modified nucleic acids are contemplated. Other embodiments provided herein include multiple vector systems for producing an interfering RNA wherein a first vector encodes the first strand of the interfering RNA and a second vector encodes the second strand of the interfering RNA.

Still other embodiments provided herein relate to tobacco cells comprising one or more of the nucleic acid constructs described herein, which encode an interfering RNA that is specific for a gene product involved in nicotine or sterol biosynthesis. In such embodiments, the interfering RNA reduces or eliminates the expression of such gene product. Additional embodiments relate to tobacco cells comprising one or more interfering RNAs that are specific for a gene product involved in nicotine biosynthesis. In certain embodiments, the interfering RNAs are synthetic interfering RNAs.

Certain embodiments provided herein relate to tobacco plants and cured tobacco products having a reduced amount or nicotine, nornicotine, TSNAs, and/or sterols. In such embodiments, reduction in nicotine, nornicotine, TSNAs, and/or sterol amounts in the tobacco plants and cured tobacco products is mediated by an interfering RNA comprising an RNA duplex wherein at least 30 consecutive nucleotides (e.g., at least or equal to 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 800, 900, 1000 consecutive nucleotides) of the RNA duplex are complementary or substantially complementary to a target mRNA that encodes a gene product involved in nicotine biosynthesis. Further aspects relate to a field or crop of tobacco plants comprising one or more of the constructs described herein. Still other aspects relate to a tobacco seed produced from one or more of the tobacco plants provided herein.

Transgenic tobacco plants produced by the methods described herein can be cured by any of the tobacco curing techniques that are known in the art. As such, some embodiments provided herein relate to cured tobacco and cure tobacco products made from the transgenic plants described herein. In some embodiments, the cured tobacco product is a blended tobacco product. In some embodiments, the cured tobacco product is processed in a microbe-free environment. In other embodiments, the cured tobacco is contacted with sterilizing vapor, heat, or radiation so as to prevent the conversion of alkaloid to TSNAs.

Some embodiments provided herein relate to methods of preparing a tobacco cell having a reduced nicotine and/or sterol content, wherein the method comprises providing a tobacco cell with one or more interfering RNAs or one or more nucleic acid constructs encoding an interfering RNA comprising an RNA duplex, which comprises a first strand having a sequence substantially similar or identical to at least a portion of the coding sequence of a target gene and/or target gene product involved in nicotine and/or sterol biosynthesis, and a second strand that is complementary or substantially complementary to the first strand. In a preferred embodiment, the target gene product involved in nicotine biosynthesis is QTPase, PMTase, or A622 and the target gene product involved in sterol biosynthesis is squalene synthase, HMG-CoA reductase, SMT2, or 14alpha demethylase.

Other embodiments provided herein relate to methods of preparing a tobacco plant having a reduced nicotine and/or sterol content comprising obtaining a tobacco cell in culture; providing to the tobacco cell one or more interfering RNAs or one or more nucleic acid constructs encoding an interfering RNA comprising an RNA duplex, which comprises a first strand having a sequence substantially similar or identical to at least a portion of the coding sequence of a target gene and/or target gene product involved in nicotine and/or sterol biosynthesis, and a second strand that is complementary or substantially complementary to the first strand; allowing expression of the interfering RNA, thereby reducing cellular nicotine and/or sterol content; and regenerating a tobacco plant from the tobacco cell. In some embodiments, the tobacco plants prepared by such method also have a reduced TSNA content and/or produce a reduced amount of PAHs upon pyrolysis, as compared to a conventional tobacco product of he same class, a reference tobacco product (e.g., IM16), or the same strain of tobacco prior to genetic modification.

As mentioned above, additional embodiments include tobacco products that have been carefully blended so that desired levels of nicotine, TSNAs, and/or sterols are obtained. For example, tobacco having a reduced level of nicotine and/or TSNAs, prepared as described above, can be blended with conventional tobacco so as to obtain virtually any amount of nicotine and/or sterols. Additionally, as mentioned above, exogenous nicotine can be added to the tobacco or tobacco product. Further, two or more varieties of tobacco (e.g., transgenic reduced alkaloid Burley, transgenic reduced alkaloid Flue-cured, and/or transgenic reduced alkaloid Oriental) can be blended so as to achieve a desired taste while maintaining nicotine levels in or delivered by the product (e.g., as measured by FTC methodology) at less than 7,000 ppm, 5,000 ppm, 3000 ppm, 2000 ppm, 1000 ppm, or 500 ppm and TSNA levels at 0.5 μg/g or less. Similarly, two or more varieties of transgenic tobacco having a reduced amount of sterols can be blended, as above, or varieties of sterol-reduced transgenic tobacco can be blended with varieties of nicotine reduced transgenic tobacco. In this manner, differences in variety, flavor, as well as amounts of nicotine and/or sterols can be incrementally adjusted. These blended tobaccos can be processed into tobacco products, which can be incorporated into tobacco use cessation kits (e.g., a multiple step nicotine reduction program, whereby a consumer's exposure to nicotine, TSNA, or PAH is gradually reduced over time by consumption of tobacco products that have increasingly smaller quantities of these compounds). Such kits and programs, are designed to reduce or eliminate nicotine dependence and reduce the potential to contribute to a tobacco related disease.

More embodiments concern methods to reduce the carcinogenic potential of tobacco products, including cigarettes, cigars, chewing tobacco, snuff and tobacco-containing gum and lozenges. Some methods, for example involve the use of the constructs described herein to obtain transgenic tobacco that comprises a reduced amount of nicotine, TSNAs, and/or sterols and the manufacture of tobacco products containing said tobacco. Accordingly, the transgenic tobacco plants, described above, are harvested, cured, and processed into tobacco products. These tobacco products have a reduced carcinogenic potential because they are prepared from tobacco that has a reduced amount of nicotine, TSNAs, and sterols. Smoke or smoke condensate generated from these tobaccos and tobacco products can also be evaluated using the assays provided herein so as to confirm that said tobaccos and tobacco products have a reduced potential to contribute to a tobacco-related disease and that said tobaccos and tobacco products are reduced risk compositions.

Yet another aspect provided herein concerns the reduction of the amount of TSNAs, preferably NNN and NNK, and polyaromatic hydrocarbons (PAHs), preferably, benz[a]pyrene and metabolites thereof in humans who smoke, consume or otherwise ingest tobacco. This method is practiced by providing a tobacco product comprising a transgenic tobacco that comprises a reduced amount of nicotine and/or a sterol to said humans, thereby lowering the amount of TSNAs and/or PAHs in said humans exposed to said tobacco product. By one approach, for example, the carcinogenic potential of side stream or main stream tobacco smoke in a human exposed to said side stream or main stream tobacco smoke is reduced by providing the cured tobacco as described above in a product that undergoes pyrolysis, wherein pyrolysis of said product results in side stream or main stream smoke comprising a reduced amount of TSNAs and/or PAHs. The section below describes several preferred approaches to develop genetically modified tobaccos and tobacco products containing genetically modified tobacco that have a reduced amount of a compound that contributes to a tobacco related disease.

Preparation of Preferred Transgenic Tobaccos

A first generation of transgenic Burley tobacco was created using a full-length antisense QPTase construct. Tobacco of the variety Burley 21 LA was transformed with the binary Agrobacterium vector pYTY32 to produce a low nicotine tobacco variety, Vector 21-41. The binary vector pYTY32 carried the 2.0 kb NtQPT1 root-cortex-specific promoter driving antisense expression of the NtQPT1 cDNA (SEQ. ID. NO. 2) and the nopaline synthase (nos) 3′ termination sequences from Agrobacterium tumefaciens T-DNA. The selectable marker for this construct was neomycin phosphotransferase (nptII) from E. coli Tn5 which confers resistance to kanamycin, and the expression nptII was directed by the nos promoter from Agrobacterium tumefaciens T-DNA. Transformed cells, tissues, and seedlings were selected by their ability to grow on Murashige-Skoog (MS) medium containing 300 μg/ml kanamycin. Burley 21 LA is a variety of Burley 21 with substantially reduced levels of nicotine as compared with Burley 21 (i.e., Burley 21 LA has 8% the nicotine levels of Burley 21, see Legg et al., Can J Genet Cytol, 13:287-91 (1971); Legg et al., J Hered, 60:213-17 (1969)).

One-hundred independent pYTY32 transformants of Burley 21 LA (T 0 ) were allowed to self. Progeny of the selfed plants (T 1 ) were germinated on medium containing kanamycin and the segregation of kanamycin resistance scored. T 1 progeny segregating 3:1 resulted from transformation at a single locus and were subjected to further analysis.

Nicotine levels of T 1 progeny segregating 3:1 were measured qualitatively using a micro-assay technique. Approximately˜200 mg fresh tobacco leaves were collected and ground in 1 ml extraction solution (Extraction solution: 1 ml Acetic acid in 100 ml H 2 O). Homogenate was centrifuged for 5 min at 14,000×g and supernatant removed to a clean tube, to which the following reagents were added: 100 μL NH 4 OAC (5 g/100 ml H 2 O+50 μL Brij 35); 500 μL Cyanogen Bromide (Sigma C-6388, 0.5 g/100 ml H 2 O+50 μL Brij 35); 400 μL Aniline (0.3 ml buffered Aniline in 100 ml NH 4 OAC+50 μL Brij 35). A nicotine standard stock solution of 10 mg/ml in extraction solution was prepared and diluted to create a standard series for calibration. Absorbance at 460 nm was read and nicotine content of test samples were determined using the standard calibration curve.

T 1 progeny that had less than 10% of the nicotine levels of the Burley 21 LA parent were allowed to self to produce T 2 progeny. Homozygous T 2 progeny were identified by germinating seeds on medium containing kanamycin and selecting clones in which 100% of the progeny were resistant to kanamycin (i.e., segregated 4:0; heterozygous progeny would segregate 3:1). Nicotine levels in homozygous and heterozygous T 2 progeny were qualitatively determined using the micro-assay and again showed levels less than 10% of the Burley 21 LA parent. Leaf samples of homozygous T 2 progeny were sent to the Southern Research and Testing Laboratory in Wilson, N.C. for quantitative analysis of nicotine levels using Gas Chromatography/Flame Ionization Detection (GC/FID). Homozygous T 2 progeny of transformant #41 gave the lowest nicotine levels (˜70 ppm), and this transformant was designated as “Vector 21-41.”

Vector 21-41 plants were allowed to self-cross, producing T 3 progeny. T 3 progeny were grown and nicotine levels assayed qualitatively and quantitatively. T 3 progeny were allowed to self-cross, producing T 4 progeny. Samples of the bulked seeds of the T 4 progeny were grown and nicotine levels tested.

In general, Vector 21-41 is similar to Burley 21 LA in all assessed characteristics, with the exception of alkaloid content and total reducing sugars (e.g., nicotine and nor-nicotine). Vector 21-41 may be distinguished from the parent Burley 21 LA by its substantially reduced content of nicotine, nor-nicotine and total alkaloids. As shown below, total alkaloid concentrations in Vector 21-41 are significantly reduced to approximately relative to the levels in the parent Burley 21 LA, and nicotine and nor-nicotine concentrations show dramatic reductions in Vector 21-41 as compared with Burley 21 LA. Vector 21-41 also has significantly higher levels of reducing sugars as compared with Burley 21 LA.

Field trials of Vector 21-41 T 4 progeny were performed at the Central Crops Research Station (Clayton, N.C.) and compared to the Burley 21 LA parent. The design was three treatments (Vector 21-41, a Burley 21 LA transformed line carrying only the NtQPTJ promoter [Promoter-Control], and untransformed Burley 21 LA [Wild-type]), 15 replicates, 10 plants per replicate. The following agronomic traits were measured and compared: days from transplant to flowering; height at flowering; leaf number at flowering; yield; percent nicotine; percent nor-nicotine; percent total nitrogen; and percent reducing sugars.

Vector 21-41 was also grown on approximately 5000 acres by greater than 600 farmers in five states (Pennsylvania, Mississippi, Louisiana, Iowa, and Illinois). The US Department of Agriculture, Agriculture Marketing Service (USDA-AMS) quantified nicotine levels (expressed as percent nicotine per dry weight) using the FTC method of 2,701 samples taken from these farms. Nicotine levels ranged from 0.01% to 0.57%. The average percent nicotine level for all these samples was 0.09%, with the median of 0.07%. Burley tobacco cultivars typically have nicotine levels between 2% and 4% dry weight (Tso, T.C., 1972, Physiology and Biochemistry of Tobacco Plants . Dowden, Hutchinson, and Ross, Inc. Stroudsbury).

A transgenic Flue-cured tobacco with a reduced amount of nicotine and TSNAs was created using an RNAi approach. FIG. 1 illustrates an RNAi construct that was used to create a reduced nicotine tobacco, wherein the root-specific promoter RD2 (Bp 1-2010) was used to drive expression of an RNAi cassette comprising an antisense full-length QPTase cDNA (Bp 2011-3409) linked to a 382 bp fragment of the cucumber aquaporin gene (Bp 3410-3792), which is linked to a sense full-length QPTase cDNA (Bp 3793-5191) and the GapC terminator (Bp5192-5688) (see SEQ. ID. No. 23). This first RNAi construct also comprises a GUS-selection cassette comprising the GapC promoter (Bp 1-1291), which drives expression of the GUS gene (Bp 1292-3103), linked to the GapC terminator (Bp 3104-3600) (see SEQ. ID. No. 34). This first RNAi construct was ligated into a binary vector, pBin19 which was then introduced into Agrobacterium tumefaciens . Leaf disks from Flue-cured variety K326 were then transformed with Agrobacterium that contained the RNAi construct comprising the RNAi cassette and the GUS selection cassette. GUS-based selection was then employed to select positively transformed plantlets (buds), which were then regenerated to plants. Leaf samples were then harvested and the alkaloid content was then determined. The alkaloid content of samples obtained from some of the transgenic lines created with this first RNAi construct was 6000 ppm. Since the total alkaloid content in tobacco is about 90% nicotine, it is understood by those skilled in the art that the transgenic Flue-cured tobacco created using the construct shown in FIG. 1 has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification. Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

FIG. 2 shows another RNAi construct that was used to generate several lines of reduced nicotine and TSNA tobacco. This RNAi construct has a QTPase inhibition cassette (SEQ. ID. No. 24) and a norflurazone selection cassette (SEQ. ID. No. 35). Starting from the right border (RB), the QPTase inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably linked to an antisense fragment (360 bp) (Bp 2011-2370) of the QTPase gene, joined to a FAD2 intron (Bp 2371-3501), which is joined to a sense fragment of the QTPase gene (360 bp) (Bp 3502-3861), which is joined to the GAD2 terminator (Bp 3862-4134). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB). Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco was transformed with the construct shown in FIG. 2 using Agrobacterium -mediated transformation and 1,140 independent lines were selected, regenerated, and transplanted in the greenhouse. Of the 1,140 independent lines, 1,097 plants were harvested and tested for alkaloid content. A total of 608 lines were identified as having less than 1,000 ppm total alkaloid and 139 lines were identified as having less than 500 ppm total alkaloid. Accordingly, the transgenic Flue-cured tobacco created using the construct shown in FIG. 2 has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco was also transformed with the construct shown in FIG. 2 using Agrobacterium -mediated transformation and 385 independent lines were selected, regenerated, and transplanted in the greenhouse. Of the 385 independent lines, 350 lines of plants were harvested and tested for alkaloid content. A total of 142 lines were identified as having less than 1,000 ppm total alkaloid and 10 lines were identified as having less than 500 ppm total alkaloid. Accordingly, it is understood by those skilled in the art that the transgenic Burley tobacco created using the construct shown in FIG. 2 also has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will be transformed with the construct shown in FIG. 2 using Agrobacterium -mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct shown in FIG. 2 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

