The present invention describes an efficient retroviral or viral based method that allows easy and quick identification of gene transfer in living, transduced mammalian cells. Retroviral and viral vector producer cells were generated containing a gene for an improved humanized red-shifted, Green Fluorescent Protein (hRGFP) which increases the resulting fluorescence yield after excitation. This humanized, red-shifted GFP (hRGFP) gene was cloned into several vectors and transfected into various packaging cell lines to produce vibrant green fluorescence after excitation with blue light at 450-490 nm. These vectors represent a substantial advance over currently available gene transfer marking systems or wild-type GFP marker systems none of which have been stably transfected into cells.

Description

23 parts

›CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of provisional application Serial. No. 60/010,371 filed Jan. 22, 1996.

›BACKGROUND OF THE INVENTION

Gene therapy involves the transfer of therapeutic genes into living cells. The potential clinical applications of gene therapy are numerous and include the treatment of a wide variety of diseases, such as those resulting from genetic defects as well as cancer and diseases caused by viral infections, such as AIDS. A number of human genetic diseases that result from a lesion in a single gene have been proposed as candidates for gene therapy. These include bone marrow disorders, erythroid cell defects, metabolic disorders resulting from defects in liver enzymes, and diseases of the central nervous system.

For some of these diseases, the introduction of a functional homolog of the defective gene and the production of even small amounts of the missing gene product would have a beneficial effect. For example, 10-20% production of the normal levels of Factor IX can alleviate severe hemophilia B. Yao, et al. (1991) B. Proc. Natl. Acad. Sci. 88:8101-8105.

Since gene therapy evolved in the early 70's there have been several clinical trials involving retroviral transfer of either therapeutic genes or suicide genes. Gene transfer of ADA gene to correct for a genetic defect, was the first gene therapy trial which began in 1990. Culver, K., et al, (1990). Transfer of a suicide gene into brain tumors followed in 1992. Culver, K., et al. (1992) Science 256:1550-1552. The gene transfer vehicle in both of these trials is a disabled retrovirus. Retroviral vectors are designed to transfer the gene of interest into target cells which must be undergoing cell division.

ADA is a rare genetic immunodeficiency disease caused when a defect occurs in both copies of the ADA gene. Children affected by this disease may have a severe combined, immunodeficiency (SCID) which could lead to death by common infections in their first months of life. Ex vivo gene transfer of the ADA gene into patients' T lymphocytes resulted in a beneficial therapy for the children in the 1990 trial. However, treatments must be repeated often to maintain sufficient levels of ADA in the bloodstream. In brain tumor trials, in situ gene transfer of the suicide gene, the HsTk gene, followed by ganciclovir treatment was used to eradicate the tumors. Although only a small portion of the tumor cells are transduced using this method, a “bystander” effect is hypothesized to help spread the killing.

In the above-described systems the therapeutic impact of gene therapy is at a minimum. Thus, there is a need to improve the efficiency of gene transfer. Currently, researchers are experimenting with alternative methods to increase transduction efficiency. However, there is a need for a quick and efficient marker gene to assess the results.

Murine retroviral vectors have emerged in the past several years as the most common vehicle to deliver marker genes. Other viral vectors such as adenoviruses, herpes viruses, adeno-associated viruses, and non-viral methods such as plasmids have also been used for gene transfer. Gene transfer systems often include markers such as β-galactosidase, luciferase, chloramphenicol acetyltransferase, and alkaline phosphatase. Detection of these markers involve either cell fixation that kills the cells and the addition of a substrate or antibody mediated detection. These methods are often time consuming and are prone to endogenous high background.

Another group of gene transfer markers convey drug resistance and thus allow positive selection of transfected cells through selection of resistant colonies. Although drug selectable markers allow the detection of living cells by expressing the transgene, they require that the cells survive in a toxic environment over a long period of time. Also, the neomycin-resistance gene, which confers resistance to the neomycin analog G418, has been shown to have deleterious effects upon the expression of other genes in retroviral vectors. Emerman, M., et al. (1986) Nucleic Acids Res. 14, 9381-9396.

A novel marker gene is now available that will alleviate these cumbersome and time consuming steps for detecting gene transfer. The Green Fluorescent Protein (GFP) is a vibrant green bioluminescent marker which offers outstanding properties. The gene has been sequenced, humanized and is commercially available through several sources, however there has been much difficulty in finding a suitable transformation vehicle that will give stable expression in mammalian cells.

It is therefore a primary objective of the present invention to provide a gene transfer marker that overcomes the deficiencies of currently available gene transfer markers as described above.

It is another objective of the present invention to provide a gene transfer marker that provides rapid identification of gene transfer in living mammalian cells.

It is a further objective of the present invention to provide a gene transfer marker that can be easily visualized.

It is yet a further objective of the present invention to provide a gene transfer marker that is stable and is effectively and efficiently transferred into living cells.

These and other objectives will become apparent from the following description.

›SUMMARY OF THE INVENTION

The present invention describes the cloning and characterization of amphotropic retroviral vectors capable of demonstrating efficient, stable transfer of humanized, red shifted GFP (hRGFP) gene into mammalian cells. Living cells transfected and/or transduced with hRGFP have a stable, bright green fluorescence after excitation with blue light.

The inventors have generated transformation vehicles containing a gene for an improved, humanized and red-shifted version of the Aequorea victoria green fluorescent protein (hRGFP) from various viral vectors. The hRGFP gene has been used to produce amphotropic vector producer cell lines that demonstrate vibrant green fluorescence after excitation with blue light. These vehicles represent a substantial improvement over currently available gene transfer marking systems. Bright, long-term expression of the hRGFP gene in living eukaryotic cells will advance the study of gene transfer, gene expression, and gene product function in vitro and in vivo, particularly for human gene therapy applications.

›BRIEF DESCRIPTION OF THE DRAWINGS · 1 of 2

The file of this patent contains at least one drawing executed in color.

FIG. 1 is the chemical structure of the GFP fluorophore. Blue light stimulates the excitation of a cyclized wild-type GFP fluorophore formed by serine-65, tyrosine-66, and glycine-67 that emits green fluorescence after stimulation. The red shifted variant contains a mutation that converts serine-65 to threonine. This results in a “red shift” of the excitation wavelength, increased amplitude of fluorescence, and a faster rate of fluorophore formation in the mutant GFP. R=H, serine; R=CH 3 , threonine.

FIG. 2 shows the retroviral constructs containing the red shift, humanized Green Fluorescent Protein. The wild-type GFP and the humanized, red shifted GFP gene were cloned into the pLNCX retroviral backbone. Plasmid pLNCG was constructed by PCR amplification of a wild-type GFP containing DNA fragment and subsequent subcloning into pLNCX. LTR, long terminal repeat; pA, polyadenylation signal; arrows indicate transcriptional start sites; Ψ + indicates the presence of the viral packaging sequence; GFP, wild-type green fluorescent protein; hRGFP, humanized, red shifted GFP.

FIGS. 3A-3C relate to GFP detection in transfected cell lines. The photographs show the expression of GFP and hRGFP in mouse PA317 packaging cells.

3 A: PA317 cells without DNA transfection visualized under 40× magnification using the Green Fluorescent Protein Longpass filter.

3 B: PA317 cells transfected with 5 μg pLNCG DNA and visualized after 48 hours under 40× magnification excited using the GFP Longpass filter set (420-470 nm).

3 C: PA317 cells transfected with 5 μg pLNChRG DNA and visualized after 40 hours under 40× magnification using the FITC filter set (450-490 nm).

FIG. 4 relates to the section of hRGFP gene activity in stable, transfected PA317 vector producer cells. The photograph shows the expression of hRGFP in mouse PA317 packaging cell after transfection with plasmid pLNChRG and selection in G418 for 2 weeks. Selected cells were trypsin digested and plated onto glass coverslips for viewing. PA317 cells were transfected with pLNChRG and visualized >24 hours after plating on glass coverslips under 40× magnification using the FITC filter set.

FIGS. 5A-5B relate to fluorescence detection in hRGFP transduced A375 melanoma and NIH3T3tk− fibroblast cells. The photograph shows expression of hRGFP in human A375 melanoma and murine NIH3T3tk− fibroblast cells after transduction with the LNChRG retroviral vector. After transduction cells were selected in G418 for 2 weeks. Selected cells were trypsin digested and plated onto glass coverslips for viewing using the FITC filter set.

5 A: A375 melanoma cells transduced with LNChRG retroviral vector and visualized under 40× magnification.

5 B: NIH3T3tk− murine fibroblast cells transduced with LNChRG retroviral vector under 40× magnification.

FIGS. 6A-6D relate to FACS analysis of pLNChRG transfected PA317 vector producer cells or pLNChRG transduced human melanoma cells. The photographs show the determination of hRGFP activity in G418 selected, stable populations of mammalian cells. Cells were trypsin digested and washed before analysis in a EPICS Profile II Analyzer.

6 A: PA317 packaging cells without transfection (negative control).

6 B: LNChRG transfected PA317 vector producer cells expressing hRGFP gene activity after excitation. Large shift in peak detected mean fluorescence corresponding with hRGFP activity.

6 C: Nontransduced A375 cells demonstrate minimal detectable fluorescence (negative control).

6 D: LNChRG transduced A375 cells expressing hRGFP gene activity after excitation. Large shift in peak detected mean fluorescence corresponding with hRGFP activity. All FACS analysis used the FL1 emission channel used to monitor green fluorescence. Count: cell number counted at given fluorescence intensity; y-axis is a log scale of mean intensity of green fluorescence detected.

FIG. 7 is a depiction of the plasmid vector pHE700-hRGFP. “a” is a HSV-1 packaging signal; Hyg + is a hygromycin resistance gene; hRGFP is a humanized red shifted fluorescent protein gene; ENBA-1 is the Epstein-Barr virus gene encoding the EBV nuclear antigen EBNA-1; OriP is the Epstein-Barr virus unique latent replication origin; OriS is a HSV-1 replication origin.

FIGS. 8A-8C depict photographs of pHE 700-hRGFP virus stock which was used to infect VA 13 normal fibroblasts (FIG. 2 A), T98G human glioblastoma cells (FIG. 2B) and SV3 CRL9.7 xeroderma pigmentosum fibroblasts (FIG. 2 C).

FIG. 9 is a depiction of the pLNChG65T vector.

FIGS. 10 ( a )- 10 ( f ) are depictions of FACS analysis of hGFP-S65T-transduced PA317 vector producer cells and human melanoma cells. All FACS analyses used the FL1 emission channel used to monitor green fluorescence. Count: Cell number counted at given fluorescence intensity; log scale represents the mean intensity of green fluorescence detected at 525 nm. 10 ( a ) is PA317 packaging cells 98.4% negative (bar 2). 10 (B) PA317-LNChG65T Cell are 95.8% positive. 10 (C) A375 cells are 99.5% negative (bar 1). 10 (D) A375-LNChG65T cells are 98.7% positive (bar 2). 10 (E) IGROV cells are 98.9% negative (bar 1). 10 (F) IGROV-LNChG65T cells are 89% positive (bar 2).

