Antisense oligonucleotides are provided which are complementary to at least a portion of HCV RNA and specifically hybridizable therewith. These oligonucleotides can be administered to inhibit the replication of Hepatitis C virus in vivo or in vitro and to treat Hepatitis C virus-associated disease. These compounds can be used either prophylactically or therapeutically to reduce the severity of diseases associated with Hepatitis C virus.

Description

19 parts

›This is a continuation of application Ser. No…

This is a continuation of application Ser. No. 08/453,085, filed May 30, 1995, now abandoned which is a continuation of application Ser. No. 07/945,289, filed Sep. 10, 1972, now abandoned.

›FIELD OF THE INVENTION

This invention relates to the design and synthesis of antisense oligonucleotides which can be administered to inhibit the replication of Hepatitis C virus in vivo or in vitro and to treat Hepatitis C virus-associated disease. These compounds can be used either prophylactically or therapeutically to reduce the severity of diseases associated with Hepatitis C virus. Oligonucleotides which are specifically hybridizable with RNA targets are disclosed.

›BACKGROUND OF THE INVENTION

The predominant form of hepatitis currently resulting from transfusions is not related to the previously characterized Hepatitis A virus or Hepatitis B virus and has been referred to as Non-A, Non-B Hepatitis (NANBH). NANBH currently accounts for over 90% of cases of post-transfusion hepatitis. Estimates of the frequency of NANBH in transfusion recipients range from 5%-13% for those receiving volunteer blood, or 25-54% for those receiving blood from commercial sources.

Acute NANBH, while often less severe than acute disease caused by Hepatitis A or Hepatitis B viruses, occasionally leads to severe or fulminant hepatitis. Of greater concern, progression to chronic hepatitis is much more common after NANBH than after either Hepatitis A or Hepatitis B infection. Chronic NANBH has been reported in 10%-70% of infected individuals. This form of hepatitis can be transmitted even by asymptomatic patients, and frequently progresses to malignant disease such as cirrhosis and hepatocellular carcinoma. Chronic active hepatitis, with or without cirrhosis, is seen in 44%-90% of posttransfusion hepatitis cases. Of those patients who developed cirrhosis, approximately one-fourth died of liver failure.

Chronic active NANBH is a significant problem to hemophiliacs who are dependent on blood products; 5%-11% of hemophiliacs die of chronic end-stage liver disease. Cases of NANBH other than those traceable to blood or blood products are frequently associated with hospital exposure, accidental needle stick, or tattooing. Transmission through close personal contact also occurs, though this is less common for NANBH than for Hepatitis B.

The causative agent of the majority of NANBH has recently been identified and is now referred to as Hepatitis C Virus (HCV). Houghton et al., EP Publication 318,216; Choo et al., Science 1989, 244, 359-362. Based on serological studies using recombinant DNA-generated antigens it is now clear that HCV is the causative agent of most cases of post-transfusion NANBH. Clones of cDNA prepared from nucleic acid isolated from concentrated virus particles were originally isolated based on their ability to encode polypeptides which reacted with sera from NANBH patients. These clones hybridized with RNA, but not DNA, isolated from infected liver tissue, indicating the presence of an RNA genome. Hybridization analyses and sequencing of the cDNA clones revealed that RNA present in infected liver and particles was the same polarity as that of the coding strand of the cDNAs; in other words, the virus genome is a positive or plus-strand RNA genome. EP Publication 318,216 (Houghton et al.) disclose partial genomic sequences of HCV-1, and teach recombinant DNA methods of cloning and expressing HCV sequences and HCV polypeptides, techniques of HCV immunodiagnostics, HCV probe diagnostic techniques, anti-HCV antibodies, and methods of isolating new HCV sequences. Houghton et al. also disclose additional HCV sequences and teach application of these sequences and polypeptides in immunodiagnostics, probe diagnostics, anti-HCV antibody production, PCR technology and recombinant DNA technology. The concept of using antisense polynucleotides as inhibitors of viral replication is disclosed, but no specific targets are taught. Oligomer probes and primers based on the sequences disclosed are also provided. EP Publication 419,182 (Miyamura et al.) discloses new HCV isolates J1 and J7 and use of sequences distinct from HCV-1 sequences for screens and diagnostics.

The only treatment regimen shown to be effective for the treatment of chronic NANBH is interferon-α. Most NANBH patients show an improvement of clinical symptoms during interferon treatment, but relapse is observed in at least half of patients when treatment is interrupted. Significant improvements in antiviral therapy are therefore greatly desired.

›OBJECTS OF THE INVENTION

It is an object of this invention to provide oligonucleotides which are capable of hybridizing with RNA of HCV to inhibit the synthesis or function of said RNA.

It is another object of this invention to provide oligonucleotides which are capable of hybridizing with RNA of HCV to inhibit replication of the virus.

It is a further object to provide oligonucleotides which can modulate the expression of HCV through antisense interaction with viral RNA.

Yet another object of this invention is to provide methods of prophylaxis, diagnostics and therapeutics for acute or chronic HCV infection.

A further object of this invention is to provide methods of prophylaxis, diagnostics and therapeutics for HCV-associated diseases.

Methods, materials and kits for detecting the presence or absence of HCV or HCV RNA in a sample suspected of containing it are further objects of the invention.

These and other objects will become apparent to persons of ordinary skill in the art from a review of the instant specification and appended claims.

›SUMMARY OF THE INVENTION

In accordance with the present invention, compositions and methods for modulating the effects of HCV infection are provided. Oligonucleotides complementary to, and specifically hybridizable with, selected sequences of HCV RNA are provided. The HCV 5′ end hairpin loop, 5′ end 6-base-pair repeats, 5′ end untranslated region, polyprotein translation initiation codon, ORF 3 translation initiation codon, 3′-untranslated region, 3′ end palindrome region, R2 sequence and 3′ end hairpin loop are preferred targets. Methods for diagnosing or treating disease states by administering oligonucleotides, either alone or in combination with a pharmaceutically acceptable carrier, to animals suspected of having HCV-associated diseases are also provided.

The relationship between the target RNA and oligonucleotides complementary to at least a portion of the target, and specifically hybridizable with it, is commonly denoted as “antisense”. The oligonucleotides are able to inhibit the function of viral RNA by interfering with its replication, transcription into mRNA, translation into protein, packaging into viral particles or any other activity necessary to its overall biological function. The failure of the RNA to perform all or part of its function results in failure of all or a portion of the normal life cycle of the virus.

It has been found that antisense oligonucleotides designed to target viruses can be effective in diminishing viral infection. It is preferred that oligonucleotides have between about 5 and about 50 nucleotide units. It is also preferred that the oligonucleotides be specifically hybridizable with the HCV 5′ end hairpin loop, 5′ end 6-base-pair repeats, 5′ end untranslated region, polyprotein translation initiation codon, ORF 3 translation initiation codon, 3′-untranslated region, 3′ end palindrome region, R2 sequence or 3′ end hairpin loop. The oligonucleotide may be modified to increase nuclease resistance and to increase its efficacy.