FIG. 3 illustrates another RNAi construct that can be used to create a reduced nicotine and TSNA transgenic tobacco. This RNAi construct has a PMTase inhibition cassette (SEQ. ID. No. 25) and a norflurazone selection cassette (SEQ. ID. No. 35). Starting from the right border (RB), the PMTase inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably linked to an antisense nucleic acid (241 bp) (Bp 2011-2251) of a PMTase gene, joined to a FAD2 intron (Bp 2252-3382), which is joined to a sense nucleic acid of the PMTase gene (241 bp) (Bp 3383-3623), which is joined to the GAD2 terminator (Bp 3624-3896). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB). Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco will be transformed with the construct shown in FIG. 3 using Agrobacterium -mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Flue-cured tobacco that will be created using the construct shown in FIG. 3 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the construct shown in FIG. 3 using Agrobacterium -mediated, Transbacter-mediated (see e.g., Broothaerts et al., Nature 433:629 (2005), herein expressly incorporated by reference in its entirety) or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct shown in FIG. 3 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will also be transformed with the construct shown in FIG. 3 using Agrobacterium -mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct shown in FIG. 3 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

FIG. 4 illustrates another RNAi construct that was used to create a reduced nicotine and TSNA transgenic tobacco. This RNAi construct has a A622 inhibition cassette (SEQ. ID. No. 26) and a norflurazone selection cassette (SEQ. ID. No. 35). Starting from the right border (RB), the A622 inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably linked to an antisense nucleic acid (628 bp) (Bp 2011-2638) of the A622 gene, joined to a FAD2 intron (Bp 2639-3769), which is joined to a sense nucleic acid of the A622 gene (628 bp) (Bp 3770-4397), which is joined to the GAD2 terminator (Bp 4398-4670). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB). Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco was transformed with the construct shown in FIG. 4 using Agrobacterium -mediated transformation and 270 independent lines were selected, regenerated, and transplanted in the greenhouse. Of the 270 independent lines, 259 plants were harvested and tested for alkaloid content. A total of 131 lines were identified as having less than 1,000 ppm total alkaloid and 45 lines were identified as having less than 500 ppm total alkaloid. Accordingly, it is understood by those skilled in the art that the transgenic Flue-cured tobacco created using the construct shown in FIG. 4 also has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Several lines that were transformed with this construct were unexpectedly found to have conventional levels of nicotine but a significantly reduced amount of nornicotine. That is, 9 lines were found to have nicotine levels ranging from 2.17 mg/g to 3.99 mg/g and nornicotine levels less than or equal to 0.00 to 0.06 mg/g (see Table 2).

Tobacco products containing the selectively reduced nornicotine transgenic tobacco described above are also embodiments provided herein. That is, tobacco products comprising a transgenic tobacco that comprises a conventional amount of nicotine (e.g., comprise or delivers according to FTC methodology at least, less than, greater than, or equal to 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg/g nicotine) and a reduced amount of nornicotine (e.g., 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.2 mg/g), as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification, are embodiments provided herein. Particularly preferred are transgenic tobacco and tobacco products made therefrom, which comprise a conventional amount of nicotine (e.g., comprises or delivers by FTC methodology at least, less than, greater than, or equal to 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg/g nicotine) and a reduced amount of nornicotine (e.g., 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.2 mg/g), as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification, and an isolated fragment of the A622 gene, in particular, comprising, consisting of, or consisting essentially of an isolated nucleic acid of SEQ. ID. No. 5, or the cassette of SEQ. ID. No. 26.

Burley tobacco will be transformed with the construct shown in FIG. 4 using Agrobacterium -mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct shown in FIG. 4 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification. It is also expected that some lines of tobacco created with the afore-mentioned nucleic acid construct will retain conventional amounts of nicotine but will comprise a reduced amount of nornicotine, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will also be transformed with the construct shown in FIG. 4 using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct shown in FIG. 4 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification. It is also expected that some lines of tobacco created with the afore-mentioned nucleic acid construct will retain conventional amounts of nicotine but will comprise a reduced amount of nornicotine, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

FIG. 5 illustrates a double-knock-out RNAi construct, which has been created to develop a reduced nicotine and TSNA transgenic tobacco. This double-knock-out RNAi construct has a QPTase/A622 inhibition cassette (SEQ. ID. No. 27) and a norflurazone selection cassette (SEQ. ID. No. 35). Starting from the right border (RB), the QPTase/A622 inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably linked to a QPTase antisense nucleic acid (360 bp) (Bp 2011-2370) of a QPTase gene, which is joined to a A622 antisense nucleic acid (628 bp) (Bp 2371-2998) of a A622 gene, which is joined to a FAD2 intron (Bp 2999-4129), which is joined to a sense nucleic acid of the A622 gene (628 bp) (Bp 4130-4757), which is joined to a sense nucleic acid of the QPTase gene (360 bp) (Bp 4758-5117), which is joined to the GAD2 terminator (Bp 5118-5390). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB). Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco will be transformed with the construct shown in FIG. 5 using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Flue-cured tobacco that will be created using the construct shown in FIG. 5 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the construct shown in FIG. 5 using Agrobacterium -mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct shown in FIG. 5 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will also be transformed with the construct shown in FIG. 5 using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct shown in FIG. 5 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

More embodiments concern an RNAi construct designed to reduce the amount of sterols in tobacco and thereby reduce production of a PAH upon pyrolysis of said transgenic tobacco. A first sterol-reducing RNAi construct has a 14alpha demethylase inhibition cassette (SEQ. ID. No. 28). The 14alpha demethylase inhibition cassette comprises a double (two promoters in tandem) 35S promoter (Bp 1-618) operably linked to an antisense 14alpha demethylase nucleic acid (Bp 619-1503), which is joined to a FAD2 intron (Bp 1504-2634), which is joined to a sense nucleic acid of the 14alpha demethylase gene (Bp 2635-3519), which is joined to the Nos terminator (Bp 3520-3773). Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco will be transformed with the 14alpha demethylase inhibition cassette using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for sterol content. It is expected that approximately 50% of the lines tested will have significantly less sterol than the parent strain of tobacco. Accordingly, it is expected that the transgenic Flue-cured tobacco that will be created using the construct above will have significantly reduced levels of sterol and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the 14alpha demethylase inhibition cassette using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for sterol content. It is expected that approximately 50% of the lines tested will have significantly less sterol than the parent strain of tobacco. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct above will have significantly reduced levels of sterol and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will be transformed with the 14alpha demethylase inhibition cassette using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for sterol content. It is expected that approximately 50% of the lines tested will have significantly less sterol than the parent strain of tobacco. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct above will have significantly reduced levels of sterol and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

More embodiments concern another RNAi construct designed to reduce the amount of a sterol in tobacco and thereby reduce production of a PAH upon pyrolysis of said transgenic tobacco. A second sterol-reducing RNAi construct has a SMT2 inhibition cassette (SEQ. ID. No. 29). The SMT2 inhibition cassette comprises a double (two promoters in tandem) 35S promoter (Bp 1-618) operably linked to an antisense SMT2 nucleic acid (Bp 619-1398), which is joined to a FAD2 intron (Bp 1399-2529), which is joined to a sense nucleic acid of the SMT2 gene (Bp 2530-3309), which is joined to the Nos terminator (Bp 3310-3563). Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco will be transformed with the SMT2 inhibition cassette using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for sterol content. It is expected that approximately 50% of the lines tested will have significantly less sterol than the parent strain of tobacco. Accordingly, it is expected that the transgenic Flue-cured tobacco that will be created using the construct above will have significantly reduced levels of sterol and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the SMT2 inhibition cassette using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for sterol content. It is expected that approximately 50% of the lines tested will have significantly less sterol than the parent strain of tobacco. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct above will have significantly reduced levels of sterol and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will be transformed with the SMT2 inhibition cassette using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for sterol content. It is expected that approximately 50% of the lines tested will have significantly less sterol than the parent strain of tobacco. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct above will have significantly reduced levels of sterols and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

More embodiments concern another RNAi construct designed to reduce the amount of a sterol in tobacco and thereby reduce production of a PAH upon pyrolysis of said transgenic tobacco. A third sterol-reducing RNAi construct has a squalene synthase inhibition cassette (SEQ. ID. No. 30). The squalene synthase inhibition cassette comprises a double (two promoters in tandem) 35S promoter (Bp 1-618) operably linked to an antisense squalene synthase nucleic acid (Bp 619-1057), which is joined to a FAD2 intron (Bp 1058-2188), which is joined to a sense nucleic acid of the squalene synthase gene (Bp 2189-2627), which is joined to the Nos terminator (Bp 2628-2881). Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco will be transformed with the squalene synthase inhibition cassette using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for sterol content. It is expected that approximately 50% of the lines tested will have significantly less sterol than the parent strain of tobacco. Accordingly, it is expected that the transgenic Flue-cured tobacco that will be created using the construct above will have significantly reduced levels of sterol and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the squalene synthase inhibition cassette using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for sterol content. It is expected that approximately 50% of the lines tested will have significantly less sterol than the parent strain of tobacco. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct above will have significantly reduced levels of sterol and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will be transformed with the squalene synthase inhibition cassette using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for sterol content. It is expected that approximately 50% of the lines tested will have significantly less sterol than the parent strain of tobacco. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct above will have significantly reduced levels of sterol and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

More embodiments concern yet another RNAi construct designed to reduce the amount of a sterol in tobacco and thereby reduce production of a PAH upon pyrolysis of said transgenic tobacco. A fourth sterol-reducing RNAi construct has a HMG-CoA reductase inhibition cassette (SEQ. ID. No. 31). The HMG-CoA reductase inhibition cassette comprises a double (two promoters in tandem) 35S promoter (Bp 1-618) operably linked to an antisense HMG-CoA reductase nucleic acid (Bp 619-1468), which is joined to a FAD2 intron (Bp 1469-2599), which is joined to a sense nucleic acid of the HMG-CoA reductase gene (Bp 2600-3449), which is joined to the Nos terminator (Bp 3450-3703). Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco (K326) was transformed with the HMG-CoA reductase inhibition cassette using Agrobacterium -mediated transformation and independent lines were selected, regenerated, and transplanted in the greenhouse. Several independent lines grown in the greenhouse were harvested and tested for the presence of various sterols (see Table 3). As shown in the table, several lines (e.g., HMGIR 1, HMGIR 2, HMGIR 3-2, HMGIR 4, HMGIR 7, HMGIR 11, HMGIR 13, HMGIR 16, HMGIR 18, HMGIR 19) were found to have significantly reduced levels of sterols, as compared to the parental strain of tobacco (i.e., tobacco of the same variety prior to genetic modification). Accordingly, embodiments include transgenic tobacco and tobacco products made therefrom comprising a reduced amount of sterols, as compared to a tobacco of the same variety, parental strain or a tobacco that has not been genetically modified. It is expected that the transgenic Flue-cured tobacco that was created using the construct above will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the HMG-CoA reductase cassette using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for sterol content. It is expected that approximately 50% of the lines tested will have significantly less sterol than the parent strain of tobacco. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct above will have significantly reduced levels of sterol and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will be transformed with the HMG-CoA reductase inhibition cassette using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for sterol content. It is expected that approximately 50% of the lines tested will have significantly less sterol than the parent strain of tobacco. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct above will have significantly reduced levels of sterol and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

More embodiments concern still another RNAi construct designed to reduce the amount of a sterol in tobacco and thereby reduce production of a PAH upon pyrolysis of said transgenic tobacco. A fifth sterol-reducing RNAi construct has a developmentally regulated SMT2 inhibition cassette (SEQ. ID. No. 32). The developmentally regulated SMT2 inhibition cassette comprises a cinnamyl alcohol dehydrogenase promoter (Bp 1-995) operably linked to an antisense SMT2 nucleic acid (Bp 996-1775), which is joined to a PAP 1 intron (Bp 1776-2955), which is joined to a sense nucleic acid of the SMT2 gene (Bp 2956-3735), which is joined to the RuBisCo small subunit terminator (Bp 3736-4286). Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in barley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco will be transformed with the developmentally regulated SMT2 inhibition cassette using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for sterol content. It is expected that approximately 50% of the lines tested will have significantly less sterol than the parent strain of tobacco. Accordingly, it is expected that the transgenic Flue-cured tobacco that will be created using the construct above will have significantly reduced levels of sterol and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the developmentally regulated SMT2 inhibition cassette using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for sterol content. It is expected that approximately 50% of the lines tested will have significantly less sterol than the parent strain of tobacco. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct above will have significantly reduced levels of sterol and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will be transformed with the developmentally regulated SMT2 inhibition cassette using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for sterol content. It is expected that approximately 50% of the lines tested will have significantly less sterol than the parent strain of tobacco. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct above will have significantly reduced levels of sterol and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

FIG. 6 illustrates a double-knock-out RNAi construct that can be used to create a reduced nicotine, TSNA, sterol transgenic tobacco that generates a reduced amount of PAH upon pyrolysis. This double-knock-out RNAi construct has a A622/SMT2 inhibition cassette (SEQ. ID. No. 33) and a norflurazone selection cassette (SEQ. ID. No. 35). Starting from the right border (RB), the A622/SMT2 inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably linked to a A622 antisense nucleic acid (628 bp) (Bp 2011-2638) of a A622 gene, which is joined to a FAD2 intron (Bp 2639-3769), which is joined to a sense nucleic acid of the A622 gene (628 bp) (Bp3770-4397), which is joined to the GAD2 terminator (Bp 4398-4670); which is joined to a cinnamyl alcohol dehydrogenase promoter (Bp 4671-5665) operably linked to an antisense SMT2 nucleic acid (Bp 5666-6445), which is joined to a PAP 1 intron (Bp 6446-7625), which is joined to a sense nucleic acid of the SMT2 gene (Bp 7626-8405), which is joined to the RuBisCo small subunit terminator (Bp 8406-8956). Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco will be transformed with the construct shown in FIG. 6 using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid and sterol content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and a reduced amount of sterols, as compared to the parental strain of tobacco, and approximately 10% of the lines tested will have less than 500 ppm total alkaloid and a reduced amount of sterols, as compared to the parental strain of tobacco. Accordingly, it is expected that the transgenic Flue-cured tobacco that will be created using the construct shown in FIG. 6 will have significantly reduced levels of nicotine, TSNA, sterol, and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the construct shown in FIG. 6 using Agrobacterium -mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid and sterol content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and a reduced amount of sterols, as compared to the parental strain of tobacco, and approximately 10% of the lines tested will have less than 500 ppm total alkaloid and a reduced amount of sterols, as compared to the parental strain of tobacco. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct shown in FIG. 6 will have significantly reduced levels of nicotine, TSNA, sterol, and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will be transformed with the construct shown in FIG. 6 using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid and sterol content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and a reduced amount of sterols, as compared to the parental strain of tobacco, and approximately 10% of the lines tested will have less than 500 ppm total alkaloid and a reduced amount of sterols, as compared to the parental strain of tobacco. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct shown in FIG. 6 will have significantly reduced levels of nicotine, TSNA, sterol, and will generate significantly less PAHs upon pyrolysis, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

FIG. 7 shows another RNAi construct that was used to generate several lines of reduced nicotine and TSNA tobacco. This RNAi construct has a QTPase inhibition cassette (SEQ. ID. No. 42) and a norflurazone selection cassette (SEQ. ID. No. 35). Starting from the right border (RB), the QPTase inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably linked to an antisense fragment (360 bp) (Bp 2011-2370) of the QTPase gene, joined to a FAD2 intron (Bp 2371-3501), which is joined to a sense fragment of the QTPase gene (360 bp) (Bp 3502-3861), which is joined to the nopaline synthase (NOS) terminator (Bp 3862-4115). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB). Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco will be transformed with the construct shown in FIG. 7 using Agrobacterium -mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid, as compared to the parental strain of tobacco, and approximately 10% of the lines tested will have less than 500 ppm total alkaloid, as compared to the parental strain of tobacco. Accordingly, it is expected that the transgenic Flue-cured tobacco that will be created using the construct shown in FIG. 7 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the construct shown in FIG. 7 using Agrobacterium -mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid, as compared to the parental strain of tobacco, and approximately 10% of the lines tested will have less than 500 ppm total alkaloid, as compared to the parental strain of tobacco. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct shown in FIG. 7 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will be transformed with the construct shown in FIG. 7 using Agrobacterium -mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct shown in FIG. 7 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