FIGS. 11 ( a ) and 11 ( b ) are photographs of transduced PBL immediately after completion of the transduction procedure.

FIG. 12 is a graph depicted FACS analysis of PBL transduced with LNChG65T Vector. Dark bars show the percentage of fluorescing cells in transduced and control PBL populations at the end of the transduction procedure

FIGS. 13 ( a ) and 13 ( b ) are photographs depicting LNChG65T transduced PBL after g418 selection and expansion.

FIGS. 14 ( a ) and 14 ( b ) are graphs depicting the determination of activity of hRGFP in G418-selected PBL populations.

FIG. 15 is a Western Blot analysis of GFP in Peripheral Blood Lymphocytes. 1° Ab anti-GFP polyclonal antibody (1:2000 Clontech Lot #62038). 2° HRP anti-rabbit IgG (1:50000 Sigma Lot #A9189).

FIGS. 16 (A)-(E) is the sequence of the plasmid vector pLEL (SEQ ID NO:2).

›BRIEF DESCRIPTION OF THE DRAWINGS · 2 of 2

FIGS. 17 (A)-(F) is the sequence of the plasmid vector pLESN SEQ ID NO:3).

FIGS. 18 (A)-(F) is the sequence of the plasmid vector pLNCE (SEQ ID NO:5).

FIGS. 19 (A)-(F) is the sequence of the plasmid vector pLNChRG (SEQ ID NO:1).

FIGS. 20 (A)-(F) is the sequence of the plasmid vector PLTKOCEGFP (SEQ ID NO:6).

FIGS. 21 (A)-(F) is the sequence of the plasmid vector pLNChG65T (SEQ ID NO:4).

›DETAILED DESCRIPTION OF THE INVENTION · 1 of 4

The following definitions that follow will aid in a fuller understanding and exemplification of the invention.

As used herein, “DNA segment” refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the segment and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector. Such segments are provided in the form of an open reading frame uninterrupted by internal nontranslated sequences, or introns, which are typically present in eukaryotic genes. Sequences of non-translated DNA may be present downstream from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.

As used herein, “nucleotide sequence” refers to a heteropolymer of deoxyribonucleotides. Generally, DNA segments encoding the proteins provided by this invention are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon.

As used herein, “recombinant expression vector” refers to a transcriptional unit comprising an assembly of (1) a genetic element or promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.

A number of species use a bioluminescent GFP to emit green light after energy transfer from either luciferases or photoproteins. Prasher, D. C. (1995) Trends Genetics 11:320-323. The GFP gene product can function as a marker in living cells and animals and does not require a substrate (other than light) to visualize gene transfer. Chalfie, M., et al. (1994) Science 263:802-805. An excellent review of recent GFP applications has been provided by Prasher (supra).

The GFP cDNA is a 730 bp gene encoding a 238 amino acid polypeptide with a molecular weight of 27 Kd protein which has been cloned from the jellyfish, A. Victoria. Prasher, D. C., et al., (1992) Gene 111:229-233. GFP acts as an energy-transfer acceptor that under physiologic conditions in A. Victoria receives energy from an activated aequorin-coelenterazine complex. Cody, C. W., et al. (1993) Biochemistry 32:1212-1218. The chromophore is produced from autocyclization of three residues, serine-65, dehydrotyrosine-66, and glycine-67. Cody, C. W., et al. (1993) (supra). GFP protein is inactive until cyclization and oxidation of these three residues to generate a p-hydroxybenzylideneimadazolidinone chromophore (see FIG. 1 ). Molecular oxygen is required for fluorophore formation. Heim, R., et al. (1994) Proc Natl Acad Sci 91:12501-12504.

GFP was found to have extremely stable fluorescence in vitro after stimulation with blue light. Prasher, D. C., et al. (1992) (supra). Upon exposure to blue light, the protein emits a beautiful bold green light. Living eukaryotic cells expressing the protein may be visualized with the aid of a fluorescent microscope containing a GFP cube with excitation at 420-470 nm and emission at 490→600 nm. Cells may be returned to tissue culture without any apparent damage (Levy, unpublished results). There is no need for fixation, staining, antibodies, or drug selection, making GFP an extremely valuable tool for following gene transfer in living systems.

The GFP fluorphore can be column purified, renatured and crystallized and still maintain its fluorescent characteristics. Ward, W. W., et al. (1982) Biochemistry 21:4535-4540. These results prompted expression studies of wild-type GFP in prokaryotic and eukaryotic cells. These basic understandings of GFP mechanistic properties have led in turn to additional modifications to extend its usefulness to other systems.

Recently, a gain of function mutant GFP gene was generated that altered the serine-65 codon to a threonine codon resulting in a protein which fluoresces quicker with emission amplitudes from 4 to 6 fold greater than the wild-type gene. Heim, R., et al. (1995) Nature 373:663-664. This improved version of GFP has been termed the red shifted GFP because the excitation peak has shifted to the red zone of 470-490 nm. The emission remains green. Recently, more involved genetic modifications of the GFP sequence have been investigated.

A few investigators have “humanized” the wild-type codons to those used more commonly in mammals. Dr. Sergei Zolotukhin and Dr. Nicholas Muzyczka, University of Florida (unpublished results). A. Victoria is classified in the phylum Ctenophora and its codon usage is significantly different from mammals. Due to differences in codon usage, mammalian cells may not efficiently translate wild-type GFP transcripts. This latest version is termed the “humanized” red shift GFP(HRGFP).

A humanized GFP is available from Clontech, Inc. therefore (see page 29). Further, those of skill in the art will appreciate that other variations may be created so that human codon usage is proscribed to achieve the desired amino acid sequence. The inventors have now cloned the HRGFP into viral and retroviral systems to provide a means of easily following gene transfer which is also quite beautiful to the eye.

The structure and life cycle of retroviruses make them ideally suited to be gene-transfer vehicles since (i) the majority of sequences coding for their structural genes are deleted and replaced by the gene(s) of interest which are transcribed under control of the retroviral regulatory sequences within its long, terminal repeat region and (ii) they replicate through a DNA intermediate that integrates into the host genome. Although the sites of integration appear to be random with respect to the host genome, the provirus integrates with a defined structure in low copy number. Most of the viral gene sequences can function when supplied in trans. For general information regarding retroviral mediated gene transfer, see McLachlin, et al. (1990) Progress in Nucleic Acid Research and Molecular Biology 38:91-135.

›DETAILED DESCRIPTION OF THE INVENTION · 2 of 4

Retroviruses are viruses which carry their genetic information in the form of RNA. Once the virus infects a cell, the RNA genome is reverse-transcribed into the DNA form, which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus and the gene order within this provirus is always maintained. The retroviral genome and the proviral DNA have three genes: the gag, the pol and the env, which are flanked by two long terminal repeat sequences (LTRs). The gag gene encodes the internal structural (nucleocapsid) proteins, the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase), and the env gene encodes viral envelope glycoproteins. The 5′ and 3′ LTRs serve to promote transcription and polyadenylation of virion RNAs. Adjacent to the 5′ LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsulation of viral RNA into particles (the Psi site). Mulligan, R. C. (1984) Proceedings of the National Academy of Sciences, U.S.A. 81:6349-6353.

In order to generate a viral particle containing the recombinant genome, it is necessary to develop cell lines that provide packaging “help”. To accomplish this, a plasmid encoding for example, the retroviral structural genes gag, pol, and env, is introduced into an otherwise untransformed tissue cell line by conventional calcium-phosphate-mediated DNA transfection. These plasmid containing cells are referred to as a packaging cell line. This plasmid containing packaging cell line can be maintained as such or a replication incompetent retroviral vector can be introduced into the cell's genome. In the latter case, the genomic RNA generated by the vector proteins of the packaging line results in the release of retroviral particles into the culture medium. A stable cell line containing the structural gene sequences of the retroviruses is a retroviral “producer cell line.”

Because genes can be introduced into progenitor cells using a retroviral vector, they can be “on” (subject to) the retroviral vector control. In such a case, the gene of interest is transcribed from a retroviral promoter. A promoter is a specific nucleotide sequence recognized by RNA polymerase molecules that start RNA synthesis. Alternatively, retroviral vectors having additional promoter elements (in addition to the promoter incorporate in the recombinant retrovirus) which are responsible for the transcription of the genetic material of interest, can be used.

Almost all retroviral vector systems consist of two components: (i) the recombinant retroviral vector molecule that carries the gene(s) of interest and (ii) construct(s) providing retroviral structural proteins in trans. Together, these two components allow the production of recombinant viral particles capable of infecting target cells.

Retroviral vectors are derivatives of wild-type, replication-competent retroviruses in which part of the retroviral coding information (gag, pol, env) has been replaced by the gene(s) to be transferred to the target cell. Usually, these vectors contain at least two heterologous genes: (i) a marker gene that confers a selectable advantage upon infected cells, allowing their easy positive selection, and (ii) the therapeutic gene to be transferred. The two genes can either be expressed from the same retroviral promoter where the natural splicing mechanism of the retrovirus is utilized to generate different RNAs from which the gene products are separately expressed, or from different promoters, one from the retrovirus itself and one introduced along with the gene construct.

Recently, dicistronic retroviral vectors have been described in which two gene products are translated from one mRNA. See for e.g. Levine, et al. (1991), Gene 108:167-174. Dicistronic retroviral vectors have been reported to produce higher virus titers, to permit the insertion of larger genes, and to show more stable expression of transferred genes when compared to a two-gene, two-promoter vector. Id.

The present invention also contemplates the use of vehicles other than retroviruses to genetically engineer or modify cell lines. Genetic information of interest can be introduced by means of any virus which can express the new genetic material in such cells. Examples of such viruses include but are not limited to SV40, herpes virus, adenovirus, and human papilloma virus.

The first generation of packaging cell line is typified by the ψ-2 and ψ-am cell lines. Mann, et al. (1983) Cell 33:153-159; Cone, R. D., et al. (1984) Proc. Natl. Acad. Sci. 81:6349-6353. These cell lines contain a Mo-MuLV provirus that carries a simple deletion in the ψ packaging signal. This packaging construct gives rise to Mo-MuLV transcripts that direct the synthesis of authentic viral gag, pol, and env proteins, but the deletion in the ψ signal precludes the packaging of the genomic-length RNA into virion particles. Ecotropic virus is produced from ψ-2 cells, whereas the ψ-am cells produce amphotropic virus that shows an extended host range. Unfortunately, such cell lines still give rise to wild-type virus at a relatively high frequency because a single recombination event between the packaging construct and the vector construct, which of necessity must carry the ψ signal, will suffice to produce a wild-type genome. Miller, A., et al. (1986) Int. J. Cancer 37:173-177.