In accordance with preferred embodiments, the viral RNA is interfered with to an extent sufficient to inhibit HCV infection and/or HCV replication. Thus, oligonucleotides which are capable of interacting with portions of HCV RNA are comprehended. Animals suspected of having HCV-associated disease are contacted with an oligonucleotide made in accordance with this invention. In particular, the present invention is believed to be effective in the treatment of acute and chronic HCV infections and HCV-associated disease, either prophylactically or therapeutically.

It is to be expected that differences in the RNA of HCV from different strains and from different types within a strain exist. Thus, it is believed, for example, that the regions of the various HCV strains serve essentially the same function for the respective strains and that interference with expression of the genetic information will afford similar results in the various strains. This is believed to be so even though differences in the nucleotide sequences among the strains exist.

Accordingly, nucleotide sequences set forth in the present specification will be understood to be representational for the particular strain being described. Homologous or analogous sequences for different strains of HCV are specifically contemplated as being within the scope of this invention.

›DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS · 1 of 2

Antisense oligonucleotides hold great promise as therapeutic agents for the treatment of many human diseases. In most cases, oligonucleotides complementary to specific RNA target sequences bind by Watson-Crick base pairing to pre-mRNA or mature mRNA, inhibiting the flow of genetic information from DNA to protein. In the case of RNA viruses such as HCV, oligonucleotides are designed to specifically hybridize to viral genomic RNA, mRNA, or replicative intermediate RNA, interfering with the function of the RNA such that viral replication or protein expression is modulated.

Numerous recent studies have documented the utility of antisense oligonucleotides as biochemical tools for studying target proteins. Rothenberg et al., J. Natl. Cancer Inst. 1989, 81, 1539-1544; Zon, G. Pharmaceutical Res. 1987, 5, 539-549. Because of recent advances in oligonucleotide chemistry, synthesis of nuclease-resistant oligonucleotides, and availability of types of oligonucleotides which exhibit enhanced cell uptake, it is now possible to consider the use of antisense oligonucleotides as a novel form of therapeutics.

For therapeutics, an animal suspected of having an HCV infection or HCV-associated disease is treated by administering oligonucleotides in accordance with this invention. Oligonucleotides may be formulated in a pharmaceutical composition, which may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the oligonucleotide. Pharmaceutical compositions may also include one or more active ingredients such as, for example, antimicrobial agents, antiinflammatory agents, anesthetics, and the like in addition to oligonucleotide.

The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.

Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms may also be useful.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.

Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

Dosing is dependent on severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with course of treatment lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved. Dosage and frequency will vary depending on, for example, body weight of patient and means of administration. Individual doses will normally range from about 0.001 mg to 500 mg, but may be higher or lower. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.

The present invention employs oligonucleotides complementary to specific regions of HCV. RNA for antisense inhibition of HCV. In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages as well as oligomers having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.

Specific examples of some preferred oligonucleotides envisioned for this invention may contain phosphorothioates, phosphotriesters, methyl phosphonates, chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are those with CH 2 —NH—O—CH 2 , CH 2 —N(CH 3 )—O—CH 2 , CH 2 —O—N(CH 3 )—CH 2 , CH 2 —N(CH 3 )—N(CH 3 )—CH 2 and O—N(CH 3 )—CH 2 —CH 2 backbones (where phosphodiester is O—P—O—CH 2 ). Also preferred are oligonucleotides having morpholino backbone structures. Summerton, J. E. and Weller, D. D. U.S. Pat. No. 5,034,506. In other preferred embodiments, such as the protein-nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone. P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science 1991, 254, 1497. Other preferred oligonucleotides may contain alkyl and halogen-substituted sugar moieties comprising one of the following at the 2′ position: OH, SH, SCH 3 , F, OCN, O(CH 2 ) n NH 2 or O(CH 2 ) n CH 3 where n is from 1 to about 10; C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF 3 ; OCF 3 ; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; 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 conjugate; 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. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.

All such oligonucleotides are comprehended by this invention so long as they function effectively to hybridize with HCV RNA. The oligonucleotides in accordance with this invention preferably comprise from about 5 to about 50 nucleic acid base units. It is more preferred that such oligonucleotides comprise from about 8 to 30 nucleic acid base units, and still more preferred to have from about 12 to 25 nucleic acid base units. As will be appreciated, a nucleic acid base unit is a base-sugar combination suitably bound to adjacent nucleic acid base unit through phosphodiester or other bonds.

›DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS · 2 of 2

The oligonucleotides used in accordance with this invention 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 Applied Biosystems. Any other means for such synthesis may also be employed, however the actual synthesis of the oligonucleotides are well within the talents of the routineer. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives.

In accordance with this invention, persons of ordinary skill in the art will understand that messenger RNA includes not only the sequence information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form regions known to such persons as the 5′-untranslated region, the 3′-untranslated region, and the 5′ cap region, as well as ribonucleotides which form various secondary structures. Thus, oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the coding ribonucleotides. In preferred embodiments, the oligonucleotide is specifically hybridizable with the HCV 5′ end hairpin loop, 5′ end 6-base-pair repeats, ORF 3 translation initiation codon (all of which are contained in the 5′-untranslated region), polyprotein translation initiation codon, 3′-untranslated region, R2 region, 3′ hairpin loop or 3′ end palindrome region.

The size of the HCV genome is approximately 9400 nucleotides, with a single translational reading frame encoding a polyprotein which is subsequently processed to several structural and non-structural proteins.

Several regions of the HCV genome have been identified as antisense targets in the present invention. It should be noted that sequence availability and nucleotide numbering schemes vary from strain to strain. The 5′ untranslated region of HCV consists of approximately 350 nucleotides upstream of the polyprotein translation initiation codon. A hairpin loop present at nucleotides 1-22 at the 5′ end of the genome (HCV-1) identified herein as the “5′ end hairpin loop” is believed to serve as a recognition signal for the viral replicase or nucleocapsid proteins. Han et al., Proc. Natl. Acad. Sci. 1991, 88, 1711-1715.

Three small (12-16 amino acids each) open reading frames (ORFs) are located in the 5′-untranslated region of HCV RNA. These ORFs may be involved in control of translation. The ORF 3 translation initiation codon as denominated herein is found at nucleotides 315-317 of HCV-1 according to the scheme of Han et al., Proc. Natl. Acad. Sci. 1991, 88, 1711-1715; and at nucleotides-127 to -125 according to the scheme of Choo et al., Proc. Natl. Acad. Sci. 1991, 88, 2451-2455 and depicted in SEQ ID NO: 25.