FIG. 8 shows another RNAi construct that was used to generate several lines of reduced nicotine and TSNA tobacco. This RNAi construct has a QTPase inhibition cassette (SEQ. ID. No. 43) and a norflurazone selection cassette (SEQ. ID. No. 35). Starting from the right border (RB), the QPTase inhibition cassette comprises a PMTasel promoter (Bp 1-711) operably linked to an antisense fragment (360 bp) (Bp 712-1071) of the QTPase gene, joined to a FAD2 intron (Bp 1072-2202), which is joined to a sense fragment of the QTPase gene (360 bp) (Bp 2203-2562), which is joined to the Gad2 terminator (Bp 2563-2835). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB). Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco was transformed with the construct shown in FIG. 8 using Agrobacterium -mediated transformation and more than about 98% of putative transformants were successfully transformed. Of the independent lines, 200 plants were regenerated, transplanted in the greenhouse, harvested and tested for alkaloid content. A total of 75 lines were identified as having less than 1,000 ppm total alkaloid and no lines were identified as having less than 500 ppm total alkaloid. Accordingly, the transgenic Flue-cured tobacco created using the construct shown in FIG. 8 has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco was also transformed with the construct shown in FIG. 8 using Agrobacterium -mediated transformation and more than about 98% of putative transformants were successfully transformed. Of the independent lines, 201 plants were regenerated, transplanted in the greenhouse, harvested and tested for alkaloid content. A total of 86 lines were identified as having less than 3,000 ppm total alkaloid and 12 lines were identified as having less than 1,000 ppm total alkaloid. Accordingly, it is understood by those skilled in the art that the transgenic Burley tobacco created using the construct shown in FIG. 8 also has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will be transformed with the construct shown in FIG. 8 using Agrobacterium -mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct shown in FIG. 8 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

FIG. 9 shows another RNAi construct that was used to generate several lines of reduced nicotine and TSNA tobacco. This RNAi construct has a PMTase inhibition cassette (SEQ. ID. No. 44) and a norflurazone selection cassette (SEQ. ID. No. 35). Starting from the right border (RB), the PMTase inhibition cassette comprises a truncated RD2 promoter (Bp 1-1061) operably linked to an antisense fragment (202 bp) (Bp 1062-1263) of the PMTase gene, joined to an Act 11 intron (Bp 1264-1418), which is joined to a sense fragment of the PMTase gene (262 bp) (Bp 1419-1620), which is joined to the Gad2 terminator (Bp 1621-1893). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB). Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco was transformed with the construct shown in FIG. 9 using Agrobacterium -mediated transformation and more than about 98% of putative transformants were successfully transformed. Of the independent lines, 100 plants were regenerated, transplanted in the greenhouse, harvested and tested for alkaloid content. A total of 86 lines were identified as having less than 1,000 ppm total alkaloid and 12 lines were identified as having less than 500 ppm total alkaloid. Accordingly, the transgenic Flue-cured tobacco created using the construct shown in FIG. 9 has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco was also transformed with the construct shown in FIG. 9 using Agrobacterium -mediated transformation and more than about 98% of putative transformants were successfully transformed. Of the independent lines, 99 plants were regenerated, transplanted in the greenhouse, harvested and tested for alkaloid content. A total of 29 lines were identified as having less than 3,000 ppm total alkaloid and no lines were identified as having less than 1,000 ppm total alkaloid. Accordingly, it is understood by those skilled in the art that the transgenic Burley tobacco created using the construct shown in FIG. 9 also has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will be transformed with the construct shown in FIG. 9 using Agrobacterium -mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct shown in FIG. 9 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

FIG. 10 shows another RNAi construct that was used to generate several lines of reduced nicotine and TSNA tobacco. This RNAi construct has a PMTase inhibition cassette (SEQ. ID. No. 45) and a norflurazone selection cassette (SEQ. ID. No. 35). Starting from the right border (RB), the PMTase inhibition cassette comprises a RD2 promoter (Bp 1-2006) operably linked to an antisense fragment (344 bp) (Bp 2007-2350) of the PMTase gene, joined to an Fad2 intron (Bp 2351-3481), which is joined to a sense fragment of the PMTase gene (344 bp) (Bp 3482-3825), which is joined to the Gad2 terminator (Bp 3826-4098) at the left border (LB). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB). Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco was transformed with the construct shown in FIG. 10 using Agrobacterium -mediated transformation and more than about 98% of putative transformants were successfully transformed. Of the independent lines, 66 plants were regenerated, transplanted in the greenhouse, harvested and tested for alkaloid content. A total of 44 lines were identified as having less than 1,000 ppm total alkaloid and 17 lines were identified as having less than 500 ppm total alkaloid. Accordingly, the transgenic Flue-cured tobacco created using the construct shown in FIG. 10 has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the construct shown in FIG. 10 using Agrobacterium -mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid, as compared to the parental strain of tobacco, and approximately 10% of the lines tested will have less than 500 ppm total alkaloid, as compared to the parental strain of tobacco. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct shown in FIG. 10 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will be transformed with the construct shown in FIG. 10 using Agrobacterium -mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct shown in FIG. 10 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

FIG. 11 shows another RNAi construct that was used to generate several lines of reduced nicotine and TSNA tobacco. This RNAi construct (SEQ ID No 49) has a QTPase inhibition cassette (SEQ. ID. No. 42) and a kanamycin selection cassette (SEQ. ID. No. 48). Starting from the right border (RB), The QPTase inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably linked to an antisense fragment (360 bp) (Bp 2011-2370) of the QTPase gene, joined to a FAD2 intron (Bp 2371-3501), which is joined to a sense fragment of the QPTase gene (360 bp) (Bp 3502-3861), which is joined to the NOS terminator (Bp 3862-4115). The selection cassette comprises the nopaline synthase (NOS) promoter (Bp 4116-4422) operably linked to a neomycin phosphotransferase (NPTII) gene (Bp 4435-5229) joined to the NOS terminator (Bp 5619-5872) at the left border (LB). Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental comprising this RNAi construct are embodiments provided herein.

Flue-cured tobacco was transformed with the construct shown in FIG. 11 using Agrobacterium -mediated transformation and more than about 98% of putative transformants were successfully transformed. Of the independent lines, 99 plants were regenerated, transplanted in the greenhouse, harvested and tested for alkaloid content. A total of 43 lines were identified as having less than 1,000 ppm total alkaloid and 15 lines were identified as having less than 500 ppm total alkaloid. Accordingly, the transgenic Flue-cured tobacco created using the construct shown in FIG. 11 has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the construct shown in FIG. 11 using Agrobacterium -mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid, as compared to the parental strain of tobacco, and approximately 10% of the lines tested will have less than 500 ppm total alkaloid, as compared to the parental strain of tobacco. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct shown in FIG. 11 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco was transformed with the construct shown in FIG. 11 using Agrobacterium -mediated transformation and more than about 98% of putative transformants were successfully transformed. Of the independent lines, 122 plants were regenerated, transplanted in the greenhouse, harvested and tested for alkaloid content. A total of 22 lines were identified as having less than 1,000 ppm total alkaloid and 6 lines were identified as having less than 500 ppm total alkaloid. Accordingly, the transgenic Flue-cured tobacco created using the construct shown in FIG. 11 has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

It should be emphasized that other promoters and terminators can be used with the nucleic acids provided herein interchangeably. Although RD2 (SEQ. ID. No. 13, 37, or 50) is a preferred root-specific promoter, there are other root-specific promoters that can be used, as well. For example, the putrescene methyl transferase 1 promoter (PMT-1) (SEQ. ID. No. 14) is a root-specific promoter that can be used in place of the RD2 promoter in any of the constructs described above. Similarly, although the actin2 promoter (SEQ. ID. No. 16) is preferred for driving expression of a norflurazone resistance gene, other constitutive promoters such as the GapC promoter (SEQ. ID. No. 15), the tobacco alcohol dehydrogenase (ADP) (SEQ. ID. No. 17) and the Arabidopsis ribosomal protein L2 (RPL2P) (SEQ. ID. No. 18) can be used to drive expression of the norflurazone resistance gene. Additionally, developmentally regulated promoters such as, cinnamyl alcohol dehydrogenase (SEQ. ID. No. 19) and metallothionein I promoter (SEQ. ID. No. 20) can be used interchangeable with the cassettes described herein.

Further, in some embodiments, a plurality of constitutive promoters, in tandem, can be used to drive expression of the norflurazone resistance gene. Additionally, a plurality of root-specific promoters can be used to drive expression one or more of the inhibition cassettes described above (e.g., the QTPase inhibition cassette, the PMTase inhibition cassette, the A622 inhibition cassette, a sterol inhibition cassette, or a double-knockout inhibition cassette). Developmentally regulated promoters, a plurality of developmentally regulated promoters, constitutive promoters, or a plurality of constitutive promoters can also be used to drive expression of one or more of the inhibition or selection cassettes described above. Accordingly, any promoter operable in tobacco can be used to drive expression of any of the inhibition cassettes or the selection cassette described herein (e.g., nos, 35S, or CAMV). Terminators, such as GAD2 terminator (SEQ. ID. No. 21), NOS terminator (SEQ ID No 38) and the FAD 2 (SEQ. ID. No. 22) or PAP1 introns can be used interchangeably, as well.

Other embodiments provided herein concern the discovery of several mutants of the phytoene desaturase gene that confer resistance to the herbicide norflurazone (e.g., SEQ. ID. Nos. 10, 11, and 12). These herbicide resistance genes were used as selectable markers in the transformations above. Typically, the selection was accomplished by introducing the transformed plant tissue to the norflurazone (e.g., 0.005 uM-0.1 uMconc). That is, the concentration of norflurazone that can be used to select positive transformants containing a norflurazone resistance gene, as described herein can be at least, less than, greater than, or equal to 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 uM. Preferably, less than or equal to 0.05 uM concentration of norflurazone is used when selecting transformants with Flue-cured tobacco and less than or equal to 0.0125 uM concentration norflurazone is used when selecting transformants with Burley tobacco. As the plantlet develops, selection was accomplished by differentiating the green shoots (positive transformants) from the yellow or white shoots (negative transformants). Once selection was made, the herbicide was removed and the plantlet was allowed to develop in the greenhouse.

The norflurazone resistant phytoene desaturase mutants (PDSM-1, PDSM-2, ansd PDSM-3) were generated by site-directed mutagenesis of particular regions of the gene believed to be involved in binding of the herbicide. Constructs carrying the various PDSM genes were then transferred to tobacco leaf disks by conventional Agrobacterium transformation and the resistance to norflurazone was analyzed at various concentrations. After several iterations, the mutants described as SEQ. ID. Nos. 10, 11, and 12, were identified as sequences that confer resistance to norflurazone. Accordingly, embodiments provided herein concern the PDSM genes described herein, their use in plants as selectable markers to identify plant cells that contain a transformed gene, whether in tissue culture or in the field, and methods of identifying new PDSM genes that confer norflurazone resistance.

In a first selection construct, the Arabidopsis phytoene desaturase gene (PDS) (SEQ. ID. No. 36) was mutated using site-directed mutagenesis, such that a T to G mutation at position 1478, resulting in a Valine to Glycine change at amino acid residue 493 was created. To generate the norflurazone resistance gene, the open reading frame of the Arabidopsis phytoene desaturase gene was amplified and cloned into the TOPO vector (Invitrogen). A single base pair change from T-G at nucleotide position 1478, leading to a Valine to Glycine change at amino acid residue 493, was introduced using QuickChange Site-directed Mutagenisis Kit (Stratgene). The point mutation was verified by sequencing and the resultant mutant was named PDSM-1 (SEQ. ID. No. 10). The 1.729 Kb PDSM1 sequence was then amplified and ligated into the binary vector pWJ001, a pCambia derivative that contained the RNAi cassettes above, which was then introduced into Agrobacterium tumefaciens . A similar approach was used to generate the PDSM-2 and PDSM-3 mutants described in the sequence listing as SEQ. ID. NOs. 11 and 12.

That is, in a second selection construct, the Arabidopsis phytoene desaturase gene (PDS) (SEQ. ID. No. 36) was mutated using site-directed mutagenesis, such that a G to C mutation at position 863, resulting in a Arginine to Proline change at amino acid residue 288 was created. To generate the norflurazone resistance gene, the open reading frame of the Arabidopsis phytoene desaturase gene was amplified and cloned into the TOPO vector (Invitrogen). A single base pair change was introduced using QuickChange Site-directed Mutagenisis Kit (Stratgene). The point mutation was verified by sequencing and the resultant mutant was named PDSM-2. The 1.729 Kb PDSM-2 sequence was then amplified and ligated into the binary vector pWJ001, a pCambia derivative that contained the RNAi cassettes above, which was then introduced into Agrobacterium tumefaciens

Further, in a third selection construct, the Arabidopsis phytoene desaturase gene (PDS) (SEQ. ID. No. 36) was mutated using site-directed mutagenesis, such that a T to C mutation at position 1226, resulting in a Leucine to Proline change at amino acid residue 409 was created. To generate the norflurazone resistance gene, the open reading frame of the Arabidopsis phytoene desaturase gene was amplified and cloned into the TOPO vector (Invitrogen). A single base pair change was introduced using QuickChange Site-directed Mutagenisis Kit (Stratgene). The point mutation was verified by sequencing and the resultant mutant was named PDSM-3. The 1.729 Kb PDSM-2 sequence was then amplified and ligated into the binary vector pWJ001, a pCambia derivative that contained the RNAi cassettes above, which was then introduced into Agrobacterium tumefaciens

Accordingly, embodiments provided herein concern methods of identifying a mutation on a phytoene desaturase gene that confers resistance to an herbicide, preferably norflurazone. By one approach, a phytoene desaturase gene is provided, preferably SEQ. ID. No. 36, a nucleotide in said gene is mutated so as to generate a mutant phytoene desaturase gene, said mutant phytoene desaturase gene is transformed to a plant cell so as to generate a plant cell comprising said mutant phytoene desaturase gene, said plant cell comprising said mutant phytoene desaturase gene is then contacted with an herbicide, preferably norflurazone, and the presence or absence of a resistance to said herbicide is identified, whereby the presence of a resistance to said herbicide identifies said mutation as one that confers resistance to said herbicide. By one approach, the entire sequence of a phytoene desaturase gene (e.g., SEQ. ID. NO. 36) is mutated one residue at a time and each mutant is screened for resistance to the herbicide. Accordingly, embodiments provided herein include compositions (e.g., nucleic acid constructs or cassettes, plant cells, plants, tobacco, or tobacco products) that comprise, consist, consist essentially of a mutant phytoene desaturase nucleic acid of SEQ. ID. NO. 10, 11, or 12 or fragment thereof at least or equal to 30, 50, 100, 200, 400, 500, 700, 900, 1000, 1200, 1400, 1600, or 1700 consecutive nucleotides of in length that confers resistance to an herbicide, in particular norflurazone. Embodiments provided herein also include compositions (e.g., nucleic acid constructs or cassettes, plant cells, plants, tobacco, or tobacco products) comprising the mutant phytoene desaturase protein or fragments thereof (e.g., at least 15, 25, 50, 100, 200, 300, 400, 500 consecutive amino acids of a protein encoded by SEQ. ID. Nos. 10, 11, or 12) that confer resistance to an herbicide, in particular norflurazone.