To reduce the risks of generating wild-type, replication-competent virus, a second generation of retroviral vector system carrying mutations in the LTR of the packaging construct has been constructed so that additional recombination events are required before a replication competent virus can be produced. The packaging cell line PA317 carries an amphotropic provirus, which in addition to the ψ region deletion, lacks part of the 5′ LTR and the 3′ LTR has been replaced with the polyadenylation signal from SV 40. Thus, at least two recombination events are required to generate replication competent virus.

›DETAILED DESCRIPTION OF THE INVENTION · 3 of 4

In gene therapy, the retroviral vector should only infect the cells in which the defect manifests itself and is thus involved in causing the disease. Thus, the expression of the introduced gene should be controlled by regulatory elements that target the expression to the relevant cell type. Replication-competent virus may have to be used for this approach, particularly if the cells of a multicellular organ or dense tumor mass are to be the target or if very high virus titers are required. Also, to prevent unsolicited replication of the retrovirus during or after gene therapy, vectors should be designed that can be conditionally silenced or inactivated.

Amphotropic MuLVs are able to infect cells of most species including rodents and humans. This observed tropism is determined by the env protein of the MuLV together with the availability of the corresponding receptor on the target cell. The env protein of ecotropic Mo-MuLV, gp70, interacts with a cationic amino acid transporter, which serves as the host cell receptor and is expressed in many tissues with the notable exceptions of liver, heart, and muscle. Kim, et al., (1991) Nature 352:725-728.

It has also been shown that it is possible to limit the infection spectrum of the Mo-MuLV virus and consequently of Mo-MuLV-based vectors. One approach involves the coupling of antibodies, directed against known proteins that are expressed on the surface of the target cell, to antibodies specific for the virus env protein via streptavidin.

A second strategy for targeting the infection spectrum of retroviral vectors involves the chemical coupling of ligands to the viral env proteins. Viral env proteins can be artificially converted to asialoglycoproteins by coupling them to lactose. Neda, et al., (1991) J. Biol. Chem. 266:14143-14146.

Another means of targeting the infection spectrum involves the co-expression of other ligands on the virus surface along with the normal env SU proteins. This strategy would allow normal viral internalization and should also result in a favored uptake by cells expressing the receptor for the co-expressed ligand.

The above disclosure reveals that the GFP has an enormous potential in the field of retroviral and viral gene therapy. Constructs using the wild-type gene in other aspects of biology have already proven to be valuable. Until now, these constructs have proven to be limited in retroviral vectors due to the low levels of expression in stable cell lines. The HS65T has overcome this barrier and provided an astounding display of bioluminescence.

The key to expression seems to lie in the 3 amino acid residues which form a cyclized chromophore. Specifically, the serine at position 65 is a site at which several amino acid replacements show increased intensity and quicken the rate of chromophore formation. The red shift may be responsible for advancing expression to this level. However, humanizing of the coding sequences seems to be providing the dominant contribution. Jellyfish are extremely divergent from mammals and consequently have different codon usage. This may present a translation challenge for mammalian cells of enough proportion to prevent a build up of detectable GFP. The HGS65T GFP gene contains 169 codon changes (Clonetech) representing 71% of the gene. The above results show that this is a superior marker gene to follow retroviral and viral transduction.

In transducing and subsequent selection, it can be determined whether the cells have completed selection or not. In addition, it is possible to determine transduction efficiency in a variety of cell lines. It is expected that the satisfactory expression of GFP is achievable in many other types of mammalian cell lines, including other types of human tumor lines, based on similarities in cell morphology and physiology. Such cell lines can be readily ascertained by those of ordinary skill in the art.

In vivo retroviral transduction experiments may also take advantage of this marker system. PA317-HGS65T VPC cells injected into an established subcutaneous tumor cell will transduce the dividing tumor cells. VPC's used in black mice systems will be destroyed by the immune system within 2 weeks after injection. The F.A.C.S. sorting capabilities also opens up some doors of opportunity for leukemia trials. Overall, this evidence demonstrated that humanized red shift GFP has the potential of becoming a major player in gene therapy.

The instant invention demonstrates the effectiveness of a humanized, red shifted mutant GFP by retroviral and viral mediated gene transfer into human tumor cells and murine fibroblasts. A few molecular genetics groups have now reported mutations of the wild-type GFP gene which can generate GFP gene products with modified excitation and emission spectra. See for e.g. Heim, R., et al. (1994) Proc Natl Acad Sci. USA 91:12501-12504. The longer wavelength excitation peak (475 nm) of native A. Victoria GFP has lower amplitude than its shorter wavelength excitation peak (470-490 nm) with fluorescence amplitudes from 4-6 fold greater than from the wild-type gene product. Heim, R. (1995) Nature 373:663-664. Interestingly, this mutant also had more rapid formation of the fluorochrome. Id. Furthermore, the mutated, red shifted GFP had its codons modified to usage common in mammals (Dr. Sergei Zolotukhi and Dr. Nicholas Muzyczka, University of Florida, unpublished results). The inventors have evaluated this humanized version of a serine-65 to threonine codon mutant that demonstrates excitation at 490 nm and emissions at 510 nm in current gene transfer experiments. Comparisons between the wild-type GFP and the humanized, serine-65 red shifted mutant (hRGFP) demonstrated substantial improvement in fluorescence expression after either transfection and retroviral mediated GFP gene transfer (Table 1).

Wild-type GFP alone and GFP containing fusion proteins have been demonstrated in mammalian cells to provide efficient marking of protein trafficking and gene expression. GFP has also been fused to chromogranin B and fluorescence could be observed in the Golgi apparatus after temperature block at 15° C. Kaether, C., et al. (1995) FEBS Lett. 369:267-271. Fluorescence can also be detected after the transient transfection of wild-type GFP, GFP fused to the PML proto-oncogene product, or GFP fused to a human glucocorticoid receptor expressed in COS-1 cells, chicken embryonic retinal cells, and EPC cells (carp). Ogawa, H., et al. (1995) Proc Natl Acad Sci. USA 92:11899-11903. Native GFP has also been fused to the microtubule associated protein 4 (MAP 4) gene and permitted the tracking of subcellular reorganization of cytoskeletal elements. Olson, K.R., et al. (1995) J Cell Biol. 130:639-650. Wild-type GFP has been fused to the N-methyl-D-aspartate (NMDA) receptor subunit green fluorescence. Marshall, J., et al. (1995) Neuron 14:211-215. Fluorescence was detected in cells transfected with GFP alone. The investigators demonstrated NMDAR-1 gene activity by patch clamp analysis after fusion gene transfer. Id. These results demonstrate that the transient transfection of wild-type GFP or chimeric proteins containing GFP can function as an excellent intracellular marker in living cells for proteins and can be used to visualize cell organelle. Of note, these experiments used transient transfection to obtain very high levels of wild-type GFP expression. The present disclosure is in agreement with these results in that transient transfection which transfers multiple transgene copies of wild-type GFP expression cassettes were easily visualized, but the inventors found that stable transduced cells with single transgene copy of wild-type GFP could never be visualized by fluorescence microscopy (Table 1). However, the present invention demonstrates that a humanized, red shifted GFP transgene in single copy can produce excellent fluorescence (FIG. 5 ).

›DETAILED DESCRIPTION OF THE INVENTION · 4 of 4

Transgenic mice have been produced expressing a slightly modified wild-type GFP expressed from the chicken β-actin promoter. Ikawa, M., et al. (1995) Develop Growth Differ. 37:455-459. The fingers or tails of these transgenic mice were distinguishable as green under a fluorescent microscope and homogenized tissue from the muscle, pancreas, lung, and kidney demonstrated fluorescence after excitation with 490 nm light. The visualization of vector gene expression in living transduced tissues with hRGFP may become an outstanding method for studying in vivo gene transfer used in human clinical trials.

The present invention demonstrates the ability to analyze and easily detect living, retroviral transduced cells without the need for cell fixation or antibodies. This allows for the clinical application of this marker in living tissue by using the appropriate enhancer/promoter or targeted transduction procedure. This may be particularly useful in bone marrow processing, lymphocyte sorting, and other applications requiring the FACS analysis of living cells. Further, since the GFP gene has not been shown to have any cytotoxicity in vitro, it is expected that the GFP gene will be safe for in vivo use. Future directions will lead to a wider range of useful GFP based mutant proteins with well defined fluorophores with characterized excitation and emission spectra, using mammalian codon usage. For example, Heim and colleagues subjected the native GFP sequence to random mutations in bacteria and developed a series of interesting mutants with altered excitation peaks and found that a substitution of Tyrosine-66 to histidine generated a mutant GFP that demonstrated blue fluorescent emission. Heim, R., et al. (1994) Proc Natl Acad Sci, USA 91:12501-12504. This may permit two color separation and analysis of living cells by FACS.

According to the invention retroviral vectors have been prepared which comprise a humanized, red shifted green fluorescent protein (hrGFP) transcription unit comprising a promoter, a gene which encodes humanized red-shifted green fluorescent protein, and a termination or polyadenylation signal sequence. Optionally the transcription unit may contain enhancer elements.

The transcription unit is a part of a retroviral vector construct, a DNA or RNA segment which comprises a viral packaging sequence and optionally a viral long terminal repeat sequence.

In a preferred embodiment the construct also comprises a selectable marker gene such as an antibiotic resistance gene including the ampicillin resistance gene, tetracycline resistance gene, neomycin resistance gene, or any other resistance gene known to those of skill in the art. See generally Maniantis “Molecular Cloning” 2nd ed., Cold Spring Harbor Press (1989) the disclosure of which is incorporated by reference. The selectable marker gene can be embodied within the GFP transcription unit, can be a separate transcription unit, or can include one or more components of the GFP transcription unit.

In a most preferred embodiment the construct comprises the elements depicted in FIG. 2, the pLNChRGFP, pLNCRGFP, pLhRGFP, pLRGFP, pLhRGFPL, plRGFPL or pLNChRGFP-B constructs.

The components described herein can be arranged in any order and vectors having the same components in a different order are intended to be within the scope of the invention. The only requirement is that transcription units including a gene the expression of which is desired must include a promoter and a termination signal in functional arrangement to the gene to be expressed after the sequence. A transcription unit can comprise more than one coding gene or can contain a second transcription unit within a larger unit that may share a common termination signal or promoter sequence. The vectors of the invention provide for stable long term transfection to recipient cells with detectable fluorescence with only single copy expression. This provides for a number of protocols including fluorescence activated cell sorting (FACS) for separation of transfected from nontransfected cells for ex vivo gene therapy techniques, which cells are harvested, transformed and then re-introduced. One such method involves bone marrow purging with transformation of lymphocytes as shown in the Examples section. Any ex vivo gene therapy application which involves selection of successfully transformed cells is applicable.

The following examples describe the cloning and characterization of ecotrophic and amphotropic retroviral vectors capable of demonstrating efficient, stable transfer of an hRGFP gene into mammalian cells. They are for informational purposes only and are not intended to limit the scope of the present invention in any manner.