The polyprotein translation initiation codon as denominated herein is an AUG sequence located at nucleotides 342-344 of HCV-1 according to Han et al., Proc. Natl. Acad. Sci. 1991, 88, 1711-1715 or at nucleotide 1-3 according to the HCV-1 numbering scheme of Choo et al., Proc. Natl. Acad. Sci. 1991, 88, 2451-2455 and SEQ ID NO: 25.

The 3′ untranslated region, as denominated herein, consists of nucleotides downstream of the polyprotein translation termination site (ending at nt 9037 according to Choo et al. and SEQ ID NO: 25; nt 9377 according to schemes of Han and Inchauspe as depicted in SEQ ID NO: 26). Nucleotides 9697-9716 (numbering scheme of Inchauspe for HCV-H which is depicted in SEQ ID NO: 26) at the 3′ terminus of the genome within the 3′ untranslated region can be organized into a stable hairpin loop structure identified herein as the 3′ hairpin loop. A short nucleotide stretch (R2) immediately upstream (nt 9691-9696 of HCV-H depicted in SEQ ID NO: 26) of the 3′ hairpin, and denominated herein “the R2 sequence”, is thought to play a role in cyclization of the viral RNA, possibly in combination with a set of 5′ end 6-base-pair repeats of the same sequence at nt 23-28 and 38-43. (Inchauspe et al., Proc. Natl. Acad. Sci. 1991, 88, 10292-10296) is identified herein as “5′ end 6-base-pair repeat”. Palindrome sequences present near the 3′ end of the genome (nucleotides 9312-9342 according to the scheme of Takamizawa et al., J. Virol. 1991, 65, 1105-1113 depicted in SEQ ID NO: 27) are capable of forming a stable secondary structure. This is referred to herein as the 3′ end palindrome region.

Oligonucleotides useful in the invention are complementary to HCV RNA. Thus, the oligonucleotides in accordance with the invention preferably have one of the sequences shown in Table 1, or an effective portion thereof. It is preferred to employ any of these oligonucleotides as set forth above or any of the similar oligonucleotides, which persons of ordinary skill in the art can prepare from knowledge of the preferred antisense targets for the modulation of HCV infection.

The oligonucleotides of this invention can be used in diagnostics, therapeutics and as research reagents and kits. Since the oligonucleotides of this invention hybridize to RNA from HCV, sandwich and other assays can easily be constructed to exploit this fact. Provision of means for detecting hybridization of oligonucleotide with HCV or HCV RNA present in a sample suspected of containing it can routinely be accomplished. Such provision may include enzyme conjugation, radiolabelling or any other suitable detection systems. Kits for detecting the presence or absence of HCV may also be prepared.

The following specific examples are given for illustrative purposes only and are not intended to limit the invention.

EXAMPLES

›Examples11

›Example 1

Oligonucleotide Synthesis

Unmodified DNA oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine. β-cyanoethyldiisopropyl-phosphoramidites are purchased from Applied Biosystems (Foster City, Calif.). For phosphorothioate oligonucleotides, the standard oxidation bottle is replaced by a 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation cycle wait step is increased to 68 seconds and is followed by the capping step.

After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Analytical gel electrophoresis is accomplished in 20% acrylamide, 8 M urea, 45 mM Tris-borate buffer, pH 7.0.

›Example 2

Transcription and Translation of HCV RNA in Genetically Engineered Cells

A recombinant DNA vector capable of expressing HCV genes in mammalian cells is constructed using standard genetic engineering methods. A cDNA fragment representing the HCV mRNA or genomic transcript is placed behind an inducible eukaryotic promotor such as the LTR from mouse mammary tumor virus in such a way that transcription of the HCV cDNA begins at the appropriate nucleotide position. At the 3′ end of the gene, a polyadenylation signal is incorporated to ensure termination at the appropriate nucleotide position. It may be advantageous to modify the coding sequence by insertion of an in-frame reporter domain (e.g., the enzymatically active domain of the firefly luciferase gene) which can simplify detection procedures for expression of the fusion protein. The vector also contains one or more selectable genetic markers such as neomycin resistance.

The described vector is introduced into mammalian cells using a standard calcium chloride transfection procedure. Cells containing transfected DNA are identified by growth in the presence of selective agents such as neomycin, and cloned by limiting dilution. Expression of HCV RNA in cloned transfectants can be verified using any one of a number of assays such as northern blots, RNA polymerase chain reaction, or nuclease protection. Protein expression can be verified using western blotting or immune precipitation with specific HCV antibodies, or by monitoring for the presence of detectable enzymatic activity resulting from the incorporation of an assayable reporter domain. If an inducible promotor such as the MMTV LTR is used in construction of the vector, a glucocorticoid inducer such as dexamethasone should be added to the transfected cells prior to assays in order to induce gene expression.

›Example 3

Evaluation of Antisense Oligonucleotide Inhibition of HCV Gene Expression from Genetically Engineered Cells

Mammalian cells transfected with expression vectors such as those described in Example 2 are incubated overnight in medium containing antisense oligonucleotides. After oligonucleotide treatment, cells are treated with dexamethasone in order to induce expression of HCV gene products. After a suitable incubation period (4-24 hours) cells are harvested, and expression of specific HCV polypeptide can be detected immunologically using specific antisera in a western blot or immunoprecipitation assay. If the cells contain a vector containing a reporter domain, such as that for firefly luciferase, fused in-frame with the HCV polyprotein, cell extracts can be harvested and evaluated for enzymatic activity of the reporter domain.

›Example 4

Transcription and Translation of HCV RNA from Cytoplasmic Virus Vectors

A cDNA fragment representing the HCV mRNA or genomic transcript is placed behind a Vaccinia virus promotor in such a way that transcription of the HCV cDNA begins at the appropriate nucleotide position. At the 3′ end of the gene, a polyadenylation signal is incorporated to ensure termination at the appropriate nucleotide position. It may be advantageous in some instances to modify the coding sequence by insertion of an in-frame reporter domain (e.g., the enzymatically active domain of the firefly luciferase gene) which can simplify detection procedures for expression of the fusion protein.

Incorporation of the expression unit into the genome of a cytoplasmic replicating DNA virus such as Vaccinia is facilitated by inclusion of sequences upstream and downstream of the expression unit which are homologous to the Vaccinia virus genome. Co-transfection of vector into Vaccinia virus-infected mammalian cells can result in homologous recombination of vector with Vaccinia. If a suitable enzymatic marker such as β-galactosidase is present at the appropriate recombination site in the virus, then recombinant plaques can be identified by a lack of color under appropriate substrate conditions. Cloned virus can be propagated in appropriate host mammalian cell lines and expression of HCV gene products verified as described in Example 2.

›Example 5

Evaluation of Antisense Oligonucleotide Inhibition of HCV Gene Expression from Cytoplasmic Virus Vectors in Mammalian Cells

Mammalian cells are incubated overnight in medium containing antisense oligonucleotides. After oligonucleotide treatment, cells are infected with recombinant Vaccinia virus expressing HCV gene products. After a suitable incubation period (4-24 hours) cells are harvested, and expression of specific HCV polypeptide can be detected immunologically using specific antisera in a western blot or immunoprecipitation assay. If the cells contain a vector containing a reporter domain, such as that for firefly luciferase, fused in-frame with the HCV polyprotein, cell extracts can be harvested and evaluated for enzymatic activity of the reporter domain.