The nucleic acid sequences, cassettes, and constructs described herein can also be altered by mutation such as substitutions, additions, or deletions that provide for sequences encoding functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same amino acid sequence can be used in some embodiments provided herein. These include, but are not limited to, nucleic acid sequences comprising all or portions of the nucleic acid embodiments described herein that complement said sequences and have been altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. In some contexts, the phrase “substantial sequence similarity” in the present specification and claims means that DNA, RNA or amino acid sequences which have slight and non-consequential sequence variations from the actual sequences disclosed and claimed herein are considered to be equivalent to the sequences provided herein. In this regard, “slight and non-consequential sequence variations” mean that “similar” sequences (i.e., the sequences that have substantial sequence similarity with the DNA, RNA, or proteins disclosed and claimed herein) will be functionally equivalent to the sequences disclosed and claimed in the present invention. Functionally equivalent sequences will function in substantially the same manner to produce substantially the same compositions as the nucleic acid and amino acid compositions disclosed and claimed herein.

Additional nucleic acid embodiments include sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identical to the nucleic acids, nucleic acid constructs, and nucleic acid cassettes provided herein. Preferably these sequences also perform the functions of the particular nucleic acid embodiment (e.g., inhibition of nicotine, nornicotine, or sterol production or confer resistance to norflurazone). Determinations of sequence similarity are made with the two sequences aligned for maximum matching; gaps in either of the two sequences being matched are allowed in maximizing matching. Gap lengths of 10 or less are preferred, gap lengths of 5 or less are more preferred, and gap lengths of 2 or less still more preferred.

Additional nucleic acid embodiments also include nucleic acids that hybridize to the nucleic acid sequences disclosed herein under low, medium, and high stringency, wherein said additional nucleic acid embodiments also perform the function of the particular embodiment (e.g., inhibit nicotine, nornicotine, or sterol production or confer resistance to norflurazone). Identification of nucleic acids that hybridize to the embodiments described herein can be determined in a routine manner. (See J. Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory)). For example, hybridization of such sequences may be carried out under conditions of reduced stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 0.3 M NaCl, 0.03 M sodium citrate, 0.1% SDS at 60 degrees C., or even 70 degrees C.). Preferably these sequences also perform the functions of the particular nucleic acid embodiment (e.g., inhibition of nicotine, nornicotine, or sterol production or confer resistance to norflurazone).

Accordingly embodiments provided herein also include compositions comprising, consisting of, or consisting essentially of: (a) the nucleic acid sequences shown in the sequence listing (SEQ. ID. NOS. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50); (b) nucleotide sequences encoding the amino acid sequences encoded by the nucleic acids of the sequence listing (SEQ. ID. NOS. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50); (c) any nucleotide sequences that hybridizes to the complement of the sequences shown in the sequence listing (SEQ. ID. NOS. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50) under stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 50 degrees C. and washing in 0.2.times.SSC/0.2% SDS at 50 degrees C.; and (d) any nucleotide sequence that hybridizes to the complement of the sequences shown in the sequence listing (SEQ. ID. NOS. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50) under less stringent conditions (e.g., hybridization in 0.5 M NaHPO4, 7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 37 degrees C. and washing in 0.2.times.SSC/0.2% SDS at 37 degrees C. Preferably these sequences also perform the functions of the particular nucleic acid embodiment (e.g., inhibition of nicotine, nornicotine, or sterol production or confer resistance to norflurazone). Embodiments provided herein also include peptides encoded by the nucleic acid sequences of (a), (b), (c), or (d), above.

The examples described herein demonstrate that several different RNAi constructs can be used to effectively reduce the levels of nicotine, nornicotine, and sterols in tobacco. Additionally, these examples demonstrate that several mutant phytoene desaturase genes, which confer resistance to the herbicide norflurazone, have been created and that selection cassettes comprising these herbicide resistant nucleic acids can be used to determine the presence of a linked gene in transformed tobacco cells. Additionally, the norflurazone resistance nucleic acids described herein can be used in a general sense (e.g., in plants other than tobacco) to efficiently select positively transformed plant cells from plant cells that do not contain a construct comprising the norflurazone resistance gene. Thus, the norflurazone selection cassette or the norflurazone resistance gene described herein can be used to confer resistance to norflurazone in plants including, but not limited to, corn ( Zea mays ), canola ( Brassica napus, Brassica rapa ssp.), alfalfa ( Medicago saliva ), rice ( Orya sativa ), rape ( Brassica napus ), rye ( Secale cereale ), sorghum ( Sorghum bicolor, Sorghum vulgare ), sunflower ( Helianthus annus ), wheat ( Triticum aestivum ), soybean ( Glycine max ), tobacco ( Nicotiana tabacum ), potato ( Solanum tuberosum ), peanuts ( Arachis hypogaea ), cotton ( Gossypium hirsutum ), sweet potato ( Ipomoea batatus ), cassava ( Manihot esculenta ), coffee ( Cofea spp.), coconut ( Cocos nucifera ), pineapple ( Ananas comosus ), citrus trees ( Citrus spp.), cocoa ( Theobroma cacao ), tea ( Camellia sinensis ), banana ( Musa spp.), avocado ( Persea americana ), fig ( Ficus casica ), guava ( Psidium guajava ), mango ( Mangifera indica ), olive ( Olea europaea ), papaya ( Carica papaya ), cashew ( Anacardium occidentale ), macadamia ( Macadamia integrifolia ), almond ( Prunus amygdalus ), sugar beets ( Beta vulgaris ), apple ( Malus pumila ), blackberry ( Rubus ), strawberry ( Fragaria ), walnut ( Juglans regia ), grape ( Vitis vinifera ), apricot ( Prunus armeniaca ), cherry ( Prunus ), peach ( Prunus persica ), plum ( Prunus domestica ), pear ( Pyrus communis ), watermelon ( Citrullus vulgaris ), duckweed ( Lemna ), oats, barley, vegetables, ornamentals, conifers, and turfgrasses (e.g., for ornamental, recreational or forage purposes). Vegetables include Solanaceous species (e.g., tomatoes; Lycopersicon esculentum ), lettuce (e.g., Lactuea sativa ), carrots ( Caucuis carota ), cauliflower ( Brassica oleracea ), celery ( apium graveolens ), eggplant ( Solanum melongena ), asparagus ( Asparagus officinalis ), ochra ( Abelmoschus esculentus ), green beans ( Phaseolus vulgaris ), lima beans ( Phaseolus limensis ), peas ( Lathyrus spp.), members of the genus Cucurbita such as Hubbard squash ( C. Hubbard ), Butternut squash ( C. moschtata ), Zucchini ( C. pepo ), Crookneck squash ( C. crookneck ), C. argyrosperma, C. argyrosperma ssp, C. digitata, C. ecuadorensis, C. foetidissima, C. lundelliana , and C. martinezii , and members of the genus Cucumis such as cucumber ( Cucumis sativus ), cantaloupe ( C. cantalupensis ), and musk melon ( C. melo ). Ornamental plants include azalea ( Rhododendron spp.), hydrangea ( Macrophylla hydrangea ), hibiscus ( Hibiscus rosasanensis ), roses ( Rosa spp.), tulips ( Tulipa spp.), daffodils ( Narcissus spp.), petunias ( Petunia hybrida ), carnation ( Dianthus caryophyllus ), poinsettia ( Euphorbia pulcherima ), and chrysanthemum. Conifers, which may be employed in practicing the present invention, include, for example, pines such as loblolly pine ( Pinus taeda ), slash pine ( Pinus elliotii ), ponderosa pine ( Pinus ponderosa ), lodgepole pine ( Pinus contorta ), and Monterey pine ( Pinus radiata ), Douglas-fir ( Pseudotsuga menziesii ); Western hemlock ( Tsuga canadensis ); Sitka spruce ( Picea glauca ); redwood ( Sequoia sempervirens ); true firs such as silver fir ( Abies amabilis ) and balsam fir ( Abies balsamea ); and cedars such as Western red cedar ( Thuja plicata ) and Alaska yellow-cedar ( Chamaecyparis nootkatensis ). Turf grass include but are not limited to zoysia grasses, bentgrasses, fescue grasses, bluegrasses, St. Augustine grasses, Bermuda grasses, buffalo grasses, ryegrasses, and orchard grasses. Also included are plants that serve primarily as laboratory models, e.g., Arabidopsis . Preferred plants for use in the present methods include (but are not limited to) legumes, solanaceous species (e.g., tomatoes), leafy vegetables such as lettuce and cabbage, turf grasses, and crop plants (e.g., tobacco, wheat, sorghum, barley, rye, rice, corn, soybean, cotton, cassaya, and the like), and laboratory plants (e.g., Arabidopsis ). While any plant may be used to carry out this aspect provided herein, tobacco plants are particularly preferred.

Further, embodiments provided herein concern the production of norflurazone-resistant or tolerant plants, which can be sprayed with the herbicide in the field. In this manner, weeds and non-transformed plants will die after contact with the herbicide but plants containing the construct harboring the norflurazone resistance gene will survive. In one embodiment, for example, a norflurazone-containing herbicide is applied to the plant comprising the DNA constructs provided herein, and the plants are evaluated for tolerance to the herbicide. Any formulation of norflurazone can be used for testing plants comprising the DNA constructs provided herein. The testing parameters for an evaluation of the norflurazone tolerance of the plant will vary depending on a number of factors. Factors would include, but are not limited to the type of norflurazone formulation, the concentration and amount of norflurazone used in the formulation, the type of plant, the plant developmental stage during the time of the application, environmental conditions, the application method, and the number of times a particular formulation is applied. For example, plants can be tested in a greenhouse environment using a spray application method. The testing range using norflurazone can include, but is not limited to 0.5 oz/acre to 500 oz/acre. That is, the amount of herbicide that can be applied to transgenic plants containing a norflurazone-resistance gene in a field can be less than, equal to, or more than 0.5, 0.6, 0.7, 0.8, 0.9.1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 250, 300, 350, 400, or 500 oz/acre. In some embodiments, the norflurazone application rate is 2.24 kg to 4.48 kg ai/hectare (2 to 4 lbs ai/acre) or 2.8 to 5.6 kg granules/hectare (2.5 to 5 lb/acre) or 234 L/hectare (25 gal/acre) in solution. Higher amounts are preferred for finer textured soils or when longer residual activity is desired.

The preferred commercially effective range can be from 25 oz/acre to 100 oz/acre of norflurazone, depending on the crop and stage of plant development. A crop can be sprayed with at least one application of a norflurazone. For testing in cotton an application of 32 oz/acre at the 3-leaf stage may be followed by additional applications at later stages in development. For wheat, corn, soybean, and tobacco an application of 32 oz/acre of norflurazone at the 3-5 leaf stage can be used. The test parameters can be optimized for each crop in order to find the particular plant comprising the constructs provided herein that confers the desired commercially effective norflurazone tolerance level. The section below describes typical curing methods which may be used to prepare the tobacco once it is harvested.

a) Heterologous Expression

Tobacco has well-established transformation procedures and well-characterized regulatory elements for the control of transgene expression. Tobacco also has a high biomass yields and rapid scalability, which makes it a very suitable platform for commercial molecular farming. Since tobacco is a non-food and non-feed crop it also carries a reduced risk that the transgenic material or recombinant proteins would contaminate animal feed and would enter the human food chain. Because conventional or wild-type tobacco has a high content of nicotine and other toxic alkaloids, however, investigators have not explored the ability to use tobacco as a bioreactor. Further, the high cost of nicotine removal has steered investigators away from this technology.

The present disclosure, however, provides several types of genetically modified tobacco that can be used as a platform into which genes encoding commercially valuable compounds can be introduced. That is, by using genetically modified tobaccos having reduced levels of nicotine, sterols, and/or TSNAs, as bioreactors, it is contemplated that many commercially valuable industrial oils, pharmaceuticals, dietary supplements can be obtained with fewer processing steps (e.g., the removal of nicotine is no longer required). Accordingly, some embodiments concern tobaccos that are genetically modified to have a reduced level of nicotine, sterols, and/or TSNAs, further comprising a heterologous gene that produces a medicinal compound, industrial oil, or dietary supplement, which can be harvested and/or isolated or purified from said tobacco. Compounds generated in this manner can be used for a variety of applications such as the preparation of immunogens, vaccines, cooking oils, pharmaceuticals and dietary supplements. Techniques for the production of medicinal compounds in low-nicotine tobacco, such as a protein, for industrial or pharmaceutical application has been described in the art. It is contemplated, that these techniques can be readily used with the tobaccos and techniques that are described herein.

As an exemplary, non-liming example, the N-terminal fragment of SARS-CoV S protein (S1) can be expressed in low-nicotine tobacco plants, as is known in the art and exemplified in Pogrebnyak et al., Proc. Natl. Acad. Sci. USA (2005) 102:9062-9067. Incorporation of the S1 fragment into plant genomes as well as its transcription can be confirmed by PCR and RT-PCR analyses. High levels of expression of recombinant S1 protein can be observed in several transgenic lines by Western blot analysis using specific antibodies. Mammals parenterally primed with tobacco-derived S1 protein can have sera containing SARS-CoV-specific IgG as detected by Western blot and ELISA analysis.

The original gene encoding the human SARS-CoV spike glycoprotein (strain TOR2, National Center for Biotechnology Information no. NC 004718) is known in the art. DNA encoding a 79-kDa S protein fragment, corresponding to amino acids 14-714, can be amplified by two consecutive rounds of PCR to generate XbaI and Sad sites at the 5′ and 3′ ends, respectively, by using the following primers: SP-F1-CCT TGC GCT TCT CAG CCA CGC AAA CTC AAG AGG ATC GCA TCA CCA TCA CCA TCA CAG TGA CCT TGA CCG GTG CAC (SEQ ID NO 51), XbaI-F2-ATA ATC TAG ATG ATC ATG GCT TCC TCC AAG TTA CTC TCC CTA GCC CTC TTC CTT GCG CTT CTC AGC CAC G (SEQ ID NO 52), and SacI-HDELR-ATT CGA GCT CTT AAA GTT CAT CAT GAG CCA TAG AAA CAG GCA TTA CT(SEQ ID NO 53). The expression cassette of SARS-CoV S1 protein can contain the plant-derived 23-a {MIMASSKLLSLALFLALLSHANS (SEQ. ID. No. 54}, signal peptide (SP), and a histidine tag {RGSHHHHHH (SEQ. ID. NO. 55} at the N-terminal portion of the resulting 79-kDa polypeptide. After addition of the plant-specific endoplasmic reticulum retention signal {HDEL (SEQ. ID. NO. 56}, the cassette can be subcloned into the XbaI/SacI site of the plant binary vector pE1801, which is known as a super promoter, followed by a tomato etch virus translation enhancer. The vector also can contain the npt II gene for kanamycin selection of transgenic plants. Plasmid pE1801-79SHDEL can be electroporated into Agrobacterium tumefaciens strain LBA4404 and used for plant transformations.

A genetically modified reduced nicotine and reduced TSNA tobacco, made as described herein, can be used as the platform. The low-nicotine/TSNA tobacco can be transformed by Agrobacterium -mediated transformation as is known in the art. Independent kanamycin-resistant (KmR) tobacco lines can be used for molecular analyses. The presence of the spike gene in transgenic plants can be confirmed by PCR using genomic DNA. KmR transgenic plants with PCR-confirmed presence of the S transgene can be further analyzed for gene-specific mRNA expression by quantitative RT-PCR as is known in the art. Western blot analysis of transgenic lines with polyclonal SARS-specific antibodies Sm and Sn can confirm the presence of SARSCoV S-specific 79-kDa protein and its derivatives. KmR T1 tobacco lines (cv. LAMD-609) can be grown hydroponically to obtain large amounts of root tissue for immunological experiments. Western blot analysis of T1 lines can reveal high levels of S protein expression comparable with the original TO transgenic lines.