›Examples14

›EXAMPLE 1 · 1 of 2

Retroviral Mediated Gene Transfer into Human Melonoma Tumor Cells and Murine Fibroblasts

Experimental Protocol

Cell Culture. A375 is a human melanoma cell line (ATCC, Manassas, Va.). PA317 is a murine amphotropic, retroviral vector packaging cell line (kindly provided by A. D. Miller, University of Washington). Cells. were grown in RPMI supplemented with 10% fetal calf serum (FCS) (all obtained from Gibco BRL) in monolayers at 37° C. and 5% CO 2 . All cells were passaged and harvested by standard trypsin (Gibco BRL) digestion at 37° C. Cells were routinely passaged at 80-90% confluence.

Plasmid preparation and digoxin probes. Plasmid pGFP-Cl containing wild-type GFP was obtained from Clontech (Palo Alto, Calif.). The plasmid pTR-UF2 containing the humanized red, shifted GFP (hRGFP) gene was kindly provided by Dr. Sergei Zolotukhin and Dr. Nicholas Muzyczka (University of Florida). Construct plasmid DNA was transformed into DH5a competent cells and colonies grown on L-broth supplemented with ampicillin (50 μg/ml) plates (LB/AMP) and transferred onto nylon membranes. The membrane was proved with a Dig-GFP probe using a digoxin probe kit (Boehringer-Mannaheim). Primers for the Dig-GFP probe amplification of a GFP fragment were 5′ primer 5′ GGG AAG CTT TTA TTA TTT GTA TAG TTC ATC CAT GCC (SEQ ID NO:7) and 3′ primer 5′ GGG AAG CTT GCG CGT ATG GGT AAA GGA GAA GAA CTT (SEQ ID NO:8). Positive colonies were grown up in LB/AMP broth and plasmid DNA was isolated using the Qiagen plasmid prep kits (Qiagen Corp., Chatsworth, Calif.).

Construction of GFP retroviral vectors. Primers were made to amplify the 5′ end of the CMV promoter/enhancer and the 3′ end of the wild-type GFP gene from the GFP-C1 vector (Clontech, Palo Alto, Calif.). The 5′ primer includes unique Xba I, Bam HI, and Not I restriction enzyme sites: 5′ GGA TCT AGA GGA TCC GCG GCC GCC TAG TTA TTA ATA GTA ATC AAT TAC GGG GTC (SEQ ID NO:9). The 3′ primer includes 3 in frame stop codons followed by a Hind III restriction enzyme site: 5′GGA AAG CTT CTA TCA TTA TTG AGC TCG AGA TCT GAG TCC GGA CTT GTA (SEQ ID NO:10). The 1.3 Kb CMV-GFP PCR product was cloned into PCR3-cloning vector (Invitrogen) to generate plasmid pPCR3CG-14. The 1.3 Kb fragment containing the CMV promoter and GFP gene was gel isolated (Jetsorb, Genomed) from the pPCR3CG-14 vector using Bam HI and Hind III restriction digest. The 800 bp retroviral CMV promoter was directionally cloned to generate the pLNCG construct. Finally, plasmid pTR-UF2 was restriction digested with Not I and the 730 bp DNA fragment containing the humanized red shift GFP open reading frame was isolated. After Klenow treatment, the blunt ended DNA was ligated into pLNCX at the Hpa I site. The resulting plasmid was designated pLNChRG.

Fluorescent detection of green fluorescent protein expressing cells. We visualized GFP expressing cells with a Nikon Labophot-2 Fluorescent Microscope (Fryer Company, Inc.). The cube used in the microscope was either the Green Fluorescent Protein Longpass 41015 filter set (excitation at 420-470 nm and emission at 490 to >600 nm) for the wild-type GFP detection (Chroma Technology Corporation) or the FITC dichromic filter set (excitation at 450-490 nm and emission at 520 nm) for the hRGFP detection (Fryer Company, Inc.). Photographs were taken using the Nikon Microflex UFX-DX and AFX-DX systems (Fryer Company, Inc.).

Transient expression of GFP. PA317 cells were seeded on a sterilized coverslip in a 6 well dish 12-24 hours before transfection. Cells were at 30-50% confluence at the time of DNA transfection. Five μg of DNA and 15 μl of DOTAP reagent (Boehringer Mannheim) was used as per the manufacturer's protocol. The mixture was added to the plates containing either RPMI 1640 with 10% FBS, L-glutamine, and penicillin/streptomycin or in serum free media. After 10-18 hours the media was replaced with RPMI with 10% FCS. The coverslip containing the cells was placed on a slide and examined for fluorescence 9-48 hours after transfection. The cells remaining in the well (after the coverslip was removed) were trypsin digested and transferred to tissue culture dishes. After attachment these cells were placed under selection with G418 (1 mg/ml) for 10-14 days.

Construction, subcloning, and titering of LNCG and LNCHRG vector producer cell lines. The plasmid pLNCG or pLNChRG were transfected with DOTAP into the amphotropic retroviral packaging line PA317. Twenty-four hours later, the cells were placed under selection with G418 (1 mg/ml) for 2 weeks. LNCG or LNChRG VPC were grown to approximately 90% confluence and supernates were removed to transduce A375 target cells. Retroviral supernates were filtered through 0.45 μm filters (Nalgene), supplemented with 10 μg/ml of protamine sulfate (Elkins-Sinn) and used to transduce A375 melanoma or NIH3T3tk− fibroblast cells. The target A375 melanoma or NIH3T3tk− cells were 40-60% confluent when transduced. Twenty-four hours after the final transduction cells were placed under G418 (1 mg/ml) selection for 2 weeks. Cells were examined under the fluorescence microscopy after reseeding the cells onto glass cover slips.

Fluorescence activated cell sorter analysis of transduced human cells. Cytometry of stable hRGFP transfected or transduced cells was performed on a Epics Profile II analyzer. Cells were analyzed using a 525 nm band pass filter set (Part #814036, Coulter Corp.). Cultures of nontransfected PA317 cells, LNChRG transfected PA317 cells, nontransduced A375 cells, or LNChRG transduced A375 cells that were 80-90% confluent were trypsin digested washed with RPMI with 10% FCS and resuspended at a concentration of approximately 1×10 6 cells/ml. All FACS analysis used the FL1 emission channel to monitor green fluorescence (normally a FITC monitor).

Transfected cell lines. PA317 retroviral packaging cells and A375 melanoma cells were transiently transfected with pLNCG or pLNChRG plasmids (FIG. 3 ). These two constructs led to significantly different levels of fluorescence after transient transfection (Table 1). Nontransfected PA317 cells did not demonstrate green fluorescence (FIG. 3 A). Transfected cells containing the wild-type GFP gene (pLNCG) exhibited a fluorescence in <2% of the cell population that was detected after 48 hours (FIG. 3 B). However, once the humanized, red shift GFP retroviral construct (pLNChRG) was transfected the results were outstanding. Fluorescence can be detected as early as 9 hours post-transfection. By 36 hours, 30-40% or more of the cells are easily visualized, and contain enough protein to produce an intense fluorescence (FIG. 3 C). Overall, with the fluorescence detection filters used, pLNChRG transfected cells had enhanced fluorescent intensity and efficiency compared to cells transfected with wild-type GFP plasmid (pLNCG). The inventors did not observe any cytopathic or growth inhibiting effect due to GFP or hRGFP in transfected cells.

›EXAMPLE 1 · 2 of 2

Stable LNCG and LNChRG retroviral vector producer cells. Stable LNCG or LNChRG PA317 VPC were generated by lipofection with the pLNCG or pLNChRG plasmids, respectively. Transfected PA317 cells were selected in media containing G418 (1 mg/ml). These stable LNCG or LNChRG PA317 VPC were examined by fluorescence microscopy. The LNCG PA317 VPC line that contains the wild-type GFP gene demonstrated no fluorescence after excitation with 420-470 nm light (data not shown). The inventors therefore analyzed the LNCG VPC line by PCR using GFP amplifiers to detect host chromosomal integration of the LNCG vector. The GFP gene was present in all lines tested, despite the fact that no fluorescence occurred (data not shown). In striking contrast, the LNChRG PA317 VPC line demonstrated vibrant green fluorescence in nearly 100% of the cells after G418 selection (FIG. 4 ). The intensity of fluorescence in the LNChRG VPC line was capable of highlighting many subcellular organelles.

Detection in LNChRG transduced A375 melanoma and NIH3T3tk-transduced cell lines. Supernates from cultures of LNCG or LNChRG PA317 VPC were collected when the cells were 90-100% confluent. Supernates were filtered and transferred into tissue culture plates containing A375 melanoma cells or NIH3T3tk-cells. Twenty-four hours after the final exposure to retroviral supernates, the target cells were placed under selection with media containing G418. With this gene transfer system, most cells will contain only one integrated copy of the retroviral vector (C. Link, unpublished results). A375 cells transduced by LNCG VPC demonstrated no evidence of fluorescence despite the fact that PCR revealed the presence of GFP in the cellular genome (data not shown). However, the neo r gene transferred by the LNCG vector was functional, since the A375 cell colonies were G418 resistant. In contrast, the LNChRG vector transduced A375 melanoma cells had bright fluorescent activity (FIG. 5 A). Similarly, murine NIH3T3tk− fibroblasts transduced with the LNChRG retroviral vector demonstrated strong fluorescence in nearly 100% of the cells (FIG. 5 B). The inventors did not observe any cytopathic or growth inhibiting effect due to GFP or hRGFP in transduced cells.

FACS analysis of GFP transfected PA317 vector producer cells and LNChRG transduced A375 melanoma cells. PA317 cells that had been transfected by the LNChRG vector and selected were analyzed by FACS. Excitation with 488 nm light resulted in light emissions at 525 nm in LNChRG containing cells. PA317 cells transfected and expressing hRGFP (FIG. 6B) were easily detected by a two log shift from nontransfected control PA317 cells (FIG. 6 A). Using the band pass filter, A375 melanoma cells transduced and selected with the LNChRG vector (FIG. 6D) were readily detected after excitation by a two log shift in detected fluorescence compared to the control nontransduced A375 cells (FIG. 6 C).

These results demonstrate the effectiveness of a humanized, red-shifted mutant GFP by retroviral mediated gene transfer into human tumor cells and murine fibroblasts.

›EXAMPLE 2 · 1 of 2

Retroviral Transduction GFP into Human Melanoma, Ovarian Carcinoma and Murine Fibroblasts

Experimental Protocol

Cell culture. A375 is a human melanoma cell line (ATCC, Manassas, Va.). PA317 is a murine amphotropic, retroviral vector packaging cell line (kindly provided by A. D. Miller, University of Washington). GPE86 is a murine ecotropic, retroviral packaging cell line (kindly provided by Arthur Banks). IGROV is an ovarian carcinoma cell line (kindly provided by Patrick Hwu). Cells were grown in RPMI supplemented with 10% fetal calf serum (FCS) (all obtained from Gibco BRL) in monolayers at 370C and 5% CO 2 . Retroviral vector producer cells (VPC) were grown in RPMI with 10% FCS in monolayers at 37° C and 5% CO 2 . All cells were passaged and harvested by standard trypsin (Gibco BRL) digestion at 37° C. Cells were routinely passaged at 80-90% confluence.