›Example 6 · 1 of 6

Evaluation of Antisense Oligonucleotide Inhibition of HCV Particle Assembly in Cells Transfected with HCV Genes or Infected with Cytoplasmic Virus Vectors Expressing HCV Genes

HCV genomic RNA and protein are expressed in cells transfected with HCV cDNA expression vectors, or in cells infected with Vaccinia virus vectors expressing the HCV cDNA. It is likely that the RNA genomes and proteins will associate to form HCV-like particles. The presence of these particles can be verified using electron microscopy. To evaluate the effects of oligonucleotides complementary to presumed packaging signals of the viral RNA on particle assembly, specific biochemical assays can be developed to measure the appearance of extracellular particles containing both HCV nucleic acid and proteins.

Mammalian cells transfected with expression vectors such as those described in Example 2 are incubated overnight in medium containing antisense oligonucleotides. After oligonucleotide treatment, cells are treated with dexamethasone in order to induce expression of HCV gene products. After a suitable incubation period (4-24 hours) extracellular fluid from treated cells is harvested, and particles are concentrated by pelleting in the ultracentrifuge. Proteins and nucleic acids are extracted from the pellet and quantitated by northern blot and western blot analysis respectively as described in Examples 4 and 5. A similar procedure could be used to monitor effects of oligonucleotide treatment on virus particle assembly resulting from infection of cells with recombinant Vaccinia virus expressing the HCV polyprotein.