Immunological assessment of the plant-expressed S protein can be performed in 6- to 8-week-old female BALB/c mice. For parenteral immunization, mice can be injected three times at 2-week intervals with an equivalent of 50 mg of dry tobacco root material per mouse. Powdered plant material can be reconstituted with saline (1/1 by weight) just before immunization. First and second immunizations can be given s.c. with complete and incomplete Freund's adjuvant, respectively; the third dose can be administered i.p. in saline. Sera can be collected retroorbitally from each mouse before and 10 days after each immunization. Four weeks after the last immunization, mice can receive an i.p. booster dose of 1 μg of commercially obtained S peptide (Cell Sciences, Canton, Mass.) without adjuvant. After 10 days, mice can be killed and exanguinated by heart puncture, and sera can be assayed by ELISA and Western blot analysis. Solid-phase ELISA can be carried out as known in the art MaxiSorp 96-well plates (Nalge Nunc) coated overnight at 4° C. with the same S peptides obtained from Cell Sciences at a concentration of 1 μg/ml in PBS. Antigen-specific antibodies can be detected by using the following antibodies: rabbit anti-mouse IgG (total) and anti-mouse IgG1 (both from BD Biosciences Pharmingen), anti-mouse IgG2a, IgG2b, IgG3, IgM, and IgA (all from Organon Teknika), and anti-mouse IgE (eBioscience, San Diego). A serum dilution with an OD450 of 0.15 units above background can be considered the ELISA titer.

III. Tobacco products

Although the modified tobaccos described herein are prefereably used to create tobacco products for human consumption (e.g., cigarettes, chew, snuff, plug, etc.) it should be realized that the tobacco described herein can be used for other applications such as animal feed, pharmaceutical production, and, in particular, the gernation of proteins (e.g., antiviral or anti-oncogenic peptides or antibodies or fragments thereof). Preferably, however, the tobacco and methods provided herein can be applied to any tobacco product, including, but not limited to pipe, cigar and cigarette tobacco and chewing tobacco in any form including leaf tobacco, shredded tobacco or cut tobacco. The term “tobacco product” includes, but is not limited to, smoking materials (e.g., cigarettes, cigars, pipe tobacco), snuff, chewing tobacco, gum, and lozenges. In numerous embodiments, the methods provided herein are applied to tobacco used to create the tobacco product.

A. Reduced Risk Tobacco Products

Provided herein are reduced risk tobacco products. A reduced risk tobacco product provided herein can be a traditionally configured tobacco product containing a reduced risk tobacco, such as a modified tobacco as provided herein. A reduced risk tobacco product provided herein also can contain conventional tobacco and be configured to reduce the risk of using the tobacco product. An example of such a reduced risk tobacco product is a cigarette containing a filter designed to reduce the risk associated with cigarette smoke. A reduced risk tobacco product also can contain a reduced risk tobacco and be configured to reduce the risk of using the tobacco product. An example of such a reduced risk tobacco product is a cigarette containing a reduced risk tobacco and a filter designed to reduce the risk associated with cigarette smoke. In some such embodiments, the reduced risk tobacco and reduced risk configuration act synergistically to reduce the overall risk of using the tobacco product.

Typical configurations of a tobacco product that reduces the risk of the tobacco product will include a filter that reduces the risk of exposure to tobacco smoke. A filter can be configured to be used with any smoking tobacco product, including cigars, pipes and cigarettes, as is known in the art. In one embodiment, the reduced risk tobacco product is a cigarette containing a filter that reduces the risk of exposure to tobacco smoke. Any of a variety of known filters that reduce the risk of exposure to tobacco smoke can be used in the reduced risk tobacco products provided herein, including, but not limited to, commercially available filters provided in cigarette products, and other filters known in the art, such as filters containing antioxidants, copper, carbon or activated charcoal, and/or paper-containing filters. One exemplary filter can be a filter containing an antioxidant or a radical scavenger. Filters containing antioxidants or radical scavengers can be prepared according to known methods, as exemplified in U.S. Pat. Nos. 6,832,612 and 6,415,798, herein expressly incorporated by reference in their entireties. Another exemplary filter is a filter that can reduce tobacco smoke-induced modulation of cell homeostasis, as can be assessed by determining, for example, modulation of the transcriptome or proteome, cell viability, or integrity of genetic material. Such filters can include a compound that sequesters or intercepts harmful components that generate DNA breaks, or enhance DNA breakage, thereby yielding a filter that removes harmful smoke components. For example, a filter can contain flat aromatic compounds that can scavenge potential carcinogens (e.g., components of tar), where exemplary flat aromatic compounds include caffeine and pontoxyfyllen. In some of the methods provided herein the filter comprises an interceptor of the carcinogen that has aromatic chemical structure: the carcinogen associates then with interceptor forming a complex that is retained in the filter.

The methods provided herein can be used for evaluating modifications to tobacco product configurations. For example, the methods provided herein can be used as assays for evaluating the effectiveness of a cigarette filler or filter. These methods also can be used to test any effect that can be resultant from a particular tobacco product configuration in combination with a modified tobacco. For example, the methods provided herein can be used to test a selected filter in combination with a modified tobacco. Accordingly, the methods provided herein provide a basis for evaluating and developing a reduced risk tobacco product. The methods provided herein further provide a basis for evaluating any further reduction in risk that can result from specific combinations of modified tobaccos and filters.

As provided herein, methods can be used for testing modulation of cell homeostasis by, for example, monitoring a molecular marker of modulation of cell homeostasis, when the cells are exposed to a tobacco composition from a modified tobacco configured in a tobacco product with a filter, or configured in a plurality of tobacco products with a plurality of different filters. Similarly, methods can be used for testing modulation of cell homeostasis when the cells are exposed to a tobacco composition from a plurality of modified tobaccos configured in a tobacco product with a filter, or configured in a plurality of tobacco products with a plurality of different filters. Accordingly, a variety of combinations of modified tobaccos and tobacco configurations can be tested for their properties of modulating cell homeostasis. The examples provided herein demonstrate that the risk-reducing properties of a tobacco and the risk-reducing properties of a filter can be interrelated such that the risk-reducing properties of a particular filter can vary depending on the type of tobacco used. The methods provided herein can be used to evaluate the degree to which a particular filter reduces the risk of one or more tobacco products, and also can be used to evaluate the ability of one or more filters to reduce the risk of a particular tobacco product. The methods provided herein also can be used to evaluate the ability of one or more filters in combination with one or more tobaccos to have additive risk-reducing properties, thereby forming an even further reduced-risk tobacco product.

B. Methods for Evaluating Tobacco and/or Tobacco Product

Methods for Determining the Risk Potential of Tobacco and Tobacco Products

Provided herein are several methods for identifying the propensity of a tobacco or tobacco product to contribute to a tobacco related disease. Generally, these approaches are practiced by providing a tobacco, obtaining smoke or a smoke condensate from the tobacco, contacting a cell with the smoke or smoke condensate, and identifying one or more attributes of the contacted cell. Tobacco products contain a number of compounds that induce various types of changes to a cell, including cell damage, change in gene expression including mRNA and/or protein expression, mutations, chromosomal aberrations, aberrant sister chromatid exchanges and micronuclei. Attributes of contacted cells indicative of such tobacco-induced cell changes can be identified in the methods provided herein, which address changes in cell homeostasis, as indicated by changes in gene expression, genetic mutations or aberrations, and modulation of cell viability and/or apoptosis. The methods provided herein can be used to determine affect of a tobacco or a tobacco product on a cell by determination of the presence, absence, or change in a molecular marker. For example, a molecular marker can be monitored, which is indicative of an affect on mRNA, protein, DNA damage, cell viability or apoptosis can be determined according to the methods provided herein or other methods generally known in the art, where monitoring of the molecular marker can be used to determine the affect of a tobacco or tobacco product on cell homeostasis. Exemplary affects of a tobacco or tobacco product on a cell include, but are not limited to, induction of a double-strand DNA break, inhibition of apoptosis, inhibition of cell proliferation, and modulation of gene expression, including modulation of the transcriptome and/or modulation of the proteome. Accordingly, the methods provided herein can be used to establish a profile for a particular tobacco by employing assay methods, including assays that identify tobacco products that modulate cell homeostasis from tobacco products that do not. For example, assays for induction of damage of cellular genetic material or assays for modulation of gene expression can be used to differentiate reduced risk tobacco products from conventional tobacco products. For example, the methods provided herein can be used to characterize a tobacco by assay methods including an assay for the induction of a double-strand DNA break, inhibition of apoptosis, inhibition of cell proliferation, modulation of transcription, or modulation of translation.

Several other assays have classically been used to analyze tobacco for the risk of adverse health effects. Traditionally, the first manner of testing consists of analysis of cigarette smoke for various components that can relate to health effects associated with smoking. A second manner of testing includes testing cigarette smoke tar on living cells. One of these tests detects changes in the genetic material of bacteria. Another test uses mouse cells grown in Petri dishes to detect potential cancer-causing activity. A third manner of testing seeks to determine if people smoke the tested tobacco cigarettes differently than the comparable brand or type currently on the market. If the way the cigarettes are smoked is different, then the other manners of testing can be repeated with the smoking machines set to reflect the change in smoking behavior. A fourth manner of testing examines the response of animals to cigarette smoke or tar. One such type of test looks for inflammation in the lungs of mice in response to cigarette smoke. A second test looks for tumor formation in the upper respiratory tract of hamsters exposed to smoke. A third test looks for the cancer producing ability of cigarette smoke tar by applying the tar to the skin of mice. Each manner of testing can include comparing tobacco cigarettes and both the effects of mainstream and sidestream smoke can be tested.

During smoking, both mainstream smoke (inhaled by the smoker) and sidestream smoke (mainly from the burning end of the cigarette) are generated. While mainstream and sidestream smoke are qualitatively similar the quantity of specific components differs between the two. Additionally, modifications to the cigarette can independently affect the composition of sidestream and mainstream smoke. It is concluded, therefore, that testing of tobacco or cigarettes can be assayed for both mainstream and sidestream smoke.

Epidemiology is not a practical approach for addressing the issue of the health effects of changes in a cigarette composition. Because people can smoke cigarettes differently (ex. longer or faster puffs) it can be important to consider whether these changes affect smoke chemistry and therefore toxicity. For example, a new, cigarette type can result in a smoker taking longer puffs, which can then change the smoke chemistry and toxicity.

Testing, however, can examine the effects on toxicity of a single design change in a cigarette or can examine the effects of a set of design changes compared to an unchanged control. Testing protocols can follow either a screening or a tradeoff approach. In the screening approach new designs can be subjected to a series of tests each with criteria for passing or failing. Designs that fail are eliminated from further testing, while those passing are subjected to additional scrutiny. In the tradeoff approach the relative changes in each test would be assessed in light of other information about the particular design.

The FTC method describes: how cigarettes are to be prepared for smoking, the type of smoking machine to use, the way the smoking machine should be operated, the method for collecting smoke products, and ways to measure moisture content, nicotine, carbon monoxide and tar. Typically in the methods provided herein, the FTC protocol for studies of cigarette smoke chemistry and toxicity are used.

Toxicity of cigarette smoke is directly related to the composition of the smoke and the composition of smoke can be changed if the way the cigarettes are smoked is changed.

There are a variety of chemical analyses that can be done to aid in the determination of the change in toxicity of a tobacco. These relate to the chemical composition of tobacco smoke. The following lists the chemical composition analysis and the health effect associated with the component or property measured: Total Particulate Matter (TPM; carcinogen), pH (effect on nicotine toxicity), Redox Potential (influence toxicity of whole smoke), Carbon Monoxide (reduces ability of blood to carry oxygen), Nitrogen Oxides (NOx; increases nitrosamine formation, inhibits enzyme function), Hydrogen Cyanide (inhibits lung clearance, lowers ability of body to use oxygen), Hydrocarbons (benzene, butadiene; suspected or known carcinogens), Aldehydes (ex. formaldehyde, acrolein; inhibit lung clearance, animal carcinogens), Volatile nitrosamines (strong animal carcinogens), Tobacco-specific nitrosamines (strong animal carcinogens), Nicotine (associated with cardiovascular disease), Phenols (enhance carcinogen action) Catechol (major carcinogen), and Polynuclear Aromatic Hydrocarbons (PNAs; major tumor initiators).

There are also a variety of known cell toxicity tests that can be performed in a relatively short time scale: bacterial mutagenicity test, animal cell test to detect potential carcinogens, and lung inflammation test in animals. One test, the Ames test, uses certain types of Salmonella bacteria to quantitatively assess the ability of a material to cause mutations, such as mutations involved in the process of carcinogenesis. In this test a solution of collected smoke particulates is mixed with the bacteria. Bacteria with the ability to grow in the absence of a particular nutrient are scored as mutants.

The potential cancer-causing ability of chemicals can also be evaluated using a cell transformation assay. In this assay, solutions of smoke particulates are given to animal cells grown in Petri dishes in the laboratory. After several weeks the cells are examined under the microscope. At this time the cells are scored for abnormal growth patterns. The number of clusters of abnormally growing cells is then compared among cigarette types.

In animal studies, inflammation of the lungs can be assessed. The changes measured in this test can be related to the development of chronic obstructive pulmonary disease. In these tests mice can be exposed to whole smoke two times per day, for any number of days according to the experimental design. At the end of the exposure period the animals would be killed and their lungs washed out to collect inflammatory cells. The numbers and kinds of the cells would be measured.

Two long-term animal tests for cancer causing ability of tobacco can be performed. In the first, test cigarette tar is applied to the back skin of mice. Skin tumors are then scored over the life of the animals. The use of this test is based on two observations: (1) in studies of tumor formation by smoke in hamsters whole smoke is active but smoke free of particulates is not and (2) a large number of known carcinogens are contained in the particulate portion of cigarette smoke.

The second test examines the tumor forming ability, of whole smoke in hamsters. A positive response can be observed in the larynx of hamsters exposed over their lifetime to whole cigarette smoke. In this test the animals are exposed twice daily to the diluted smoke of one cigarette every day for their entire lives. Tumor formation is the endpoint measured in this assay. Because the test is so labor intensive it is recommended only as a last step in a series of tests.

These known methods for assaying tobacco toxicity have limitations in terms of time length and/or expense relative to the assay methods provided herein. Accordingly, there is a long felt need for more rapid and less costly methods of analysis of tobacco products of different compositions. Despite the inefficiencies of the approaches above, it is contemplated herein that these methods for assaying tobacco toxicity can be used alone or in conjunction with the methods provided herein so as to provide additional information regarding the properties of the tobacco being characterized.

In the methods provided herein, one or more cells can be contacted with a tobacco composition such as tobacco smoke (TS), a tobacco smoke condensate (TSC), or total particulate matter (TPM), where exemplary TS and TSC are cigarette smoke (CS) and cigarette smoke condensate (CSC). Preparation of the tobacco composition used in the methods provided herein can be performed in accordance with the teachings herein and the knowledge and skill in the art. For example, TS can be collected using a smoking machine such as an INBIFO-Condor smoking machine, and TSC can be collected using cold traps, and TPM can be collected using a filter, as is known in the art. For example, CSC for testing can be prepared by passing smoke through a series of cold traps containing glass beads upon which CSC condenses; the CSC can then be collected by washing the beads with acetone as described in Mathewson, H. D. Beitrage zur Tabforschung. 3(6):430-7. September 1966. In addition, cells can be contacted with smoke provided in diluted form, where diluted smoke can be produced in a dilution chamber, as known in the art. For example, a smoking setup can contain a dilution chamber where the concentration of the smoke being applied to the cells can be varied by dilution with air in order to produce different dosages and intensities of smoke. The dilution chamber can be located between the burning cigarette and the cell exposure chamber. In addition, cigarette particulate matter for testing can be prepared by passing smoke through a glass fiber filter which is subsequently washed with solvent to collect the sample as described in Coresta Recommended Method No. 23 (August 1991). Although the description herein provides several methods in the context of characterizing tobacco and tobacco products that undergo pyrolysis (e.g., cigarettes, pipe tobacco, and cigars), similar approaches can be applied to the evaluate snuff, chew, and other tobacco products that do not undergo pyrolysis. Accordingly, the methods provided herein are not limited to smoke or smoke condensate, but can be applied to any tobacco composition known in the art. The preparation and analysis of compositions from such non-pyrolysis tobacco products is straightforward given the teachings provided herein or otherwise known in the art. Methods for contacting cells with compositions from such non-pyrolysis tobacco products also is straightforward given the teachings provided herein or otherwise known in the art.