Plasmid preparation. Plasmid phGFP-S65T was obtained from Clontech (FIG. 7 and Table 2) (Palo Alto, Calif.). The plasmid was kindly provided by Dr. Paul Kitts and Dr. Steve Kain. Cloned construct plasmid DNA was transformed into DH5α competent cells and colonies grown on L-broth supplemented with ampicillin (50 μg/ml) plates (LB/AMP). Positive colonies were grown up in LB/AMP broth and plasmid DNA was isolated using the Qiagen plasmid prep kits (Qiagen Corp., Chatsworth, Calif.).

Construction of GFP retroviral vector. Plasmid phGFP-S65T was restriction digested with Hind III and Not I. The 750 bp fragment containing GFP was gel isolated (Jetsorb, Genomed). Plasmid pLNCX was restriction digested with Hind III and Hpa I. The GFP fragment was then ligated into pLNCX followed by a klenow reaction to blunt the 3′ end of the GFP. A second ligation was then performed to ligate the 3′ end into the Hpa I site to produce plasmid pLNChGS65T. Positive clones drop out a 300, 500, and 600 bp fragment following restriction digest with Ban HI and Nco I.

Transient expression of GFP. PA317 cells were seeded on a sterilized coverslip in a 6 well dish 12-24 hours before transfection. Cells were at 30-50% confluence at the time of DNA transfection. Five μg of DNA and 15 μl of DOTAP reagent (Boehringer Mannheim) was used as per the manufacturer's protocol. The mixture was added to the plates containing RPMI 1640 with 10% FBS, L-glutamine, and penicillin/streptomycin. After 10-18 hours the media was replaced with fresh RPMI media. The coverslip containing the cells was inverted, placed on a slide and examined for fluorescence 9-48 hours after transfection.

Production of the vector producer cell line—PA317-HGS65T VPC. GPE86 cells were seeded in a 6 well dish 24 hours before transfection. Cells were at 30-50% confluence at the time of DNA transfection. Five μg of DNA and 15 μl of DOTAP reagent (Boehringer Mannheim) was used per the manufacturer's protocol. The mixture was added to the dishes containing RPMI 1640 with 10% FBS, L-glutamine, and penicillin/streptomycin. After 12 hours the cells were rinsed once and 1 ml of RPMI media was placed in each well. Retroviral supernate was collected 24 hours later, sterile filtered and 10 μg/ml protamine sulfate solution was added. This supernate was transferred to a 6 well dish containing PA317 cells which were at 30-50% confluence. After an additional 24 hours cells from each well were trypsin digested and transferred to a 10 cm tissue culture dish (Falcon). G418 (1 mg/ml) was added after another 24 hours for 10-14 days.

Transduction of A375 and IGROV cell lines. PA317-HGS65T VPC cells were grown to 80-90% confluency in a T175 flask. The media was then replaced with 20 ml fresh media. 24 hours later the retroviral supernate was collected, filtered (0.45 μm costar) and supplemented with 10 μg/ml protamine sulfate. A375 and IGROV cells were seeded in a 10 cm tissue culture dish and in 6 well dishes containing sterile coverslips. 10 ml of supernate was added to the culture dish, and 2 ml was added to each well. 24 hours later the supernate was removed and replaced with G418 (1 mg/ml). Coverslips were removed 72 hours post transduction and later for fluorescent microscopy and photography.

Fluorescent detection of green fluorescent protein expressing cells. The inventors visualized GFP expressed cells with a Nikon Labophot-2 Fluorescent Microscope (Fryer Company, Inc.). The cube used in the microscope was the FITC dichromic filter set (excitation at 450-490 nm and emission at 520 nm) for the hRGFP detection. Photographs were taken using the Nikon Microflex AFX-DX systems (Fryer Company, Inc.).

Fluorescence activated cell sorted analysis of and transduced cells. Cytometry of transduced cells was performed on a Epics Profile II Analyzer. Cells were analyzed using a 525 nm band pas filter set (Part #814036, Coulter Corp.). Cultures of nontransduced PA317, A375, and IGROV cells, as well as transduced PA317, A375, and IGROV cells that were 80-90% confluent were trypsin digested washed with RPMI with 10% FCS and resuspended at a concentration of approx. 1×10 6 cells/ml. All FACS analysis used the FL1 emission channel to monitor green fluorescence (normally a FITC monitor).

Results

Transfected cell lines. PA317 retroviral packaging cells were transiently transfected with pLNChGS65T. The construct fluoresces at many different levels. Nontransfected PA317 cells did not demonstrate green fluorescence. Fluorescence can be detected as early as 9 hours post-transfection. By 36 hours, 30-40% or more of the cells are easily visualized, and contain enough protein to produce an intense fluorescence. We did not observe any cytopathic or growth inhibiting effect due to pLNChGsS65T in transfected cells.

Transduced expression of GFP. After selection nearly 100% of PA317-HGS65T VPC were fluorescing with great intensity comparable to that of the transfected cells. A375 and IGROV cells were examined for fluorescence 72 hours post transduction for transduction efficiency. Both cell lines appear to be 50% fluorescent. After only 7 days of selection, A375 cells were at approximately 90% fluorescence. 75% of the total IGROV cells were fluorescent at 4 days post selection.

›EXAMPLE 2 · 2 of 2

FACS analysis of GFP transduced cell lines. PA317, A375, and IGROV cells that had been transduced by LNCHGS65T VPC and selected were analyzed by FACS. Emission was detected at 525 nm. PA317-HGS65T were easily distinguished by a two log shift from nontransfected control PA317 cells by FACS analysis. Using the same 525 nm band pass filter, A375 melanoma cells transduced, not selected were readily detected after excitation with 488 nm light by a two log shift in detected fluorescence compared to the control nontransduced A375 cells IGROV cells transduced, not selected were readily detected after excitation with 488 nm light by a two log shift in detected fluorescence compared to the control nontransduced IGROV cells. These results demonstrate that GFP gene fluorescence can be quantified with available instrumentation.

›EXAMPLE 3

Tracking and Quantitation of Retroviral-Mediated Transfer Using a Completely Humanized, Red-Shifted Green Fluorescent Protein Gene

Cell Culture. The following cell lines were used: A375, a human melanoma cell line (ATCC, Manassas, Va., USA); IGROV, an ovarian carcinoma cell line (kindly provided by Patrick Hwu, National Cancer Institute); PA317, a murine amphotropic, RV-packaging cell line (kindly provided by A.D. Miller, University of Washington); and GPE86, a murine ecotropic, RV-packaging cell line (kindly provided by Arthur Banks, Columbia University). Cells were grown in RPMI supplemented with 10% fetal calf serum (FCS) (both obtained from Life Technologies, Gaithersburg, Md., USA) in monolayers at 37° C. and 5% CO 2 . All cells were passaged and harvested by standard trypsin (Life Technologies) digestion at 37° C. Cells were routinely passaged at 80%-90% confluence.

Construction of GFP Retroviral Vector. Plasmid phGFP-S65T was obtained from CLONTECH Laboratories (Palo Alto, Calif., USA). Cloned construct plasmid DNA was transformed into MAX Efficiency DH5α™ Competent Cells (Life Technologies), and colonies were grown on L-broth supplemented with ampicillin (50 μg/mL) plates (LB/AMP). Positive colonies were grown in LB/AMP broth, and plasmid DNA was isolated using the plasmid kit from Qiagen (Chatsworth, Calif., USA). Plasmid phGFP-S65T was restriction-digested with HindIII and NotI to obtain the 750-bp hGFP-S65T open reading fragment, which was then gel-isolated (Jetsorb©; Genomed, Raleigh, N.C., USA). Plasmid pLNCX was restriction-digested with HindIII and HpaI. The hGFP-S65T fragment was then ligated into linearized pLNCX, followed by treatment with a Klenow enzyme to remove the NotI overhang. This 3′ end was then ligated to the HpaI site to produce plasmid pLNChG65T.

Construction of the LNChG65T Vector-Producer Cell Line. GPE86 cells were plated in a 6-well dish (Falcon®) 24 hours before transfection. Cells that were at 30%-50% confluence were transfected with 5 μg of pLNChG65T plasmid DNA and 15 μL of DOTAP reagent (Boehringer Mannheim, Indianapolis, Ind., USA) according to the manufacturer's protocol. After 12 hours, the cells were rinsed, and 1 mL of fresh medium was placed in each well. RV supernatant was collected 24 hours later and filtered (0.45 μm; Corning Costar, Cambridge, Mass., USA); then 10 μg/mL of protamine sulfate solution were added. This supernatant was transferred to a 6-well dish containing PA317 cells that were at 30%-50% confluence. After 24 hours, both cell lines were trypsin-digested and transferred to a 10-cm tissue culture dish (Falcon). Cells were selected in 1 mg/mL G-418 (Genteticin®; Life Technologies for 10-14 days.

Transduction of A375 and IGROV Human Tumor Cell Lines. LNChG65T VPC cells were grown to 80%-90% confluence in Nunclon™ T-175 flasks (Allegiance Health Care, McGaw Park, Ill., USA). The medium was replaced with 30 mL of fresh medium, and 24 hours later the RV supernatant was collected, filtered and supplemented with 10 μg/mL protamine sulfate. A 375 and IGROV cells were plated onto 10-cm tissue culture dishes and in 6-well dishes containing sterile coverslips. Ten milliliters of the supernatant were added to the culture dishes, and 2 mL were added to each well of the 6-well plates. The supernatant was removed 24 hours later and replaced with fresh medium containing G-418 (1 mg/mL) and selected for 7-14 days.

Fluorescent Detection of GFP-Expressing Cells. The hGFP-S65T-expressing cells were visualized with a Nikon Labophot-2™ fluorescent microscope (Melville, N.Y., USA) . The filter cube used in the microscope was the FITC dichroic filter set (excitation at 450-490 nm and emission at 520 nm). The coverslips from the 6-cell dishes were inverted and placed on a glass slide for viewing. Photographs were taken using the Microflex™ AFX-DX systems (Nikon).

Fluorescence-Activated Cell Sorter (FACS) Analysis of LNChG65T-Transduced Mammalian Cells. Cytometry of transduced cells was performed on an EPICS® Profile II analyzer (Coulter, Miami, Fla., USA) with an excitation source of 488 nm. Cells were analyzed using a 525-nm bandpass filter set (Part No. 814036; Coulter). Cultures of cells that were 80%-90% confluent were trypsin-digested, washed with RPMI with 10% FCS and resuspended at a concentration of approximately 1×10 6 cells/mL. All FACS analyses used the FL1 emission channel to monitor green fluorescence (normally at FITC monitor).