ATGGTGGAGT GTCGCCCCGT C 21

GGAGTGATCT ATGGTGGAGT G 21

GATTCGTGCT CATGGTGCAC G 21

TCCAGGCATT GAGCGGGTTG A 21

TGGCCTGGAG TGTTTATCTC C 21

GGGGTAGGCA TCTACCTGCT C 21

CGCCCCCATC AGGGGGCTGG C 21

TTCATGGTGG AGTGTCGCCC C 21

GTTCCTCACA GGGGAGTGAT T 21

TACTAACGCC ATGGCTAGAC G 21

CTATGGCTCT CCCGGGAGGG G 21

CCACTATGGC TCTCCCGGGA G 21

CGGTGTACTC ACCGGTTCCG C 21

CTGGCAATTC CGGTGTACTC A 21

GGGGCACGCC CAAATCTCCA G 21

CCTTTCGCGA CCCAACACTA C 21

CCCTATCAGG CAGTACCACA A 21

CTCCCGGGGC ACTCGCAAGC A 21

CATGGTGCAC GGTCTACGAG A 21

GATTCGTGCT CATGGTGCAC G 21

TTTAGGATTC GTGCTCATGG T 21

GAGTGGTTAG CCCAATCTTC A 21

TATTGGCCTG GAGTGGTTAG C 21

AGGGAATGGC CTATTGGCCT G 21

gccagccccc tgatgggggc gacactccac catgaatcac tcccctgtga 50

ggaactactg tcttcacgca gaaagcgtct agccatggcg ttagtatgag 100

tgtcgtgcag cctccaggac cccccctccc gggagagcca tagtggtctg 150

cggaaccggt gagtacaccg gaattgccag gacgaccggg tcctttcttg 200

gatcaacccg ctcaatgcct ggagatttgg gcgtgccccc gcaagactgc 250

tagccgagta gtgttgggtc gcgaaaggcc ttgtggtact gcctgatagg 300

gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac catgagcacg 350

aatcctaaac ctcaaaaaaa aaacaaacgt aacaccaacc gtcgcccaca 400

ggacgtcaag ttcccgggtg gcggtcagat cgttggtgga gtttacttgt 450

tgccgcgcag gggccctaga ttgggtgtgc gcgcgacgag aaagacttcc 500

gagcggtcgc aacctcgagg tagacgtcag cctatcccca aggctcgtcg 550

gcccgagggc aggacctggg ctcagcccgg gtacccttgg cccctctatg 600

gcaatgaggg ctgcgggtgg gcgggatggc tcctgtctcc ccgtggctct 650

cggcctagct ggggccccac agacccccgg cgtaggtcgc gcaatttggg 700

taaggtcatc gataccctta cgtgcggctt cgccgacctc atggggtaca 750

taccgctcgt cggcgcccct cttggaggcg ctgccagggc cctggcgcat 800

ggcgtccggg ttctggaaga cggcgtgaac tatgcaacag ggaaccttcc 850

tggttgctct ttctctatct tccttctggc cctgctctct tgcttgactg 900

tgcccgcttc ggcctaccaa gtgcgcaact ccacggggct ttaccacgtc 950

accaatgatt gccctaactc gagtattgtg tacgaggcgg ccgatgccat 1000

cctgcacact ccggggtgcg tcccttgcgt tcgtgagggc aacgcctcga 1050

ggtgttgggt ggcgatgacc cctacggtgg ccaccaggga tggcaaactc 1100

cccgcgacgc agcttcgacg tcacatcgat ctgcttgtcg ggagcgccac 1150

cctctgttcg gccctctacg tgggggacct atgcgggtct gtctttcttg 1200

tcggccaact gttcaccttc tctcccaggc gccactggac gacgcaaggt 1250

tgcaattgct ctatctatcc cggccatata acgggtcacc gcatggcatg 1300

ggatatgatg atgaactggt cccctacgac ggcgttggta atggctcagc 1350

tgctccggat cccacaagcc atcttggaca tgatcgctgg tgctcactgg 1400

ggagtcctgg cgggcatagc gtatttctcc atggtgggga actgggcgaa 1450

ggtcctggta gtgctgctgc tatttgccgg cgtcgacgcg gaaacccacg 1500

tcaccggggg aagtgccggc cacactgtgt ctggatttgt tagcctcctc 1550

gcaccaggcg ccaagcagaa cgtccagctg atcaacacca acggcagttg 1600

gcacctcaat agcacggccc tgaactgcaa tgatagcctc aacaccggct 1650

ggttggcagg gcttttctat caccacaagt tcaactcttc aggctgtcct 1700

gagaggctag ccagctgccg accccttacc gattttgacc agggctgggg 1750

ccctatcagt tatgccaacg gaagcggccc cgaccagcgc ccctactgct 1800

ggcactaccc cccaaaacct tgcggtattg tgcccgcgaa gagtgtgtgt 1850

ggtccggtat attgcttcac tcccagcccc gtggtggtgg gaacgaccga 1900

caggtcgggc gcgcccacct acagctgggg tgaaaatgat acggacgtct 1950

tcgtccttaa caataccagg ccaccgctgg gcaattggtt cggttgtacc 2000

tggatgaact caactggatt caccaaagtg tgcggagcgc ctccttgtgt 2050

catcggaggg gcgggcaaca acaccctgca ctgccccact gattgcttcc 2100

gcaagcatcc ggacgccaca tactctcggt gcggctccgg tccctggatc 2150

acacccaggt gcctggtcga ctacccgtat aggctttggc attatccttg 2200

taccatcaac tacaccatat ttaaaatcag gatgtacgtg ggaggggtcg 2250

aacacaggct ggaagctgcc tgcaactgga cgcggggcga acgttgcgat 2300

ctggaagaca gggacaggtc cgagctcagc ccgttactgc tgaccactac 2350

acagtggcag gtcctcccgt gttccttcac aaccctacca gccttgtcca 2400

ccggcctcat ccacctccac cagaacattg tggacgtgca gtacttgtac 2450

ggggtggggt caagcatcgc gtcctgggcc attaagtggg agtacgtcgt 2500

tctcctgttc cttctgcttg cagacgcgcg cgtctgctcc tgcttgtgga 2550

tgatgctact catatcccaa gcggaggcgg ctttggagaa cctcgtaata 2600

cttaatgcag catccctggc cgggacgcac ggtcttgtat ccttcctcgt 2650

gttcttctgc tttgcatggt atttgaaggg taagtgggtg cccggagcgg 2700

tctacacctt ctacgggatg tggcctctcc tcctgctcct gttggcgttg 2750

ccccagcggg cgtacgcgct ggacacggag gtggccgcgt cgtgtggcgg 2800

tgttgttctc gtcgggttga tggcgctgac tctgtcacca tattacaagc 2850

gctatatcag ctggtgcttg tggtggcttc agtattttct gaccagagtg 2900

gaagcgcaac tgcacgtgtg gattcccccc ctcaacgtcc gaggggggcg 2950

cgacgccgtc atcttactca tgtgtgctgt acacccgact ctggtatttg 3000

acatcaccaa attgctgctg gccgtcttcg gacccctttg gattcttcaa 3050

gccagtttgc ttaaagtacc ctactttgtg cgcgtccaag gccttctccg 3100

gttctgcgcg ttagcgcgga agatgatcgg aggccattac gtgcaaatgg 3150

tcatcattaa gttaggggcg cttactggca cctatgttta taaccatctc 3200

actcctcttc gggactgggc gcacaacggc ttgcgagatc tggccgtggc 3250

›Example 6 · 2 of 6

tgtagagcca gtcgtcttct cccaaatgga gaccaagctc atcacgtggg 3300

gggcagatac cgccgcgtgc ggtgacatca tcaacggctt gcctgtttcc 3350

gcccgcaggg gccgggagat actgctcggg ccagccgatg gaatggtctc 3400

caaggggtgg aggttgctgg cgcccatcac ggcgtacgcc cagcagacaa 3450

ggggcctcct agggtgcata atcaccagcc taactggccg ggacaaaaac 3500

caagtggagg gtgaggtcca gattgtgtca actgctgccc aaaccttcct 3550

ggcaacgtgc atcaatgggg tgtgctggac tgtctaccac ggggccggaa 3600

cgaggaccat cgcgtcaccc aagggtcctg tcatccagat gtataccaat 3650