The tobacco derived composition (i) can originate in a tobacco product, which can be either pure tobacco or a tobacco formulation (such as a cigarette, cigar, pipe or chewing tobacco) having multiple compositional elements, for example but not limited to structural elements, flavor chemicals and/or other additives, and (ii) can have multiple components (e.g., smoke or a smoke condensate, also referred to collectively as “smoke products”) or can be a single known or unidentified component (e.g., a single chemical compound). The composition can be “derived” from tobacco or a tobacco formulation (i) by simple physical separation; (ii) as a product of combustion or heating, (iii) by solvent extraction, (iv) by chemical reaction(s) or (v) by enzyme activity (e.g., smoke concentrate treated with a microsomal cellular fraction or purified cytochrome P450).

In some methods provided herein, cells are contacted with TS, such as CS. The contacting of the cells with the CS, CSC, TS, TSC or TPM can be accomplished using any method known to one of skill in the art, including but not limited to, placing said cells into a smoking machine or smoke chamber (e.g., CULTEX®) for a period of time to allow the cells to be contacted with smoke, and/or providing a CSC or TSC to the media for a designated period of time (e.g., in 0.5% dimethylsulfoxide or other formulation). The contacting can be for any amount of time, however, preferably the cells are contacted for an amount of time that does not result in nonviability of more than 50% of the cells. In some embodiments, the amount of time can be varied and the results are compared. In a further embodiment, the cells are treated for an amount of time in which the gene expression is modulated, but the majority of cells are still viable. That is, in some embodiments, the cells are treated to a point in which the cells are at least, equal to, or more than 1% viable, including but not limited to 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, and 100% viable.

In a another embodiment, the amount of time for contacting a cell with the CS, CSC, TS, TSC or TPM is any amount selected from the group consisting of about at least, equal to, or more than 1 seconds to about 24 hours, including but not limited to at least, equal to, or more than 1 second, 15 seconds, 30 seconds, 45 seconds, 1 minute, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 60 minutes, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, 12.5 hours, 13 hours, 13.5 hours, 14 hours, 14.5 hours, 15 hours, 15.5 hours, 16 hours, 16.5 hours, 17 hours, 17.5 hours, 18 hours, 18.5 hours, 19 hours, 19.5 hours, 20 hours, 20.5 hours, 21 hours, 22 hours 23 hours and 24 hours. In a further embodiment, the cells are contacted for less than and including about 20 minutes. In yet another embodiment, the cells are contacted for about 2 to about 20 minutes.

The amount of smoke with which the cells are contacted can be any of a variety of amounts according to the desired level of exposure. For example, smoke exposure can be performed in accordance with FTC parameters: 2.0 second puff duration, 35 mL puff every 60 seconds. Puff duration, volume and frequency can be increased or decreased to achieve different levels of smoke exposure, as desired. Similarly, smoke condensate or other tobacco compositions can be contacted with cells at a variety of different concentrations and for a variety of different durations, as desired. For example, smoke condensate at 20 mg/mL can be contacted with cells for any of the above-provided amounts of time, as desired.

Tobacco smoke or smoke products can be treated prior to contacting the cells with the smoke or smoke product. For example, the smoke or smoke concentrate can be contacted with a filter, such as a filter provided herein, for example by obtaining smoke or smoke condensate from a cigarette after passing through a filter attached to the tobacco product, such as a cigarette.

The cells suitable for use in the methods provided herein include human as well as non-human cells, but are preferably human pulmonary cells (e.g., lung or bronchial cell), although cells of other systems impacted by smoking, including but not limited to cells of the upper aerodigestive tract (e.g., oral cavity including cheek, pharynx, larynx, and esophagus), bladder, stomach, kidney, pancreas, and blood (e.g., lymphocytes, monocytes, neutrophils, esoinophils or basophils, or neoplastic blood cells such as myeloid leukemia cells); cells of the cardiovascular system (including endothelial cells, smooth muscle cells (e.g. from vessel walls, myocardial cells, etc.) and cells of the female reproductive system (e.g. cells of the uterus, cervix, fallopian tubes, ovary, and placenta), can also be used. The cells can be normal or can be neoplastic, metaplastic, dysplastic or malignant. The cells can be collected from a living organism (e.g., a pulmonary lavage specimen, tissue section such as a lung or bronchial section, oral mucosa sample, cheek swab, or sputum sample), can be primary cell cultures, or can be established cell cultures. In some embodiments, the cells can be obtained from a living organism, including a human, after the organism is contacted with a tobacco composition, for example, after a human consumes a cigarette. Cells collected from a living organism can be collected using any of a variety of known methods known in the art, according to the cell type to be collected (e.g., a cheek scrape or lung lavage). In specific, non-limiting embodiments, the cells can be NHBE cells, or can be human epithelian pulmonary type II cells, such as A549 cells, or can be cells obtained from a human primary culture.

Many embodiments described herein employ NHBE cells that are maintained in culture, and other embodiments employ human lung carcinoma cells (A549 cells). Although NHBE and A549 cells are preferred for the methods described herein, it should be understood that many other cells that are typically contacted with tobacco or TS during the process of smoking (e.g., lung cells, bronchial cells, cells of the oral mucosa, pharynx, larynx, and tongue) can also be used. Additionally, many immortal cell lines can be used with the methods described herein. Preferred cells for use with the embodied approaches include, but are not limited to, human bronchial cells (e.g., BEP2D or 16HBE140 cells), human bronchial epithelial cells (e.g., HBEC cells, 1198, or 1170-I cells), NHBE cells, BEAS cells (e.g., BEAS-2B), NCI-H292 cells, non-small cell lung cancer (NSCLC) cells or human alveolar cells (e.g., H460, H1792, SK-MES-1, Calu, H292, H157, H1944, H596, H522, A549, and H226), tongue cells (e.g., CAL 27), and mouth cells (e.g., Ueda-1)). Many of such cultures are available commercially or through a public repository (e.g., ATCC). Further, several techniques exist that allow for one to generate primary cultures of said cells and these primary cultures can be used with the methods described herein.

Conventional approaches in tissue culture can be used to establish and maintain said cells in preparation for the methods described herein. That is, the cells may be grown in culture by any method known to one of skill in the art and with the appropriate media and conditions. The cells grown in culture may require feeder layers, for example. The cells may be grown to confluence or may be grown to less than confluence before, during, or after treatment. In some embodiments the cells are grown to between about 10% and about 90% confluence, including but not limited to, at least, equal to, or more than 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99% confluence before contact with CS, CSC, TS, or TSC.

In some embodiments, the cells contacted and assayed in accordance with the methods provided herein are manipulated to control and/or modify the percentage of cells that are in one or more phases of the cell cycle. For example, the cells can be manipulated such that at least 50% of the cells of the population of cells are in the S phase. The cells used herein can be manipulated to control the population of cells in one or more of G0, G1, S, G2, or M phases of the cell cycle. For example, cells can be manipulated such that at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of the population of cells are in G0, G1, S, G2, or M phase. In another example, cells can be manipulated such that greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of the population of cells are in G0, G1, S, G2, or M phase. In another example, cells can be manipulated such that 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of the population of cells are in G0, G1, S, G2, or M phase. The section below describes several preferred methods for characterizing tobacco and tobacco products in greater detail.

Exemplary Assays

The methods provided herein for characterizing tobacco or a tobacco product can be used in a variety of applications, including, but not limited to, the comparison of two or more tobaccos or two or more tobacco products, identifying a modulation of cell homeostasis, identifying an induction of damage of cellular genetic material, or identifying a modulation of gene expression including mRNA and/or protein expression. The methods provided herein for characterizing tobacco also can be used for identifying a tobacco product that has a reduced potential to contribute to a tobacco-related disease, and making a tobacco product that has a reduced potential to contribute to a tobacco-related disease. In addition to methods provided herein for characterizing tobacco or a tobacco product, additional methods known in the art for characterizing tobacco or a tobacco product can be used in the place of, or in conjunction with, the methods provided herein.

The methods of identifying a tobacco, identifying a compound in tobacco, identifying a tobacco product, and making a tobacco product provided herein, can in addition be utilized in methods of identifying two or more tobaccos, identifying a compound in two or more tobaccos, identifying a tobacco product by comparing two or more tobacco products, and making a tobacco product by comparing two or more tobacco products. In some embodiments, the two or more tobaccos or tobacco products can be compared for their effect on cell homeostasis, gene expression including mRNA and/or protein expression, or damage to genetic material of cells. In some embodiments, at least one tobacco or tobacco product can be a reduced risk tobacco or tobacco product, respectively. In some embodiments, at least one tobacco can be a modified tobacco, such as a chemically modified tobacco or a genetically modified tobacco. In some embodiments, at least one tobacco can be a reduced risk tobacco product, such as a tobacco product configured to contain a filter that reduces the risk of tobacco exposure such as a filter provided herein. In one example, two or more tobaccos can be compared to identify a compound in tobacco that modulates gene expression including mRNA and/or protein expression. In one example, two or more tobacco products can be compared to identify a compound in tobacco that modulates gene expression including mRNA and/or protein expression.

In some embodiments, a second tobacco product (e.g., a cigarette) is compared to a first tobacco product (e.g., a cigarette) using the methods above so as to identify which of the two tobacco products is less likely to contribute to a tobacco-related disease. For example, a first population of isolated human cells of the mouth, tongue, oral cavity, or lungs (e.g., NHBE cells), is contacted with a CS from a first tobacco product (e.g., a “reduced risk full flavor” cigarette) in an amount and for a time sufficient to modulate cell homeostasis, such as inducing damage of cellular genetic material or modulating gene expression including mRNA and/or protein expression. A second population of isolated human cells of the mouth, tongue, oral cavity, or lungs (e.g., NHBE cells), preferably the same type of cell as used in the analysis of the first tobacco product, is also contacted with a CS from a second tobacco product (e.g., a cigarette) in an amount and for the same amount of time as used with the first product or for a time sufficient to modulate cell homeostasis by, for example, inducing damage of cellular genetic material or modulating gene expression including mRNA and/or protein expression.

The data obtained from the analysis of the first tobacco product can be compared to the data obtained from the analysis of the second tobacco product so as to identify, for example, an increased risk tobacco or a compound in tobacco. The data also can be used to identify a reduced risk tobacco. The data further can be used to identify a tobacco product configuration, such as a filter, with increased risk or with reduced risk. Thus, by analyzing the differences between the tobacco products, one can identify a tobacco product that has less potential to contribute to a tobacco related disease or to identify, for example, a first tobacco product that has a reduced risk to contribute to a tobacco-related disease, as compared to a second tobacco product or vice versa. By one technique, a tobacco product that is less likely to contribute to a tobacco-related disease is identified because it causes less modulation of cell homeostasis. By another technique, a tobacco product that is less likely to contribute to a tobacco-related disease is identified because it causes less modulation of cell homeostasis under the same level of damage induced to cellular genetic material. In another technique, a tobacco product that is less likely to contribute to a tobacco-related disease is identified because it induces less damage to cellular genetic material. In another technique, a tobacco product that is less likely to contribute to a tobacco-related disease is identified because it induces fewer or smaller in degree changes in gene expression such as changes in the transcriptome or changes in the proteome.

The methods provided herein can be used not only to identify a tobacco product that has a reduced potential to contribute to a tobacco-related disease, as compared to a second tobacco product, but also to develop tobacco products that have a reduced potential to contribute to a tobacco-related disease, as compared to a second tobacco product. For example, by screening modified tobacco (e.g., chemically or genetically modified tobacco) or a tobacco product with a modified configuration in accordance with the methods disclosed herein, one can rapidly determine whether the modified tobacco or modified tobacco product has an increased or decreased potential to contribute to a tobacco-related disease, as compared to the tobacco or tobacco product that is not modified.

More embodiments concern methods to identify components of a tobacco product that contribute to a tobacco-related disease, the selective removal or inhibition of production of these components, and the determination that the removal of the component(s) results in a reduced risk tobacco product. Such a determination that the removal of the component(s) results in a reduced risk tobacco product can be indicated by, for example, a molecular marker that is associated with a tobacco-related disease. Exemplary molecular markers of a tobacco-related disease include, but are not limited to a molecular marker indicative of apoptosis, a molecular marker indicative of double-stranded DNA breaks, an overexpressed or underexpressed mRNA, an overexpressed or underexpressed polypeptide. In one example methods are provided to identify components of a tobacco product that contribute to a tobacco-related disease, the selective removal or inhibition of production of these components, and the determination that the removal of the component(s) modulates expression of a gene that is associated with a tobacco-related disease in a manner that reduces the potential for the tobacco product to contribute to a tobacco related disease. It is contemplated that particular components of tobacco products are the factors that modulate responses in human cells that contribute to tobacco-related disease. It is further contemplated that modification of the tobacco product will, concomitantly, result in a modulation of the response in human cells contacted with the smoke from said modified tobacco product, which modulates the likelihood to contribute to a tobacco-related disease relative to an unmodified tobacco product. For example, modification of genes that contribute to the production of these toxic components in tobacco (e.g., genetic engineering or chemical treatment) will, concomitantly, result in a modulation of the response in human cells contacted with the smoke from said modified tobacco, which modulates the likelihood to contribute to a tobacco-related disease relative to tobacco prior to modification of the component-producing gene. Accordingly, by selectively removing the components from a tobacco product (e.g., by modifying a tobacco or a tobacco product configuration) that induce the events that contribute to tobacco-related disease in a human, one can develop tobacco products that are less likely to contribute to a tobacco-related disease.

Methods for Identifying a Tobacco or Tobacco Product that Modulates Cell Homeostasis

Provided herein are methods for identifying a tobacco that modulates cell homeostasis by providing a tobacco, obtaining a tobacco composition from the tobacco, contacting a cell with the tobacco composition, and identifying the presence or absence of a modulation of cell homeostasis after contact with the tobacco composition. In some embodiments, the methods provided herein can be used to identify a tobacco that affects cell homeostasis as can be monitored by, for example, determining that the tobacco induces double strand DNA breaks, modulates apoptosis, modulates cell proliferation, or induces expression of a gene that is silent during homeostasis or repress a gene that is active during homeostasis. In some embodiments, the tobacco composition can be smoke or smoke condensate. By modulation of homeostasis of a cell is meant the change in the state of a cell upon contact of the cell by a tobacco or tobacco composition (e.g., tobacco smoke or tobacco smoke condensate), where the state of the cell can be the cell cycle, the apoptotic state, the expression levels of one or more genes as represented by mRNA levels and/or protein levels or post-translational modifications to proteins. Typically modulation of cell homeostasis can be monitored by measurement of one or more molecular markers of the state of a cell. It is contemplated herein that any tobacco-induced change in cell homeostasis can serve as an indicator that said tobacco or tobacco composition may contribute to a tobacco-related disease. Because it is known that tobacco or tobacco compositions can have a plurality of adverse affects on cells, it is contemplated that typically a less pronounced modulation of cell homeostasis after the cell is contacted with a tobacco or tobacco composition (e.g., tobacco smoke or tobacco smoke condensate), the greater propensity that the tobacco or tobacco composition will present a reduced level of risk for developing a tobacco related disease relative to a conventional or reference tobacco. Accordingly, it is contemplated herein that a tobacco or tobacco composition characterized as reduced risk tobacco or tobacco composition is one that, upon contact with a cell, does not modulate cell homeostasis or modulates cell homeostasis to a lesser degree than the cell homeostasis modulation induced by a conventional or reference tobacco.