Results

Transfected Cell Lines. GPE86 RV-packaging cells were transfected with the construct pLN-ChG65T (FIG. 9 ). Once selected, nearly 100% of the cells emitted green fluorescence after excitation (data not shown). No cytopathic or growth-inhibiting effects due to the expression of pLNChG65T in transfected cells were observed.

Transduced Expression of GFP. The LNChG65T vector-producer cell line (VPC) exhibited intense fluorescence. A375-LNChG65T-transduced cells, examined 72 hours after transduction, demonstrated fluorescence in approximately 50% of the cells. After 7 days of G-418 selection, approximately 90% of the A375 (data not shown) and 70% of the IGROV cells demonstrated fluorescent activity. By 14 days after selection, both of the transduced cell lines demonstrated nearly 100% fluorescence. No significant background fluorescence was detected in nontransduced cells.

FACS Analysis of GFP-Transduced Cell Lines. PA317, A375 and IGROV cells transduced by the LNChG65T RV vector and selected in G-418 were analyzed by FACS. The PA317- and A375-transduced Cells (FIGS. 10B and D) were distinguished by up to a three log shift in mean fluorescent intensity in comparison to nontransduced control cells (FIGS. 10 A and C). Transduced IGROV cells also showed a significant shift in fluorescence intensity.

›EXAMPLE 4 · 1 of 9

Construction of a Vector Producer Cell Line. The hG65T gene was cloned into the retroviral vector pLNCX to produce the pLNChG65T vector (FIG. 9 ). The vector DNA was transfected with DOTAP into the ecotropic retroviral packaging cell line GP-E86. The supernatant from transfected GP-E86 cells was transferred onto murine amphotropic packaging cell line PA317. 24 hours later, cells were placed under G418 selection for two weeks. The fresh supernatants from LNChG65T vector producer cell line were used to transduce PBL.

PBL Transduction. Human peripheral blood mononuclear cells were isolated from health donors by density gradient centrifugation. Cells were cultured in RPMI 1640 medium, supplemented with 5% human AB serum in the presence of PHA at 1 μg/ml and hIL-2 at 100 U/ml. After 72 hours of incubation, cells were washed and cultured in phosphate-depleted RPMI 1640 medium containing 500 u/ml hIL-2 for 12 hours. PBL were pelleted and resuspended in supernatant from LNChG65T vector producer cells containing 500 U/ml hIL-2 and 10 mg/ml protamine. After 12 hours of incubation at 37° C., 5% CO 2 , the phosphate-depletion-transduction cycle was repeated twice.

GFP expressing PBL were visualized 24-72 hours after transduction (FIG. 11 ). Immediately after the completion of transduction procedure PBL population was analyzed for GFP expression and phenotypes using FACS (Epics Profile II Analyzer, standard FITC filter). The population of PBL cultured under the same conditions, but not exposed to viral supernatant served as a control.

The results of FACS analysis show that about 30% of PBL population incubated with LNChG65T viral supernatant expressed GFP (FIG. 12) and the ratio of cells with different phenotypes was not affected by the transduction procedure (35.0% CD4 + , 59% CD8 + cells).

Transduced PBL were placed under G418 selection for 7 days and then expanded for 5 days in RPMI 1640 containing 5% human serum and 500 U/ml IL-2.

Microphotographs and FACS analysis of G418-selected PBL population are shown in FIGS. 13 and 14. Western analysis demonstrated high levels of GFP protein produced in the transduced cells (FIG. 15)

Alloreactivity of LNChG65T Transduced PBL. The capacity of LNChG65T transduced PBL to respond to an immunological stimulus was tested. The assay used the incorporation of [ 3 H]-thymidine to determine the proliferation rates after stimulation with irradiated LCL/HA lymphoblastoid cells or irradiated, pooled human peripheral blood mononuclear cells (PBMC). Control and transduced PBL cultures were washed twice and incubated in medium without IL-2 for 12 hours prior to stimulation with allogeneic cells. The cell mixtures were cultured in humidified 5% CO2 at 37° C. for 3 days and pulsed with 1 μCi of [ 3 H]-thymidine for the last 18 hours.

Incorporated radioactivity was measured by scintillation counting and was expressed as cpm of radioactivity.

These results demonstrate that transduced peripheral blood lymphocytes remain alloreactive compared to non-transduced control cells.

Conclusion. The results of this study show that the GFP gene can be successfully transferred and stably expressed at high level in human PBL by means of retroviral vector delivery system. GFP expression does not affect the capacity of PBL to respond to an immunological stimulus.

The ability to efficiently sort transduced, living human lymphocytes without prolonged drug selection will allow clinical investigation into a variety of therapeutic gene transfer and marking studies in humans.

GAATTCATAC CAGATCACCG AAAACTGTCC TCCAAATGTG TCCCCCTCAC ACTCCCAAAT 60

TCGCGGGCTT CTGCCTCTTA GACCACTCTA CCCTATTCCC CACACTCACC GGAGCCAAAG 120

CCGCGGCCCT TCCGTTTCTT TGCTTTTGAA AGACCCCACC CGTAGGTGGC AAGCTAGCTT 180

AAGTAACGCC ACTTTGCAAG GCATGGAAAA ATACATAACT GAGAATAGAA AAGTTCAGAT 240

CAAGGTCAGG AACAAAGAAA CAGCTGAATA CCAAACAGGA TATCTGTGGT AAGCGGTTCC 300

TGCCCCGGCT CAGGGCCAAG AACAGATGAG ACAGCTGAGT GATGGGCCAA ACAGGATATC 360

TGTGGTAAGC AGTTCCTGCC CCGGCTCGGG GCCAAGAACA GATGGTCCCC AGATGCGGTC 420

CAGCCCTCAG CAGTTTCTAG TGAATCATCA GATGTTTCCA GGGTGCCCCA AGGACCTGAA 480

AATGACCCTG TACCTTATTT GAACTAACCA ATCAGTTCGC TTCTCGCTTC TGTTCGCGCG 540

CTTCCGCTCT CCGAGCTCAA TAAAAGAGCC CACAACCCCT CACTCGGCGC GCCAGTCTTC 600

CGATAGACTG CGTCGCCCGG GTACCCGTAT TCCCAATAAA GCCTCTTGCT GTTTGCATCC 660

GAATCGTGGT CTCGCTGTTC CTTGGGAGGG TCTCCTCTGA GTGATTGACT ACCCACGACG 720

GGGGTCTTTC ATTTGGGGGC TCGTCCGGGA TTTGGAGACC CCTGCCCAGG GACCACCGAC 780

CCACCACCGG GAGGTAAGCT GGCCAGCAAC TTATCTGTGT CTGTCCGATT GTCTAGTGTC 840

TATGTTTGAT GTTATGCGCC TGCGTCTGTA CTAGTTAGCT AACTAGCTCT GTATCTGGCG 900

GACCCGTGGT GGAACTGACG AGTTCTGAAC ACCCGGCCGC AACCCTGGGA GACGTCCCAG 960

GGACTTTGGG GGCCGTTTTT GTGGCCCGAC CTGAGGAAGG GAGTCGATGT GGAATCCGAC 1020

CCCGTCAGGA TATGTGGTTC TGGTAGGAGA CGAGAACCTA AAACAGTTCC CGCCTCCGTC 1080

TGAATTTTTG CTTTCGGTTT GGAACCGAAG CCGCGCGTCT TGTCTGCTGC AGCGCTGCAG 1140

CATCGTTCTG TGTTGTCTCT GTCTGACTGT GTTTCTGTAT TTGTCTGAAA ATTAGGGCCA 1200

GACTGTTACC ACTCCCTTAA GTTTGACCTT AGGTCACTGG AAAGATGTCG AGCGGATCGC 1260

TCACAACCAG TCGGTAGATG TCAAGAAGAG ACGTTGGGTT ACCTTCTGCT CTGCAGAATG 1320

GCCAACCTTT AACGTCGGAT GGCCGCGAGA CGGCACCTTT AACCGAGACC TCATCACCCA 1380

GGTTAAGATC AAGGTCTTTT CACCTGGCCC GCATGGACAC CCAGACCAGG TCCCCTACAT 1440

CGTGACCTGG GAAGCCTTGG CTTTTGACCC CCCTCCCTGG GTCAAGCCCT TTGTACACCC 1500

TAAGCCTCCG CCTCCTCTTC CTCCATCCGC CCCGTCTCTC CCCCTTGAAC CTCCTCGTTC 1560

GACCCCGCCT CGATCCTCCC TTTATCCAGC CCTCACTCCT TCTCTAGGCG CCGGAATTCC 1620

GATCTGATCA AGAGACAGGA TGAGGATCGT TTCGCATGAT TGAACAAGAT GGATTGCACG 1680

CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA 1740

TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG 1800

TCAAGACCGA CCTGTCCGGT GCCCTGAATG AACTGCAGGA CGAGGCAGCG CGGCTATCGT 1860

GGCTGGCCAC GACGGGCGTT CCTTGCGCAG CTGTGCTCGA CGTTGTCACT GAAGCGGGAA 1920

GGGACTGGCT GCTATTGGGC GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC 1980

CTGCCGAGAA AGTATCCATC ATGGCTGATG CAATGCGGCG GCTGCATACG CTTGATCCGG 2040

CTACCTGCCC ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT ACTCGGATGG 2100

AAGCCGGTCT TGTCGATCAG GATGATCTGG ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG 2160

›EXAMPLE 4 · 2 of 9

AACTGTTCGC CAGGCTCAAG GCGCGCATGC CCGACGGCGA GGATCTCGTC GTGACCCATG 2220

GCGATGCCTG CTTGCCGAAT ATCATGGTGG AAAATGGCCG CTTTTCTGGA TTCATCGACT 2280

GTGGCCGGCT GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC CGTGATATTG 2340

CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT ATCGCCGCTC 2400

CCGATTCGCA GCGCATCGCC TTCTATCGCC TTCTTGACGA GTTCTTCTGA GCGGGACTCT 2460

GGGGTTCGAA ATGACCGACC AAGCGACGCC CAACCTGCCA TCACGAGATT TCGATTCCAC 2520

CGCCGCCTTC TATGAAAGGT TGGGCTTCGG AATCGTTTTC CGGGACGCCG GCTGGATGAT 2580

CCTCCAGCGC GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCCGGGCTCG ATCCCCTCGC 2640