gtagaccaag accttgtggg ctggcccgct ccgcaaggta gccgctcatt 3700

gacaccctgc acttgcggct cctcggacct ttacctggtc acgaggcacg 3750

ccgatgtcat tcccgtgcgc cggcggggtg atagcagggg cagcctgctg 3800

tcgccccggc ccatttccta cttgaaaggc tcctcggggg gtccgctgtt 3850

gtgccccgcg gggcacgccg tgggcatatt tagggccgcg gtgtgcaccc 3900

gtggagtggc taaggcggtg gactttatcc ctgtggagaa cctagagaca 3950

accatgaggt ccccggtgtt cacggataac tcctctccac cagtagtgcc 4000

ccagagcttc caggtggctc acctccatgc tcccacaggc agcggcaaaa 4050

gcaccaaggt cccggctgca tatgcagctc agggctataa ggtgctagta 4100

ctcaacccct ctgttgctgc aacactgggc tttggtgctt acatgtccaa 4150

ggctcatggg atcgatccta acatcaggac cggggtgaga acaattacca 4200

ctggcagccc catcacgtac tccacctacg gcaagttcct tgccgacggc 4250

gggtgctcgg ggggcgctta tgacataata atttgtgacg agtgccactc 4300

cacggatgcc acatccatct tgggcatcgg cactgtcctt gaccaagcag 4350

agactgcggg ggcgagactg gttgtgctcg ccaccgccac ccctccgggc 4400

tccgtcactg tgccccatcc caacatcgag gaggttgctc tgtccaccac 4450

cggagagatc cctttttacg gcaaggctat ccccctcgaa gtaatcaagg 4500

gggggagaca tctcatcttc tgtcattcaa agaagaagtg cgacgaactc 4550

gccgcaaagc tggtcgcatt gggcatcaat gccgtggcct actaccgcgg 4600

tcttgacgtg tccgtcatcc cgaccagcgg cgatgttgtc gtcgtggcaa 4650

ccgatgccct catgaccggc tataccggcg acttcgactc ggtgatagac 4700

tgcaatacgt gtgtcaccca gacagtcgat ttcagccttg accctacctt 4750

caccattgag acaatcacgc tcccccagga tgctgtctcc cgcactcaac 4800

gtcggggcag gactggcagg gggaagccag gcatctacag atttgtggca 4850

ccgggggagc gcccctccgg catgttcgac tcgtccgtcc tctgtgagtg 4900

ctatgacgca ggctgtgctt ggtatgagct cacgcccgcc gagactacag 4950

ttaggctacg agcgtacatg aacaccccgg ggcttcccgt gtgccaggac 5000

catcttgaat tttgggaggg cgtctttaca ggcctcactc atatagatgc 5050

ccactttcta tcccagacaa agcagagtgg ggagaacctt ccttacctgg 5100

tagcgtacca agccaccgtg tgcgctaggg ctcaagcccc tcccccatcg 5150

tgggaccaga tgtggaagtg tttgattcgc ctcaagccca ccctccatgg 5200

gccaacaccc ctgctataca gactgggcgc tgttcagaat gaaatcaccc 5250

tgacgcaccc agtcaccaaa tacatcatga catgcatgtc ggccgacctg 5300

gaggtcgtca cgagcacctg ggtgctcgtt ggcggcgtcc tggctgcttt 5350

ggccgcgtat tgcctgtcaa caggctgcgt ggtcatagtg ggcagggtcg 5400

tcttgtccgg gaagccggca atcatacctg acagggaagt cctctaccga 5450

gagttcgatg agatggaaga gtgctctcag cacttaccgt acatcgagca 5500

agggatgatg ctcgccgagc agttcaagca gaaggccctc ggcctcctgc 5550

agaccgcgtc ccgtcaggca gaggttatcg cccctgctgt ccagaccaac 5600

tggcaaaaac tcgagacctt ctgggcgaag catatgtgga acttcatcag 5650

tgggatacaa tacttggcgg gcttgtcaac gctgcctggt aaccccgcca 5700

ttgcttcatt gatggctttt acagctgctg tcaccagccc actaaccact 5750

agccaaaccc tcctcttcaa catattgggg gggtgggtgg ctgcccagct 5800

cgccgccccc ggtgccgcta ctgcctttgt gggcgctggc ttagctggcg 5850

ccgccatcgg cagtgttgga ctggggaagg tcctcataga catccttgca 5900

gggtatggcg cgggcgtggc gggagctctt gtggcattca agatcatgag 5950

cggtgaggtc ccctccacgg aggacctggt caatctactg cccgccatcc 6000

tctcgcccgg agccctcgta gtcggcgtgg tctgtgcagc aatactgcgc 6050

cggcacgttg gcccgggcga gggggcagtg cagtggatga accggctgat 6100

agccttcgcc tcccggggga accatgtttc ccccacgcac tacgtgccgg 6150

agagcgatgc agctgcccgc gtcactgcca tactcagcag cctcactgta 6200

acccagctcc tgaggcgact gcaccagtgg ataagctcgg agtgtaccac 6250

tccatgctcc ggttcctggc taagggacat ctgggactgg atatgcgagg 6300

tgttgagcga ctttaagacc tggctaaaag ctaagctcat gccacagctg 6350

cctgggatcc cctttgtgtc ctgccagcgc gggtataagg gggtctggcg 6400

agtggacggc atcatgcaca ctcgctgcca ctgtggagct gagatcactg 6450

gacatgtcaa aaacgggacg atgaggatcg tcggtcctag gacctgcagg 6500

aacatgtgga gtgggacctt ccccattaat gcctacacca cgggcccctg 6550

tacccccctt cctgcgccga actacacgtt cgcgctatgg agggtgtctg 6600

cagaggaata tgtggagata aggcaggtgg gggacttcca ctacgtgacg 6650

ggtatgacta ctgacaatct caaatgcccg tgccaggtcc catcgcccga 6700

atttttcaca gaattggacg gggtgcgcct acataggttt gcgcccccct 6750

gcaagccctt gctgcgggag gaggtatcat tcagagtagg actccacgaa 6800

tacccggtag ggtcgcaatt accttgcgag cccgaaccgg acgtggccgt 6850

gttgacgtcc atgctcactg atccctccca tataacagca gaggcggccg 6900

ggcgaaggtt ggcgagggga tcacccccct ctgtggccag ctcctcggct 6950

agccagctat ccgctccatc tctcaaggca acttgcaccg ctaaccatga 7000

ctcccctgat gctgagctca tagaggccaa cctcctatgg aggcaggaga 7050

tgggcggcaa catcaccagg gttgagtcag aaaacaaagt ggtgattctg 7100

gactccttcg atccgcttgt ggcggaggag gacgagcggg agatctccgt 7150

acccgcagaa atcctgcgga agtctcggag attcgcccag gccctgcccg 7200

tttgggcgcg gccggactat aaccccccgc tagtggagac gtggaaaaag 7250

cccgactacg aaccacctgt ggtccatggc tgtccgcttc cacctccaaa 7300

gtcccctcct gtgcctccgc ctcggaagaa gcggacggtg gtcctcactg 7350

aatcaaccct atctactgcc ttggccgagc tcgccaccag aagctttggc 7400

agctcctcaa cttccggcat tacgggcgac aatacgacaa catcctctga 7450

gcccgcccct tctggctgcc cccccgactc cgacgctgag tcctattcct 7500

ccatgccccc cctggagggg gagcctgggg atccggatct tagcgacggg 7550

tcatggtcaa cggtcagtag tgaggccaac gcggaggatg tcgtgtgctg 7600

ctcaatgtct tactcttgga caggcgcact cgtcaccccg tgcgccgcgg 7650

aagaacagaa actgcccatc aatgcactaa gcaactcgtt gctacgtcac 7700

cacaatttgg tgtattccac cacctcacgc agtgcttgcc aaaggcagaa 7750

gaaagtcaca tttgacagac tgcaagttct ggacagccat taccaggacg 7800

tactcaagga ggttaaagca gcggcgtcaa aagtgaaggc taacttgcta 7850

tccgtagagg aagcttgcag cctgacgccc ccacactcag ccaaatccaa 7900