Also provided herein are methods of identifying a compound in tobacco that modulates cell homeostasis by providing a first tobacco, obtaining a tobacco composition from the first tobacco, contacting a first population of cells with the tobacco composition from the first tobacco, identifying the degree of modulation of cell homeostasis in the first population of cells after contact with the tobacco composition from the first tobacco, providing a second tobacco that has been modified to reduce a compound in the second tobacco, obtaining a tobacco composition from the second tobacco, contacting a second population of cells with the tobacco composition from the second tobacco, and identifying the degree of modulation of cell homeostasis after contact with the tobacco composition from the second tobacco, where an identification of a reduction in the degree of modulation of cell homeostasis after contact with the tobacco composition from the second tobacco identifies the compound as one that modulates cell homeostasis. In some embodiments, the methods provided herein can be used to identify a compound in tobacco that modulates apoptosis. In some embodiments, the methods provided herein can be used to identify a compound in tobacco that modulates cell proliferation. In some embodiments, the tobacco composition can be smoke or smoke condensate.

Also provided herein are methods of identifying a tobacco product that has a reduced potential to contribute to a tobacco-related disease by providing a first tobacco product, obtaining a tobacco composition from the first tobacco product, contacting a first population of cells with the tobacco composition from the first tobacco product, identifying the degree of modulation of cell homeostasis in the first population of cells after contact with the tobacco composition from the first tobacco product, providing a second tobacco product, obtaining a tobacco composition from the second tobacco product, contacting a second population of cells with the tobacco composition from the second tobacco product, and identifying the degree of modulation of cell homeostasis after contact with the tobacco composition from the second tobacco product, where an identification of a reduction in the degree of modulation of cell homeostasis after contact with the tobacco composition from the second tobacco product as compared to the degree of modulation of cell homeostasis after contact with the tobacco composition from the first tobacco product identifies the second tobacco product as one that has a reduced potential to contribute to a tobacco-related disease. In some embodiments, the second tobacco product has been modified to reduce a compound in the second tobacco. In some embodiments, the second tobacco product can be genetically modified to reduce the expression of at least one gene that regulates production of the compound. In some embodiments of the methods provided herein, the degree of modulation of cell homeostasis can be determined by identifying the degree of modulation of apoptosis. In some embodiments of the methods provided herein, the degree of modulation of cell homeostasis can be determined by identifying the degree of modulation of cell proliferation. In some embodiments, the tobacco composition can be smoke or smoke condensate.

Also provided herein are methods of making a tobacco product that has a reduced potential to contribute to a tobacco-related disease by providing a first tobacco, obtaining a tobacco composition from the first tobacco, contacting a first population of cells with the tobacco composition from the first tobacco, identifying the degree of modulation of cell homeostasis in the first population of cells after contact with the tobacco composition from the first tobacco, providing a second tobacco, obtaining a tobacco composition from the second tobacco, contacting a second population of cells with the tobacco composition from the second tobacco, identifying the degree of modulation of cell homeostasis after contact with the tobacco composition from the second tobacco product, where an identification of a reduction in the degree of modulation of cell homeostasis after contact with the tobacco composition from the second tobacco as compared to the degree of modulation of cell homeostasis after contact with the tobacco composition from the first tobacco identifies the second tobacco as one that has a reduced potential to contribute to a tobacco-related disease, and incorporating the second tobacco, which has a reduced potential to contribute to a tobacco-related disease, into a tobacco product. In some embodiments, the second tobacco has been modified to reduce a compound in the second tobacco. In some embodiments, the second tobacco can be genetically modified to reduce the expression of at least one gene that regulates production of the compound. In some embodiments of the methods provided herein, the degree of modulation of cell homeostasis can be determined by identifying the degree of modulation of apoptosis. In some embodiments of the methods provided herein, the degree of modulation of cell homeostasis can be determined by identifying the degree of modulation of cell proliferation. In some embodiments, the tobacco composition can be smoke or smoke condensate.

The methods provided herein can be used to determine the effect of a tobacco product or a compound from a tobacco product, on cell homeostasis. Cells of an organism contacted with a tobacco composition, e.g., mammalian epithelial cells, can undergo apoptosis and can proliferate at particular levels under “normal” conditions, where “normal” as used in this context refers to conditions in which cells are not contacted with tobacco or a tobacco composition and are not otherwise placed under atypical (e.g., stressful) environmental conditions. Environmental conditions, for example, contacting the cells with a tobacco composition, can modulate apoptosis of the contacted cells and also can modulate the proliferation of the contacted cells. Such modulation can result in processes that can directly lead to cellular events in tobacco-related disease (e.g., apoptosis can be decreased, which can lead to neoplastic cell growth) or can indirectly lead to cellular events in tobacco-related disease (e.g., apoptosis can be increased, which can trigger a cell growth response in an organism, which can lead to neoplastic cell growth). The methods provided herein can be used to examine the affect of a tobacco product or a compound from a tobacco product, on cell homeostasis by, for example, determining the affect of a tobacco or tobacco compound on apoptosis in a cell or a cell population, or, for example, determining the affect of a tobacco or tobacco compound on cell proliferation of a cell or a cell population. In some embodiments, a first tobacco that causes a lesser degree of modulation of cell homeostasis relative to a second tobacco can be characterized as a reduced risk tobacco. In some embodiments, a first tobacco that causes a lesser degree of inhibition of apoptosis relative to a second tobacco can be characterized as a reduced risk tobacco. In some embodiments, a first tobacco that causes a lesser degree of inhibition of cell proliferation relative to a second tobacco can be characterized as a reduced risk tobacco. Any of a variety of known methods for determining modulation of cell homeostasis by, for example, modulating apoptosis, modulating cell proliferation, modulating gene expression (e.g., mRNA or protein levels) as exemplified herein, can be used in the methods provided herein.

Also provided herein are methods for determining cell response to cell damage. Cells can be exposed to environmental input, such as a tobacco composition, that causes cell damage. The response of these cells to the environmental input-mediated damage can be indicative of the likelihood of the environmental input leading to an environmental input-related disease. In one embodiment, cells can be contacted with a tobacco composition, and the response of the cells to the contact by the tobacco composition can indicate the likelihood of the tobacco composition leading to a tobacco-related disease.

As provided herein, cells contacted by different environmental inputs, for example, different tobacco compositions, can respond differently to cell damage caused by the environmental input, where some cell responses are more indicative of leading to a disease state compared to other cell responses. Thus, contemplated herein, two or more tobacco compositions can be compared and characterized according to the cell responses in reaction to damage induced by exposure to the tobacco compositions. In such methods, exposure conditions can be manipulated such that the amount of damage to the cells is equivalent for each different tobacco composition, resulting in a determination of different characteristic cell responses to the same amount of cell damage.

Accordingly, methods are provided herein for comparing two or more tobacco compositions by contacting a first tobacco composition with a first population of cells, and contacting a second composition with a second population of cells, where the two different contacting steps are performed in such a manner that the first and second population of cells undergo equivalent amount of cell damage, and then determining the degree of modulation of cell homeostasis in the first and second populations of cells, where the tobacco composition that is characterized by the lowest degree of cell modulation can be identified as a tobacco with reduced likelihood of causing a tobacco-related disease. In such methods, damage to the cells caused by the tobacco compositions can be measured by, for example, measuring the degree of damage to the genetic material of the cells, in accordance with the methods provided herein or otherwise known in the art. Also in such methods, the degree of modulation of cell homeostasis can be determined by the degree of modulation of apoptosis or cell proliferation relative to cells not contacted by a tobacco composition or relative to cells contacted by a tobacco composition from a tobacco, such as a reduced risk tobacco with a known degree of modulation of cell homeostasis. The following section describes several methods for differentiating tobaccos and tobacco products that induce genetic damage from those that do not.

Analysis of Changes to Cell Homeostasis: Identifying a Tobacco that Induces Genetic Damage

Provided herein are methods of identifying a tobacco that induces genetic damage by providing a tobacco, obtaining a tobacco composition from the tobacco, contacting a cell with the tobacco composition, and identifying the presence or absence of damage of cellular genetic material in the cell after contact with the tobacco composition. In some embodiments, the methods provided herein can be used to identify a tobacco that induces a double-strand DNA break. In some embodiments, the tobacco composition can be smoke or smoke condensate.

Also provided herein are methods of identifying a compound in tobacco that induces damage of cellular genetic material by providing a first tobacco, obtaining a tobacco composition from the first tobacco, contacting a first population of cells with the tobacco composition from the first tobacco, identifying the amount of damage of cellular genetic material in the first population of cells after contact with the tobacco composition from the first tobacco, providing a second tobacco that has been modified to reduce a compound in the second tobacco, obtaining a tobacco composition from the second tobacco, contacting a second population of cells with the tobacco composition from the second tobacco, and identifying the amount of damage of cellular genetic material after contact with the tobacco composition from the second tobacco, where an identification of a reduction in the amount of damage of cellular genetic material after contact with the tobacco composition from the second tobacco identifies the compound as one that induces the damage of cellular genetic material. In some embodiments, the methods provided herein can be used to identify a compound in tobacco that induces a double-strand DNA break. In some embodiments, the tobacco composition can be smoke or smoke condensate.

Also provided herein are methods of identifying a tobacco product that has a reduced potential to contribute to a tobacco-related disease by providing a first tobacco product, obtaining a tobacco composition from the first tobacco product, contacting a first population of cells with the tobacco composition from the first tobacco product, identifying the amount of damage of cellular genetic material in the first population of cells after contact with the tobacco composition from the first tobacco product, providing a second tobacco product, obtaining a tobacco composition from the second tobacco product, contacting a second population of cells with the tobacco composition from the second tobacco product, and identifying the amount of damage of cellular genetic material after contact with the tobacco composition from the second tobacco product, where an identification of a reduction in the amount of damage of cellular genetic material after contact with the tobacco composition from the second tobacco product as compared to the amount of damage of cellular genetic material after contact with the tobacco composition from the first tobacco product identifies the second tobacco product as one that has a reduced potential to contribute to a tobacco-related disease. In some embodiments, the second tobacco product has been modified to reduce a compound in the second tobacco. In some embodiments, the second tobacco product can be genetically modified to reduce the expression of at least one gene that regulates production of the compound. In some embodiments of the methods provided herein, the amount of damage of cellular genetic material can be determined by identifying the induction of double-strand DNA breaks. In some embodiments, the tobacco composition can be smoke or smoke condensate.

Also provided herein are methods of making a tobacco product that has a reduced potential to contribute to a tobacco-related disease by providing a first tobacco, obtaining a tobacco composition from the first tobacco, contacting a first population of cells with the tobacco composition from the first tobacco, identifying the amount of damage of cellular genetic material in the first population of cells after contact with the tobacco composition from the first tobacco, providing a second tobacco, obtaining a tobacco composition from the second tobacco, contacting a second population of cells with the tobacco composition from the second tobacco, identifying the amount of damage of cellular genetic material after contact with the tobacco composition from the second tobacco product, where an identification of a reduction in the amount of damage of cellular genetic material after contact with the tobacco composition from the second tobacco as compared to the amount of damage of cellular genetic material after contact with the tobacco composition from the first tobacco identifies the second tobacco as one that has a reduced potential to contribute to a tobacco-related disease, and incorporating the second tobacco, which has a reduced potential to contribute to a tobacco-related disease, into a tobacco product. In some embodiments, the second tobacco has been modified to reduce a compound in the second tobacco. In some embodiments, the second tobacco can be genetically modified to reduce the expression of at least one gene that regulates production of the compound. In some embodiments of the methods provided herein, the amount of damage of cellular genetic material can be determined by identifying the induction of double-strand DNA breaks. In some embodiments, the tobacco composition can be smoke or smoke condensate.

Also provided herein are methods, compositions and kits for evaluating the ability of a tobacco-derived substance to produce DSBs in chromosomal DNA. The presence of DSBs is detected using an appropriate marker, which, in preferred embodiments provided herein, is phosphorylated histone H2AX (also referred to herein as “γH2AX”). The presence of DSBs also can be detected by detecting (i) activation of one or more of the protein kinases that are responsible for H2AX phosphorylation (e.g., ATM, ATR and/or DNA-PK); (ii) appearance of nuclear foci that are induced by histone H2AX phosphorylation; or (iii) activation of one or more protein components of nuclear foci induced by H2AX phosphorylation that are associated with DNA repair. The term “activation” in regard to proteins activated by DSBs refers to a chemical modification such as phosphorylation, acetylation, ubiquitinylation or poly(ADP)ribosylation, and/or a change in protein conformation, occurring in response to formation of DSBs. Activated proteins can be detected, for example, immunocytochemically.

Some of the assays provided concern methods of detecting, quantifying, identifying and/or evaluating (e.g., for harmfulness) a tobacco-derived substance in the course of research or in the environment via its promotion of DSBs in the chromosomal DNA of a test cell. A correlation with harmful potential is drawn based upon the known relationship between DSBs and genetic mutations (including cancer-causing and teratogenic mutations) as well as cell damage and death.

Accordingly, one set of preferred embodiments provided herein are methods of detecting a harmful tobacco-derived substance comprising the steps of (a) exposing a test cell (or test cell population) to a tobacco test composition; (b) measuring the degree of H2AX phosphorylation in the test cell or cell population; and (c) comparing the degree of H2AX phosphorylation determined in the test cell or cell population to the degree of H2AX phosphorylation in a control cell or control cell population; wherein a higher degree of H2AX phosphorylation in the test cell compared to the control cell indicates the presence of a harmful tobacco derived substance in the tobacco test composition. The presence of DSBs also can be detected by detecting (i) activation of one or more of the protein kinases that are responsible for H2AX phosphorylation (e.g., ATM, ATR and/or DNA-PK); (ii) appearance of nuclear foci that are induced by histone H2AX phosphorylation; or (iii) activation of one or more protein components of nuclear foci induced by H2AX phosphorylation that are associated with DNA repair.

Another set of non-limiting embodiments, provided herein include methods for identifying one or more harmful components of TS comprising the steps of: (a) exposing a first test cell population to a first smoke product generated from a first tobacco composition; (b) exposing a second test cell population to a second smoke product generated from a second tobacco composition, wherein the first and second smoke products are prepared using essentially equivalent protocols; (c) measuring the degree of H2AX phosphorylation in the first and second test cell populations; and (d) comparing the degree of H2AX phosphorylation in the first and second test cell populations; (e) identifying the tobacco composition associated with a greater degree of H2AX phosphorylation in steps (a)-(d); and (f) comparing the components of the first and second tobacco composition to identify one or more component present in the tobacco composition of step (e) but absent in the other tobacco composition. Methods for detecting activation of protein kinases such as ATM, ATR and/or DNA-PK as well as formation of nuclear foci and protein components of the nuclear foci can be performed according to the same steps. According to such embodiments, the first tobacco composition can differ from the second tobacco composition in its ingredients and/or in the way it was processed. The information obtained by this method can be used to develop a tobacco product that lacks or has lower levels of the identified harmful component(s), which can render the product lower-risk. Alternatively, the information can be used in an environmental context: for example, air purifiers can be modified to extract the harmful component from smoke-contaminated air.

Another set of non-limiting embodiments provided herein concern methods for identifying one or more harmful components of TS comprising the steps of: (a) exposing a first test cell population to a first smoke product generated from a tobacco composition; (b) exposing a second test cell population to a second smoke product generated from the tobacco composition, wherein the first and second smoke products are prepared differently; (c) measuring the degree of H2AX phosphorylation in the first and second test cell populations; (d) comparing the degree of H2AX phosphorylation in the first and second test cell populations; and (e) identifying the method of smoke product preparation associated with a greater degree of H2AX phosphorylation in steps (a)-(d); wherein the method of smoke product preparation identified in step (e) has greater harmful potential. Methods for detecting activation of protein kinases such as ATM, ATR and/or DNA-PK as well as formation of nuclear foci and protein components of the nuclear foci can be performed according to the same steps. In such embodiments, the methods of smoke product preparation can differ in the rate of combustion of the tobacco composition (including whether the tobacco composition is burned or heated), or can differ in the filtering of the smoke product (e.g., unfiltered, filtered with a traditional filter, or filtered with a filter containing an antioxidant), or can differ by other known methods of altering tobacco smoke products. The components of the different smoke products can be compared to identify one or more harmful components. As above, the identification of a harmful component can facilitate the development of lower-risk tobacco products and/or environmental safeguards.