GAGTTGGTTC AGCTGCTGCC TGAGGCTGGA CGACCTCGCG GAGTTCTACC GGCAGTGCAA 2700

ATCCGTCGGC ATCCAGGAAA CCAGCAGCGG CTATCCGCGC ATCCATGCCC CCGAACTGCA 2760

GGAGTGGGGA GGCACGATGG CCGCTTTGGT CGAGGCGGAT CCGGCCATTA GCCATATTAT 2820

TCATTGGTTA TATAGCATAA ATCAATATTG GCTATTGGCC ATTGCATACG TTGTATCCAT 2880

ATCATAATAT GTACATTTAT ATTGGCTCAT GTCCAACATT ACCGCCATGT TGACATTGAT 2940

TATTGACTAG TTATTAATAG TAATCAATTA CGGGGTCATT AGTTCATAGC CCATATATGG 3000

AGTTCCGCGT TACATAACTT ACGGTAAATG GCCCGCCTGG CTGACCGCCC AACGACCCCC 3060

GCCCATTGAC GTCAATAATG ACGTATGTTC CCATAGTAAC GCCAATAGGG ACTTTCCATT 3120

GACGTCAATG GGTGGAGTAT TTACGGTAAA CTGCCCACTT GGCAGTACAT CAAGTGTATC 3180

ATATGCCAAG TACGCCCCCT ATTGACGTCA ATGACGGTAA ATGGCCCGCC TGGCATTATG 3240

CCCAGTACAT GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA TTAGTCATCG 3300

CTATTACCAT GGTGATGCGG TTTTGGCAGT ACATCAATGG GCGTGGATAG CGGTTTGACT 3360

CACGGGGATT TCCAAGTCTC CACCCCATTG ACGTCAATGG GAGTTTGTTT TGGCACCAAA 3420

ATCAACGGGA CTTTCCAAAA TGTCGTAACA ACTCCGCCCC ATTGACGCAA ATGGGCGGTA 3480

GGCATGTACG GTGGGAGGTC TATATAAGCA GAGCTCGTTT AGTGAACCGT CAGATCGCCT 3540

GGAGACGCCA TCCACGCTGT TTTGACCTCC ATAGAAGACA CCGGGACCGA TCCAGCCTCC 3600

GCGGCCCCAA GCTTGCCGCC ACCATGGTGA GCAAGGGCGA GGAGCTCTTC ACCGGGGTGG 3660

TGCCCATCCT GGTCGAGCTG GACGGCGACG TGAACGGCCA CAAGTTCAGC GTGTCCGGCG 3720

AGGGCGACCC CGATGCCACC TACGGCAAGC TGACCCTGAA GTTCATCTGC ACCACCGGCA 3780

AGCTGCCCGT GCCCTGGCCC ACCCTCGTCA CCACCTTCAC CTACGGCGTG CAGTGCTTCA 3840

GCCGCTACCC CGACCACATG AAGCAGCACG ACTTCTTCAA GTCCGCCATG CCCGAAGGCT 3900

ACGTCCAGGA GCGCACCATC TTCTTCAAGG ACGACGGCAA CTACAAGACC CGCGCCGAGG 3960

TGAAGTTCGA GGGCGACACC CTGGTGAACC GCATCGAGCT GAAGGGCATC GACTTCAAGG 4020

AGGACGGCAA CATCCTGGGG CACAAGCTGG AGTACAACTA CAACAGCCAC AACGTCTATA 4080

TCATGGCCGA CAAGCAGAAG AACGGCATCA AGGTGAACTT CAAGATCCCC CACAACATCG 4140

AGGACGGCAG CGTGCAGCTC GCCGACCACT ACCAGCAGAA CACCCCCATC GGCGACGGCC 4200

CCGTGCTGCT GCCCGACAAC CACTACCTGA GCACCCAGTC CGCCCTGAGC AAAGACCCCA 4260

ACGAGAAGCG CGATCACATG GTCCTGCTGG AGTTCGTGAC CGCCGCCGGG ATCACTCACG 4320

GCATGGACGA GCTGTACAAG TAAAGCGGCC AACATCGATA AAATAAAAGA TTTTATTTAG 4380

TCTCCAGAAA AAGGGGGGAA TGAAAGACCC CACCTGTAGG TTTGGCAAGC TAGCTTAAGT 4440

AACGCCATTT TGCAAGGCAT GGAAAAATAC ATAACTGAGA ATAGAGAAGT TCAGATCAAG 4500

GTCAGGAACA GATGGAACAG CTGAATATGG GCCAAACAGG ATATCTGTGG TAAGCAGTTC 4560

CTGCCCCGGC TCAGGGCCAA GAACAGATGG AACAGCTGAA TATGGGCCAA ACAGGATATC 4620

TGTGGTAAGC AGTTCCTGCC CCGGCTCAGG GCCAAGAACA GATGGTCCCC AGATGCGGTC 4680

CAGCCCTCAG CAGTTTCTAG AGAACCATCA GATGTTTCCA GGGTGCCCCA AGGACCTGAA 4740

ATGACCCTGT GCCTTATTTG AACTAACCAA TCAGTTCGCT TCTCGCTTCT GTTCGCGCGC 4800

TTCTGCTCCC CGAGCTCAAT AAAAGAGCCC ACAACCCCTC ACTCGGGGCG CCAGTCCTCC 4860

GATTGACTGA GTCGCCCGGG TACCCGTGTA TCCAATAAAC CCTCTTGCAG TTGCATCCGA 4920

CTTGTGGTCT CGCTGTTCCT TGGGAGGGTC TCCTCTGAGT GATTGACTAC CCGTCAGCGG 4980

GGGTCTTTCA TTTGGGGGCT CGTCCGGGAT CGGGAGACCC CTGCCCAGGG ACCACCGACC 5040

CACCACCGGG AGGTAAGCTG GCTGCCTCGC GCGTTTCGGT GATGACGGTG AAAACCTCTG 5100

ACACATGCAG CTCCCGGAGA CGGTCACAGC TTGTCTGTAA GCGGATGCCG GGAGCAGACA 5160

AGCCCGTCAG GGCGCGTCAG CGGGTGTTGG CGGGTGTCGG GGCGCAGCCA TGACCCAGTC 5220

ACGTAGCGAT AGCGGAGTGT ATACTGGCTT AACTATGCGG CATCAGAGCA GATTGTACTG 5280

AGAGTGCACC ATATGCGGTG TGAAATACCG CACAGATGCG TAAGGAGAAA ATACCGCATC 5340

AGGCGCTCTT CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA 5400

GCGGTATCAG CTCACTCAAA GGCGGTAATA CGGTTATCCA CAGAATCAGG GGATAACGCA 5460

GGAAAGAACA TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA GGCCGCGTTG 5520

CTGGCGTTTT TCCATAGGCT CCGCCCCCCT GACGAGCATC ACAAAAATCG ACGCTCAAGT 5580

CAGAGGTGGC GAAACCCGAC AGGACTATAA AGATACCAGG CGTTTCCCCC TGGAAGCTCC 5640

CTCGTGCGCT CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC CTTTCTCCCT 5700

TCGGGAAGCG TGGCGCTTTC TCATAGCTCA CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC 5760

GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CCCCCCGTTC AGCCCGACCG CTGCGCCTTA 5820

TCCGGTAACT ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC ACTGGCAGCA 5880

GCCACTGGTA ACAGGATTAG CAGAGCGAGG TATGTAGGCG GTGCTACAGA GTTCTTGAAG 5940

TGGTGGCCTA ACTACGGCTA CACTAGAAGG ACAGTATTTG GTATCTGCGC TCTGCTGAAG 6000

CCAGTTACCT TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC CACCGCTGGT 6060

AGCGGTGGTT TTTTTGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG ATCTCAAGAA 6120

GATCCTTTGA TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGGG 6180

ATTTTGGTCA TGAGATTATC AAAAAGGATC TTCACCTAGA TCCTTTTAAA TTAAAAATGA 6240

AGTTTTAAAT CAATCTAAAG TATATATGAG TAAACTTGGT CTGACAGTTA CCAATGCTTA 6300

ATCAGTGAGG CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT TGCCTGACTC 6360