gtttggttat ggggcaaaag acgtccgttg ccatgccaga aaggccgtaa 7950

cccacatcaa ctccgtgtgg aaagaccttc tggaagacaa tgtaacacca 8000

atagacacta ccatcatggc taagaacgag gttttctgcg ttcagcctga 8050

gaaggggggt cgtaagccag ctcgtctcat cgtgttcccc gatctgggcg 8100

tgcgcgtgtg cgaaaagatg gctttgtacg acgtggttac aaagctcccc 8150

ttggccgtga tgggaagctc ctacggattc caatactcac caggacagcg 8200

ggttgaattc ctcgtgcaag cgtggaagtc caagaaaacc ccaatggggt 8250

tctcgtatga tacccgctgc tttgactcca cagtcactga gagcgacatc 8300

cgtacggagg aggcaatcta ccaatgttgt gacctcgacc cccaagcccg 8350

›Example 6 · 3 of 6

cgtggccatc aagtccctca ccgagaggct ttatgttggg ggccctctta 8400

ccaattcaag gggggagaac tgcggctatc gcaggtgccg cgcgagcggc 8450

gtactgacaa ctagctgtgg taacaccctc acttgctaca tcaaggcccg 8500

ggcagcctgt cgagccgcag ggctccagga ctgcaccatg ctcgtgtgtg 8550

gcgacgactt agtcgttatc tgtgaaagcg cgggggtcca ggaggacgcg 8600

gcgagcctga gagccttcac ggaggctatg accaggtact ccgccccccc 8650

tggggacccc ccacaaccag aatacgactt ggagctcata acatcatgct 8700

cctccaacgt gtcagtcgcc cacgacggcg ctggaaagag ggtctactac 8750

ctcacccgtg accctacaac ccccctcgcg agagctgcgt gggagacagc 8800

aagacacact ccagtcaatt cctggctagg caacataatc atgtttgccc 8850

ccacactgtg ggcgaggatg atactgatga cccatttctt tagcgtcctt 8900

atagccaggg accagcttga acaggccctc gattgcgaga tctacggggc 8950

ctgctactcc atagaaccac ttgatctacc tccaatcatt caaagactcc 9000

atggcctcag cgcattttca ctccacagtt actctccagg tgaaattaat 9050

agggtggccg catgcctcag aaaacttggg gtaccgccct tgcgagcttg 9100

gagacaccgg gcccggagcg tccgcgctag gcttctggcc agaggaggca 9150

gggctgccat atgtggcaag tacctcttca actgggcagt aagaacaaag 9200

ctcaaactca ctccaatagc ggccgctggc cagctggact tgtccggctg 9250

gttcacggct ggctacagcg ggggagacat ttatcacagc gtgtctcatg 9300

cccggccccg ctggatctgg ttttgcctac tcctgcttgc tgcaggggta 9350

ggcatctacc tcctccccaa ccgatgaagg ttggggtaaa cactccggcc 9400

t 9401

gccagccccc tgatgggggc gacactccac catgaatcac tcccctgtga 50

ggaactactg tcttcacgca gaaagcgtct agccatggcg ttagtatgag 100

tgtcgtgcag cctccaggac cccccctccc gggagagcca tagtggtctg 150

cggaaccggt gagtacaccg gaattgccag gacgaccggg tcctttcttg 200

gataaacccg ctcaatgcct ggagatttgg gcgtgccccc gcaagactgc 250

tagccgagta gtgttgggtc gcgaaaggcc ttgtggtact gcctgatagg 300

gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac catgagcacg 350

aatcctaaac ctcaaagaaa aaccaaacgt aacaccaacc gtcgcccaca 400

ggacgtcaag ttcccgggtg gcggtcagat cgttggtgga gtttacttgt 450

tgccgcgcag gggccctaga ttgggtgtgc gcgcgacgag gaagacttcc 500

gagcggtcgc aacctcgagg tagacgtcag cctatcccca aggcacgtcg 550

gcccgagggc aggacctggg ctcagcccgg gtacccttgg cccctctatg 600

gcaatgaggg ttgcgggtgg gcgggatggc tcctgtctcc ccgtggctct 650

cggcctagct ggggccccac agacccccgg cgtaggtcgc gcaatttggg 700

taaggtcatc gataccctta cgtgcggctt cgccgacctc atggggtaca 750

taccgctcgt cggcgcccct cttggaggcg ctgccagggc cctggcgcat 800

ggcgtccggg ttctggaaga cggcgtgaac tatgcaacag ggaaccttcc 850

tggttgctct ttctctatct tccttctggc cctgctctct tgcctgactg 900

tgcccgcttc agcctaccaa gtgcgcaatt cctcggggct ttaccatgtc 950

accaatgatt gccctaactc gagtgttgtg tacgaggcgg ccgatgccat 1000

cctgcacact ccggggtgtg tcccttgcgt tcgcgagggt aacgcctcga 1050

ggtgttgggt ggcggtgacc cccacggtgg ccaccaggga cggcaaactc 1100

cccacaacgc agcttcgacg tcatatcgat ctgcttgtcg ggagcgccac 1150

cctctgctcg gccctctacg tgggggacct gtgcgggtct gtctttcttg 1200

ttggtcaact gtttaccttc tctcccaggc accactggac gacgcaagac 1250

tgcaattgtt ctatctatcc cggccatata acgggtcatc gcatggcatg 1300

gaatatgatg atgaactggt cccctacggc agcgttggtg gtagctcagc 1350

tgctccgaat cccacaagcc atcatggaca tgatcgctgg cgcccactgg 1400

ggagtcctgg cgggcataaa gtatttctcc atggtgggga actgggcgaa 1450

ggtcctggta gtgctgctgc tatttgccgg cgtcgacgcg gaaacccacg 1500

tcaccggggg aaatgccggc cgcaccacgg ctgggcttgt tggtctcctt 1550

acaccaggcg ccaagcagaa catccaactg atcaacacca acggcagttg 1600

gcacatcaat agcacggcct tgaactgcaa tgaaagcctt aacaccggct 1650

ggttagcagg gctcttctat cagcacaaat tcaactcttc aggctgtcct 1700

gagaggttgg ccagctgccg acgccttacc gattttgccc agggctgggg 1750

tcctatcagt tatgccaacg gaagcggcct cgacgaacgc ccctactgct 1800

ggcactaccc tccaagacct tgtggcattg tgcccgcaaa gagcgtgtgt 1850

ggcccggtat attgcttcac tcccagcccc gtggtggtgg gaacgaccga 1900

caggtcgggc gcgcctacct acagctgggg tgcaaatgat acggatgtct 1950

tcgtccttaa caacaccagg ccaccgctgg gcaattggtt cggttgtacc 2000

tggatgaact caactggatt caccaaagtg tgcggagcgc ccccttgtgt 2050

catcggaggg gtgggcaaca acaccttgct ctgccccact gattgcttcc 2100

gcaaatatcc ggaagccaca tactctcggt gcggctccgg tcccaggatt 2150

acacccaggt gcatggtcga ctacccgtat aggctttggc actatccttg 2200

taccatcaat tacaccatat tcaaagtcag gatgtacgtg ggaggggtcg 2250

agcacaggct ggaagcggcc tgcaactgga cgcggggcga acgctgtgat 2300

ctggaagaca gggacaggtc cgagctcagc ccgttgctgc tgtccaccac 2350

acagtggcag gtccttccgt gttctttcac gaccctgcca gccttgtcca 2400

ccggcctcat ccacctccac cagaacattg tggacgtgca gtacttgtac 2450

ggggtagggt caagcatcgc gtcctgggcc attaagtggg agtacgtcgt 2500

tctcctgttc cttctgcttg cagacgcgcg cgtctgttcc tgcttgtgga 2550

tgatgttact catatcccaa gcggaggcgg ctttggagaa cctcgtaata 2600

ctcaatgcag catccctggc cgggacgcat ggtcttgtgt ccttcctcgt 2650

gttcttctgc tttgcgtggt atctgaaggg taggtgggtg cccggagcgg 2700