Also provided herein are methods for comparing the harmful potentials of a first and a second tobacco composition comprising the steps of: (a) exposing a first test cell population to a first smoke product generated from the first tobacco composition; (b) exposing a second test cell population to a second smoke product generated from the second tobacco composition, wherein the first and second smoke products are prepared using essentially equivalent protocols; (c) measuring the degree of H2AX phosphorylation in the first and second test cell populations; and (d) comparing the degree of H2AX phosphorylation in the first and second test cell populations; wherein the tobacco composition which generated the smoke product that produced a higher degree of H2AX phosphorylation has greater harmful potential. Methods for detecting activation of protein kinases such as ATM, ATR and/or DNA-PK as well as formation of nuclear foci and protein components of the nuclear foci can be performed according to the same steps.

Accordingly, the methods provided herein include one or more steps of determining whether damage of cellular genetic material has occurred. Typically, such methods include assays for damage to the genomic DNA of the cell. Any of a variety of methods known in the art for assaying damage of cellular genetic material, such as genomic DNA, can be used in the methods provided herein. Exemplary known assays include assays for double-strand DNA breaks, assays for single-strand DNA breaks, and assays for modulated properties of DNA resultant from damage, such as assays for micronuclei and assays for chromosome exchange. Assays for DNA breaks are known in the art, as exemplified in U.S. Pat. Pub. No. 20040132004 and U.S. Pat. No. 6,309,838, all of which are hereby expressly incorporated by reference in their entireties.

In one example, the methods provided herein can include detection of double-strand DNA breaks by detection of phosphorylation of histone H2AX. Mammalian cells respond to agents that introduce DNA double-stranded breaks with the immediate and substantial phosphorylation of histone H2AX. While not wishing to be bound to the following theory, which is only offered to explain one possible mechanism, H2AX is thought to be involved in the recognition of regions of chromatin containing a DNA double-stranded break. Formation of the phosphorylated H2AX protein, termed gamma-H2AX, can be detected as an indicator of DNA double-stranded breaks. Known antibodies or antigenically-reactive fragments thereof that specifically bind to a C-terminal phosphorylated serine in an H2AX histone protein can be used for the detection of gamma-H2AX, and, thus can be used to indicate the presence of double stranded breaks in a cell. Thus, in the methods provided herein, the presence or absence of DNA damage can be detected by detecting the presence or absence of phosphorylation of histone H2AX. For example, the presence or absence of phosphorylation of histone H2AX can be identified with an antibody or fragment thereof, which binds to phosphorylated H2AX but not unphosphorylated H2AX. Antibodies and fragments thereof, and related methods for selectively detecting gamma-H2AX, are known in the art, as exemplified in U.S. Pat. Nos. 6,362,317 and 6,884,873, all of which hereby expressly incorporated by reference in their entireties.

In some embodiments provided herein, the methods include assaying a cell for double-strand DNA breaks (DSBs). DSBs are generated by a variety of genotoxic agents, and are among the most critical lesions that lead either to apoptosis, mutations or the loss of significant sections of chromosomal material. Detection of DSBs upon cell exposure to a potential carcinogen, therefore, provides the means to assess the potential hazard of the exposure in terms of cancer induction. In one embodiment, a sensitive assay of DSBs detection based on analysis of histone H2AX phosphorylation can be used. Histone H2AX, a variant of a family of at least eight protein species of the nucleosome core histone H2A, becomes phosphorylated in live cells upon induction of DNA double strand breaks (DSBs). The phosphorylation of H2AX on Ser 139 at sites flanking the DSBs is carried out by ATM-, ATR-, and/or DNA-dependent protein kinases (DNA-PKs). The phosphorylated form of H2AX is denoted γH2AX.

The availability of antibodies to γH2AX allow for immunocytochemical detection of DSBs. After induction of DSBs, the appearance of γH2AX in chromatin manifests in the form of discrete foci, each focus considered to represent a single DSB. Checkpoint and DNA repair proteins such as Rad50, Rad51 and Brcal co-localize with γH2AX. The intensity of γH2AX immunofluorescence (IF) measured by cytometry was reported to strongly correlate with the dose of ionizing radiation and thus with the number of the induced DSBs. However, because untreated cells, particularly cells replicating DNA, express γH2AX, to obtain a stoichiometric relationship between DSBs and the intensity of γH2AX IF, it is necessary to compensate for the extent of this “programmed” H2AX phosphorylation. Following compensation, the γH2AX IF measured by cytometry offers a sensitive and convenient means to detect and measure DSBs in individual cells following radiation. In fact, γH2AX IF can be a surrogate for cell killing in viability assays of radiated cells.

γH2AX antibody (“Ab”) in conjunction with multiparameter flow-and laser scanning cytometry can be used in assays of DSBs, to detect and measure their induction in individual, live cancer cells exposed to antitumor drugs in vitro. The intensity of γH2AX IF correlates well with the drug concentration and duration of cell exposure to the drug, indicating a relationship between the incidence of DSBs induced by these drugs and γH2AX IF intensity. Multiparameter analysis of γH2AX IF and cellular DNA content made it possible to relate the abundance of DSBs (extent of DNA damage) to the position of the cell in the cycle.

The ability of the tobacco-derived substance to promote the formation of DSBs is measured using an appropriate DSB marker, which is preferably γH2AX (phosphorylated histone H2AX), but which can be another associated molecule, such as, but not limited to, Rad50, Rad51 and Brcal, and other proteins that are characteristic of nuclear foci formation. Formation of DSBs also can be detected by detecting activate protein kinases associated with DSBs such as ATM, ATR or DNA-PK. The presence of such markers can be determined using a marker-specific antibody (or derivative or fragment thereof), preferably an antibody (or fragment or derivative thereof) specific for γH2AX, or an antibody (or fragment or derivative thereof) specific for Rad50, Rad51 or Brcal, or ATM, ATR or DNA-PK. The presence of such markers can be determined using a marker-specific antibody (or derivative or fragment thereof), preferably an antibody (or fragment or derivative thereof) specific for a polypeptide encoded by a gene provided in Tables 1 and 2. The genes provided in Table 4 encode polypeptides that are involved in homologous recombination processes in the cell, and these genes can be activated in response to cellular damage of genetic material. Accordingly, detection of one or more products of the genes of Table 4 can be indicative of cellular damage of genetic material, for example, double-strand DNA breaks. The genes provided in Table 5 encode polypeptides that are involved in non-homologous nucleic acid end-joining processes in the cell, and these genes can be activated in response to cellular damage of genetic material. Accordingly, detection of one or more products of the genes of Table 5 can be indicative of cellular damage of genetic material, for example, double-strand DNA breaks. Provided herein is an exemplary use of antibody directed to γH2AX; analogous methods can be applied using antibodies directed to Rad50, Rad51, Brcal, ATM, ATR or DNA-PK, or the products of the genes listed in Tables 1 and 2. In preferred non-limiting embodiments provided herein, antibody binding can be detected by immunofluorescence-based techniques. Various antibodies for Rad50, Rad51, Brcal, ATM, ATR, DNA-PK, and the products of the genes listed in Tables 1 and 2 are known in the art and can be readily obtained for use in accordance with the methods provided herein; for example, Anti-phospho-ATM (Ser1981), is available from Upstate USA as clone 10H11.E12. Such techniques can optionally be used in conjunction with automated cytometry, such as, for example, flow and/or laser scanning cytometry.

The term “immunofluorescence-based techniques” or “immunocytochemical-based techniques” encompasses various forms of such assays, as are known in the art. For example, and not by way of limitation, an immunofluorescence-based technique can use an unlabelled primary antibody and a fluorescently labeled secondary antibody (as illustrated, for example, in Example 1); or can use a primary antibody that carries a fluorescent tag to detect the phosphorylated H2AX molecule directly; or the primary antibody can carry a biotin molecule while the secondary antibody can carry both an avidin molecule (which binds specifically to biotin) and a fluorescence molecule. In the biotin/avidin approach, the binding of the secondary antibody is based on binding of biotin by avidin rather than the binding of an antibody of one species directed against a protein of another species. Other variations of such techniques that would be known to the skilled artisan as “immunfluorescence-based techniques” or “immunocytochemical-based techniques” can be used according to the invention. Likewise, detection can be made using analogous methods that utilize a modality other than fluorescence, such as chromogenic or colorimetric assays, radiologic assays, and so forth.

Techniques such as immunocytochemical-based techniques can be used in conjunction with methods for counting cells, sorting cells, or other method for further characterizing cells. Exemplary methods include, but are not limited to, flow cytometry, laser scanning cytometry, fluorescence image analysis, chromogenic product imaging, fluorescence microscopy or transmission microscopy.

The “degree of phosphorylation of H2AX” as used herein refers to the relative, rather than absolute, amount of γH2AX. This is because γH2AX is produced during normal progression of the cell cycle. As discussed in Example 1, allowance can be made for normally occurring phosphorylation of H2AX. For example, the data can preferably be subjected to two normalization processes. First, allowance can be made for the normally occurring “programmed” phosphorylation of H2AX. Second, correction can be made for the fact that histone content is exactly doubled over the course of a cell cycle, doubling the size of the target (histone). In a specific non-limiting embodiment, a data value from cells with twice the DNA content (e.g., G2 and mitotic cells) with twice the histone target can be divided by 2 while a data value from cells in S phase having an intermediate in histone content can be divided by 1.5. In this manner, the amount of γH2AX detected beyond what occurs in an untreated control cell or cell population is normalized to a unit of histone so that one can refer to the “degree of histone H2AX phosphorylation” on a per unit of histone basis.

In another example, the methods provided herein can include detection of DNA breaks and other forms of genomic damage by Comet assay. Comet assay can be used to detect damaged DNA pulled from the nucleus of cells exposed to an electric field. Comet assay is a fluorescent microscopic method to examine DNA damage and repair at individual cell level. For example, cells can be embedded in agarose on a microscope slide and lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix, and electrophoresis at high pH can result in structures resembling comets, observed by fluorescence microscopy. The intensity of the comet tail relative to the head reflects the number of DNA breaks. This assay can be used for detecting various forms of DNA damage (e.g., single- and double-strand breaks, oxidative DNA base damage, and DNA-DNA/DNA-protein/DNA-Drug cross-linking) and DNA repair in many eukaryotic cell types. Comet assay not only provides an estimate of how much damage is present in cells, but what form it takes. Although it is primarily a method for measuring DNA breaks, modifications of the methods, for example, by introducing lesion-specific endonucleases, allows detection of, for example, pyrimidine dimers, oxidized bases, and alkylation damage. Thus, in the methods provided herein, the presence or absence of DNA damage can be identified by, for example, the presence or absence of comet tails when cells are analyzed using the Comet assay. Various methods for performing Comet assays are known in the art, as exemplified in Collins, (2004) Mol. Biotechnology. 26:249-261, Tice, et al. (2000) Environ. Mol. Mutagen. 35:206-221 and Gichner et al. (2004) Mutation Res. 559:49-57, all of which are hereby expressly incorporated by reference in their entireties.

In another example, the methods provided herein can include detection of double-strand DNA breaks by TUNEL assay. TUNEL assay can be used to measure double-strand breaks by incorporation of labeled nucleotides at the site of double-strand breaks using terminal transferase. The labeled nucleotides can then be detected with antibodies. TUNEL assay is frequently used to detect apoptosis-induced DNA fragmentation through a quantitative fluorescence assay. In one exemplary protocol, terminal deoxynucleotidyl transferase (TdT) catalyzes the incorporation of bromo-deoxyuridine (BrdU) residues into the fragmenting nuclear DNA at the 3′-hydroxyl ends by nicked end labeling. A TRITC-conjugated anti-BrdU antibody can then label the 3′-hydroxyl ends for detection. The TUNEL assay distinguishes two populations of cells: non-apoptotic cells (TUNEL-negative) and apoptotic cells (TUNEL-positive). Thus, in the methods provided herein, the presence or absence of DNA damage can identified by, for example, detecting the presence or absence of labeled nucleotides at the site of double-strand breaks, incorporated by, for example, terminal transferase. A variety of methods of performing TUNEL assays is known in the art, as exemplified in Doolin et al., J. Burn Care Rehabil. 20: 374-376, 1999; Kalyuzhny (2002) Methods Mol. Biol. 203:219-34; Lawry, Methods Mol. Med. (2004) 88:183-90; U.S. Pat. No. 6,506,609 and U.S. Pat. Pub. No. 20030017462, all of which are hereby expressly incorporated by reference in their entireties.

In another example, the methods provided herein can include detection of double-strand DNA breaks by sister chromatid exchange assay. Sister chromatid exchange assays detect late damage when genetic material is exchanged between sister chromatids. Sister chromatid exchange refers to a reciprocal interchange of the two chromatid arms within a single chromosome. This exchange can be visualized during the metaphase portion of the cell cycle and can be mediated by the enzymatic incision, translocation and ligation of at least two DNA helices. Thus, in the methods provided herein, the presence or absence of DNA damage can identified by, for example, detecting the presence or absence of interchange of chromatid arms within a single chromosome by, for example, sister chromatid exchange assay. A variety of methods for performing sister chromatid exchange assays are known in the art, as exemplified in 40 C.F.R. §79.65, 40 C.F.R. §798.5915, Renqing et al., (2000) Toxicology Letters 115:23-32, Deen et al. and Cancer Res. (1986) 46:1599-602, all of which are hereby expressly incorporated by reference in their entireties.

In another example, the methods provided herein can include detection of double-strand DNA breaks by micronuclei assays. Micronuclei assays can be used to detect late damage occurring after cells attempt to divide so that non-centromeric DNA forms as micronuclei in daughter cells. The test is based on the observation that a secondary nucleus (micronucleus) is formed around a chromosomal fragment, outside the main nucleus of a dividing cell. A micronucleus may also be produced due to a lagging whole chromosome formed as a result of a chromosome loss at anaphase. Thus, in the methods provided herein, the presence or absence of micronuclei can be identified. Micronuclei can be detected by microscopic methods, flow cytometric methods and automated image recognition methods, as known in the art and exemplified in Offer et al., FASEB J. (2005) 19:485-7; Smolewski et al., Cytometry (2001) 45:19-26; Driessens et al., Ann N Y Acad. Sci. (2003) 1010:775-9; and U.S. Pat. Pub. No. 20050002552, all of which are hereby expressly incorporated by reference in their entireties.

In another example, the methods provided herein can include detection of chromosomal translocations. Chromosomal translocations can occur as a result of DNA damage. Methods for detecting chromosomal translocations can include fluorescence in situ hybridization methods (FISH), in which probe hybridization patterns in cells containing chromosomal translocation are altered relative to wild type. Thus, in the methods provided herein, the presence or absence of DNA damage can identified by, for example, detecting the presence or absence of chromosomal translocations by, for example, FISH. Methods for detecting chromosomal translocations are known in the art, as exemplified by U.S. Pat. Nos. 5,997,869, 6,576,421, and 6,416,948, and U.S. Pat. Pub. Nos. 20040235039 and 20020192692, all of which are hereby expressly incorporated by reference in their entireties.

The example below provides one non-limiting specific example of the DSB detection methods and materials. Variations of the assay method used in terms of materials, assay times, instrumentation and protocols would be apparent to the skilled artisan for detecting and/or quantifying DSBs, for example via γH2AX.