CCCGTCGTGT AGATAACTAC GATACGGGAG GGCTTACCAT CTGGCCCCAG TGCTGCAATG 6420

ATACCGCGAG ACCCACGCTC ACCGGCTCCA GATTTATCAG CAATAAACCA GCCAGCCGGA 6480

AGGGCCGAGC GCAGAAGTGG TCCTGCAACT TTATCCGCCT CCATCCAGTC TATTAATTGT 6540

TGCCGGGAAG CTAGAGTAAG TAGTTCGCCA GTTAATAGTT TGCGCAACGT TGTTGCCATT 6600

GCTGCAGGCA TCGTGGTGTC ACGCTCGTCG TTTGGTATGG CTTCATTCAG CTCCGGTTCC 6660

CAACGATCAA GGCGAGTTAC ATGATCCCCC ATGTTGTGCA AAAAAGCGGT TAGCTCCTTC 6720

GGTCCTCCGA TCGTTGTCAG AAGTAAGTTG GCCGCAGTGT TATCACTCAT GGTTATGGCA 6780

GCACTGCATA ATTCTCTTAC TGTCATGCCA TCCGTAAGAT GCTTTTCTGT GACTGGTGAG 6840

TACTCAACCA AGTCATTCTG AGAATAGTGT ATGCGGCGAC CGAGTTGCTC TTGCCCGGCG 6900

TCAACACGGG ATAATACCGC GCCACATAGC AGAACTTTAA AAGTGCTCAT CATTGGAAAA 6960

CGTTCTTCGG GGCGAAAACT CTCAAGGATC TTACCGCTGT TGAGATCCAG TTCGATGTAA 7020

CCCACTCGTG CACCCAACTG ATCTTCAGCA TCTTTTACTT TCACCAGCGT TTCTGGGTGA 7080

GCAAAAACAG GAAGGCAAAA TGCCGCAAAA AAGGGAATAA GGGCGACACG GAAATGTTGA 7140

ATACTCATAC TCTTCCTTTT TCAATATTAT TGAAGCATTT ATCAGGGTTA TTGTCTCATG 7200

AGCGGATACA TATTTGAATG TATTTAGAAA AATAAACAAA TAGGGGTTCC GCGCACATTT 7260

CCCCGAAAAG TGCCACCTGA CGTCTAAGAA ACCATTATTA TCATGACATT AACCTATAAA 7320

›EXAMPLE 4 · 3 of 9

AATAGGCGTA TCACGAGGCC CTTTCGTCTT CAA 7353

GAATTGCTAG CAATTGCTAG CAATTGCTAG CAATTCATAC CAGATCACCG AAAACTGTCC 60

TCCAAATGTG TCCCCCTCAC ACTCCCAAAT TCGCGGGCTT CTGCCTCTTA GACCACTCTA 120

CCCTATTCCC CACACTCACC GGAGCCAAAG CCGCGGCCCT TCCGTTTCTT TGCTTTTGAA 180

AGACCCCACC CGTAGGTGGC AAGCTAGCTT AAGTAACGCC ACTTTGCAAG GCATGGAAAA 240

ATACATAACT GAGAATAGAA AAGTTCAGAT CAAGGTCAGG AACAAAGAAA CAGCTGAATA 300

CCAAACAGGA TATCTGTGGT AAGCGGTTCC TGCCCCGGCT CAGGGCCAAG AACAGATGAG 360

ACAGCTGAGT GATGGGCCAA ACAGGATATC TGTGGTAAGC AGTTCCTGCC CCGGCTCGGG 420

GCCAAGAACA GATGGTCCCC AGATGCGGTC CAGCCCTCAG CAGTTTCTAG TGAATCATCA 480

GATGTTTCCA GGGTGCCCCA AGGACCTGAA AATGACCCTG TACCTTATTT GAACTAACCA 540

ATCAGTTCGC TTCTCGCTTC TGTTCGCGCG CTTCCGCTCT CCGAGCTCAA TAAAAGAGCC 600

CACAACCCCT CACTCGGCGC GCCAGTCTTC CGATAGACTG CGTCGCCCGG GTACCCGTAT 660

TCCCAATAAA GCCTCTTGCT GTTTGCATCC GAATCGTGGT CTCGCTGTTC CTTGGGAGGG 720

TCTCCTCTGA GTGATTGACT ACCCACGACG GGGGTCTTTC ATTTGGGGGC TCGTCCGGGA 780

TTTGGAGACC CCTGCCCAGG GACCACCGAC CCACCACCGG GAGGTAAGCT GGCCAGCAAC 840

TTATCTGTGT CTGTCCGATT GTCTAGTGTC TATGTTTGAT GTTATGCGCC TGCGTCTGTA 900

CTAGTTAGCT AACTAGCTCT GTATCTGGCG GACCCGTGGT GGAACTGACG AGTTCTGAAC 960

ACCCGGCCGC AACCCTGGGA GACGTCCCAG GGACTTTGGG GGCCGTTTTT GTGGCCCGAC 1020

CTGAGGAAGG GAGTCGATGT GGAATCCGAC CCCGTCAGGA TATGTGGTTC TGGTAGGAGA 1080

CGAGAACCTA AAACAGTTCC CGCCTCCGTC TGAATTTTTG CTTTCGGTTT GGAACCGAAG 1140

CCGCGCGTCT TGTCTGCTGC AGCGCTGCAG CATCGTTCTG TGTTGTCTCT GTCTGACTGT 1200

GTTTCTGTAT TTGTCTGAAA ATTAGGGCCA GACTGTTACC ACTCCCTTAA GTTTGACCTT 1260

AGGTCACTGG AAAGATGTCG AGCGGATCGC TCACAACCAG TCGGTAGATG TCAAGAAGAG 1320

ACGTTGGGTT ACCTTCTGCT CTGCAGAATG GCCAACCTTT AACGTCGGAT GGCCGCGAGA 1380

CGGCACCTTT AACCGAGACC TCATCACCCA GGTTAAGATC AAGGTCTTTT CACCTGGCCC 1440

GCATGGACAC CCAGACCAGG TCCCCTACAT CGTGACCTGG GAAGCCTTGG CTTTTGACCC 1500

CCCTCCCTGG GTCAAGCCCT TTGTACACCC TAAGCCTCCG CCTCCTCTTC CTCCATCCGC 1560

CCCGTCTCTC CCCCTTGAAC CTCCTCGTTC GACCCCGCCT CGATCCTCCC TTTATCCAGC 1620

CCTCACTCCT TCTCTAGGCG CCGGAATTCG TTGCTACCGG TCGCCAACAT GGTGAGCAAG 1680

GGCGAGGAGC TGTTCACCGG GGTGGTGCCC ATCCTGGTCG AGCTGGACGG CGACGTGAAC 1740

GGCCACAAGT TCAGCGTGTC CGGCGAGGGC GAGGGCGATG CCACCTACGG CAAGCTGACC 1800

CTGAAGTTCA TCTGCACCAC CGGCAAGCTG CCCGTGCCCT GGCCCACCCT CGTGACCACC 1860

CTGACCTACG GCGTGCAGTG CTTCAGCCGC TACCCCGACC ACATGAAGCA GCACGACGTC 1920

TTCAAGTCCG CCATGCCCGA AGGCTACGTC CAGGAGCGCA CCATCTTCTT CAAGGACGAC 1980

GGCAACTACA AGACCCGCGC CGAGGTGAAG TTCGAGGGCG ACACCCTGGT GAACCGCATC 2040

GAGCTGAAGG GCATCGACTT CAAGGAGGAC GGCAACATCC TGGGGCACAA GCTGGAGTAC 2100

AACTACAACA GCCACAACGT CTATATCATG GCCGACAAGC AGAAGAACGG CATCAAGGTG 2160

AACTTCAAGA TCCGCCACAA CATCGAGGAC GGCAGCGTGC AGCTCGCCGA CCACTACCAG 2220

CAGAACACCC CCATCGGCGA CGGCCCCGTG CTGCTGCCCG ACAACCACTA CCTGAGCACC 2280

CAGTCCGCCC TGAGCAAAGA CCCCAACGAG AAGCGCGATC ACATGGTCCT GCTGGAGTTC 2340

GTGACCGCCG CCGGGATCAC TCACGGCATG GACGAGCTGT ACAAGTAGCG GCCATGCTAA 2400

CTCGAGGATC CGAAAGACCC CACCTGTAGG TTTGGCAAGC TAGCTTAAGT AACGCCATTT 2460

TGCAAGGCAT GGAAAAATAC ATAACTGAGA ATAGAGAAGT TCAGATCAAG GTCAGGAACA 2520

GATGGAACAG CTGAATATGG GCCAAACAGG ATATCTGTGG TAAGCAGTTC CTGCCCCGGC 2580

TCAGGGCCAA GAACAGATGG AACAGCTGAA TATGGGCCAA ACAGGATATC TGTGGTAAGC 2640

AGTTCCTGCC CCGGCTCAGG GCCAAGAACA GATGGTCCCC AGATGCGGTC CAGCCCTCAG 2700

CAGTTTCTAG AGAACCATCA GATGTTTCCA GGGTGCCCCA AGGACCTGAA ATGACCCTGT 2760

GCCTTATTTG AACTAACCAA TCAGTTCGCT TCTCGCTTCT GTTCGCGCGC TTCTGCTCCC 2820

CGAGCTCAAT AAAAGAGCCC ACAACCCCTC ACTCGGGGCG CCAGTCCTCC GATTGACTGA 2880

GTCGCCCGGG TACCCGTGTA TCCAATAAAC CCTCTTGCAG TTGCATCCGA CTTGTGGTCT 2940

CGCTGTTCCT TGGGAGGGTC TCCTCTGAGT GATTGACTAC CCGTCAGCGG GGGTCTTTCA 3000

TTTGGGGGCT CGTCCGGGAT CGGGAGACCC CTGCCCAGGG ACCACCGACC CACCACCGGG 3060

AGGTAAGCTG GCTGCCTCGC GCGTTTCGGT GATGACGGTG AAAACCTCTG ACACATGCAG 3120

CTCCCGGAGA CGGTCACAGC TTGTCTGTAA GCGGATGCCG GGAGCAGACA AGCCCGTCAG 3180

GGCGCGTCAG CGGGTGTTGG CGGGTGTCGG GGCGCAGCCA TGACCCAGTC ACGTAGCGAT 3240

AGCGGAGTGT ATACTGGCTT AACTATGCGG CATCAGAGCA GATTGTACTG AGAGTGCACC 3300

ATATGCGGTG TGAAATACCG CACAGATGCG TAAGGAGAAA ATACCGCATC AGGCGCTCTT 3360

CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA GCGGTATCAG 3420

CTCACTCAAA GGCGGTAATA CGGTTATCCA CAGAATCAGG GGATAACGCA GGAAAGAACA 3480

TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT 3540

TCCATAGGCT CCGCCCCCCT GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC 3600

GAAACCCGAC AGGACTATAA AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT 3660

CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG 3720

TGGCGCTTTC TCATAGCTCA CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA 3780

AGCTGGGCTG TGTGCACGAA CCCCCCGTTC AGCCCGACCG CTGCGCCTTA TCCGGTAACT 3840

ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA 3900

ACAGGATTAG CAGAGCGAGG TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA 3960

ACTACGGCTA CACTAGAAGG ACAGTATTTG GTATCTGCGC TCTGCTGAAG CCAGTTACCT 4020

TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC CACCGCTGGT AGCGGTGGTT 4080

TTTTTGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA 4140

TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA 4200

TGAGATTATC AAAAAGGATC TTCACCTAGA TCCTTTTAAA TTAAAAATGA AGTTTTAAAT 4260

CAATCTAAAG TATATATGAG TAAACTTGGT CTGACAGTTA CCAATGCTTA ATCAGTGAGG 4320

CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT TGCCTGACTC CCCGTCGTGT 4380

AGATAACTAC GATACGGGAG GGCTTACCAT CTGGCCCCAG TGCTGCAATG ATACCGCGAG 4440

ACCCACGCTC ACCGGCTCCA GATTTATCAG CAATAAACCA GCCAGCCGGA AGGGCCGAGC 4500

GCAGAAGTGG TCCTGCAACT TTATCCGCCT CCATCCAGTC TATTAATTGT TGCCGGGAAG 4560

CTAGAGTAAG TAGTTCGCCA GTTAATAGTT TGCGCAACGT TGTTGCCATT GCTGCAGGCA 4620

TCGTGGTGTC ACGCTCGTCG TTTGGTATGG CTTCATTCAG CTCCGGTTCC CAACGATCAA 4680

GGCGAGTTAC ATGATCCCCC ATGTTGTGCA AAAAAGCGGT TAGCTCCTTC GGTCCTCCGA 4740

TCGTTGTCAG AAGTAAGTTG GCCGCAGTGT TATCACTCAT GGTTATGGCA GCACTGCATA 4800

ATTCTCTTAC TGTCATGCCA TCCGTAAGAT GCTTTTCTGT GACTGGTGAG TACTCAACCA 4860

AGTCATTCTG AGAATAGTGT ATGCGGCGAC CGAGTTGCTC TTGCCCGGCG TCAACACGGG 4920

ATAATACCGC GCCACATAGC AGAACTTTAA AAGTGCTCAT CATTGGAAAA CGTTCTTCGG 4980

GGCGAAAACT CTCAAGGATC TTACCGCTGT TGAGATCCAG TTCGATGTAA CCCACTCGTG 5040

CACCCAACTG ATCTTCAGCA TCTTTTACTT TCACCAGCGT TTCTGGGTGA GCAAAAACAG 5100

GAAGGCAAAA TGCCGCAAAA AAGGGAATAA GGGCGACACG GAAATGTTGA ATACTCATAC 5160

›EXAMPLE 4 · 4 of 9

TCTTCCTTTT TCAATATTAT TGAAGCATTT ATCAGGGTTA TTGTCTCATG AGCGGATACA 5220

TATTTGAATG TATTTAGAAA AATAAACAAA TAGGGGTTCC GCGCACATTT CCCCGAAAAG 5280

TGCCACCTGA CGTCTAAGAA ACCATTATTA TCATGACATT AACCTATAAA AATAGGCGTA 5340

TCACGAGGCC CTTTCGTCTT CAA 5363