tctacgccct ctacgggatg tggcctctcc tcctgctcct gctggcgttg 2750

cctcagcggg catacgcact ggacacggag gtggccgcgt cgtgtggcgg 2800

cgttgttctt gtcgggttaa tggcgctgac tctgtcgcca tattacaagc 2850

gctatatcag ctggtgcatg tggtggcttc agtattttct gaccagagta 2900

gaagcgcaac tgcacgtgtg ggttcccccc ctcaacgtcc ggggggggcg 2950

cgatgccgtc atcttactca cgtgtgtagt acacccggcc ctggtatttg 3000

acatcaccaa actactcctg gccatcttcg gacccctttg gattcttcaa 3050

gccagtttgc ttaaagtccc ctacttcgtg cgcgttcaag gccttctccg 3100

gatctgcgcg ctagcgcgga agatagccgg aggtcattac gtgcaaatgg 3150

ccatcatcaa gttaggggcg cttactggca cctgtgtgta taaccatctc 3200

gctcctcttc gagactgggc gcacaacggc ctgcgagatc tggccgtggc 3250

tgtggaacca gtcgtcttct cccgaatgga gaccaagctc atcacgtggg 3300

gggcagatac cgccgcgtgc ggtgacatca tcaacggctt gcccgtctct 3350

gcccgtaggg gccaggagat actgcttggg ccagccgacg gaatggtctc 3400

caaggggtgg aggttgctgg cgcccatcac ggcgtacgcc cagcagacga 3450

gaggcctcct agggtgtata atcaccagcc tgactggccg ggacaaaaac 3500

caagtggagg gtgaggtcca gatcgtgtca actgctaccc agaccttcct 3550

ggcaacgtgc atcaatgggg tatgctggac tgtctaccac ggggccggaa 3600

cgaggaccat cgcatcaccc aagggtcctg tcatccagac gtataccaat 3650

gtggatcaag acctcgtggg ctggcccgct cctcaaggtt cccgctcatt 3700

gacaccctgc acctgcggct cctcggacct ttacctggtc acgaggcacg 3750

ccgatgtcat tcccgtgcgc cggcgaggtg atagcagggg tagcctgctt 3800

tcgccccggc ccatttccta cttgaaaggc tcctcggggg gtccgctgtt 3850

gtgccccacg ggacacgccg tgggcctatt cagggccgcg gtgtgcaccc 3900

gtggagtggc taaggcggtg gactttatcc ctgtggagaa cctagagaca 3950

accatgagat ccccggtgtt cacggacaac tcctctccac cagcagtgcc 4000

ccagagcttc caggtggccc acctgcatgc tcccaccggc agcggtaaga 4050

›Example 6 · 4 of 6

gcaccaaggt cccggctgcg tacgcagcca agggctacaa ggtgttggtg 4100

ctcaacccct ctgttgctgc aacactgggc tttggtgctt acatgtccaa 4150

ggcccatggg gttgatccta atatcaggac cggggtgaga acaattacca 4200

ctggcagccc catcacgtac tccacctacg gcaagttcct tgccgacgcc 4250

gggtgctcag gaggtgctta tgacataata atttgtgacg agtgccactc 4300

cacggatgcc acatccatct cgggcatcgg cactgtcctt gaccaagcag 4350

agactgcggg ggcgagactg gttgtgctcg ccactgctac ccctccgggc 4400

tccgtcactg tgtcccatcc taacatcgag gaggttgctc tgtccaccac 4450

cggagagatc cccttttacg gcaaggctat ccccctcgag gtgatcaagg 4500

ggggaagaca tctcatcttc tgccactcaa agaagaagtg cgacgagctc 4550

gccgcgaagc tggtcgcatt gggcatcaat gccgtggcct actaccgcgg 4600

tcttgacgtg tctgtcatcc cgaccagcgg cgatgttgtc gtcgtgtcga 4650

ccgatgctct catgactggc tttaccggcg acttcgactc tgtgatagac 4700

tgcaacacgt gtgtcactca gacagtcgat tttagccttg accctacctt 4750

taccattgag acaaccacgc tcccccagga tgctgtctcc aggactcaac 4800

gccggggcag gactggcagg gggaagccag gcatctatag atttgtggca 4850

ccgggggagc gcccctccgg catgttcgac tcgtccgtcc tctgtgagtg 4900

ctatgacgcg ggctgtgctt ggtatgagct cacgcccgcc gagactacag 4950

ttaggctacg agcgtacatg aacaccccgg ggcttcccgt gtgccaggac 5000

catcttggat tttgggaggg cgtctttacg ggcctcactc atatagatgc 5050

ccactttcta tcccagacaa agcagagtgg ggagaacttt ccttacctgg 5100

tagcgtacca agccaccgtg tgcgctaggg ctcaagcccc tcccccatcg 5150

tgggaccaga tgcggaagtg tttgatccgc cttaaaccca ccctccatgg 5200

gccaacaccc ctgctataca gactgggcgc tgttcagaat gaagtcaccc 5250

tgacgcaccc aatcaccaaa tacatcatga catgcatgtc ggccgacctg 5300

gaggtcgtca cgagcacctg ggtgctcgtt ggcggcgtcc tggctgctct 5350

ggccgcgtat tgcctgtcaa caggctgcgt ggtcatagtg ggcaggatcg 5400

tcttgtccgg gaagccggca attatacctg acagggaggt tctctaccag 5450

gagttcgatg agatggaaga gtgctctcag cacttaccgt acatcgagca 5500

agggatgatg ctcgctgagc agttcaagca gaaggccctc ggcctcctgc 5550

agaccgcgtc ccgccatgca gaggttatca cccctgctgt ccagaccaac 5600

tggcagaaac tcgaggtctt ttgggcgaag cacatgtgga atttcatcag 5650

tgggatacaa tacttggcgg gcctgtcaac gctgcctggt aaccccgcca 5700

ttgcttcatt gatggctttt acagctgccg tcaccagccc actaaccact 5750

ggccaaaccc tcctcttcaa catattgggg gggtgggtgg ctgcccagct 5800

cgccgccccc ggtgccgcta ccgcctttgt gggcgctggc ttagctggcg 5850

ccgcactcga cagcgttgga ctggggaagg tcctcgtgga cattcttgca 5900

ggctatggcg cgggcgtggc gggagctctt gtggcattca agatcatgag 5950

cggtgaggtc ccctccacgg aggacctggt caatctgctg cccgccatcc 6000

tctcacctgg agcccttgca gtcggtgtgg tctttgcatc aatactgcgc 6050

cggcgtgttg gcccgggcga gggggcagtg caatggatga accggctaat 6100

agccttcgcc tcccggggga accatgtttc ccccacacac tacgtgccgg 6150

agagcgatgc agccgcccgc gtcactgcca tactcagcag cctcactgta 6200

acccagctcc tgaggcgact gcatcagtgg ataagctcgg agtgtaccac 6250

tccatgctcc ggttcctggc taagggacat ctgggactgg atatgcgagg 6300

tgctgagcga ctttaagacc tggctgaaag ccaagctcat gccacaactg 6350

cctgggattc cctttgtgtc ctgccagcgc gggtataggg gggtctggcg 6400

aggagacggc attatgcaca ctcgctgcca ctgtggagct gagatcactg 6450

gacatgtcaa aaacgggacg atgaggatcg tcggtcctag gacctgcaag 6500

aacatgtgga gtgggacgtt cttcattaat gcctacacca cgggcccctg 6550

tactcccctt cctgcgccga actataagtt cgcgctgtgg agggtgtctg 6600

cagaggaata cgtggagata aggcgggtgg gggacttcca ctacgtatcg 6650

ggcatgacta ctgacaatct caaatgcccg tgccagatcc catcgcccga 6700

atttttcaca gaattggacg gggtgcgcct acataggttt gcgccccctt 6750

gcaagccctt gctgcgggag gaggtatcat tcagagtagg actccacgag 6800

tacccggtgg ggtcgcaatt accttgcgag cccgaaccgg acgtagccgt 6850

gttgacgtcc atgctcactg atccctccca tataacagca gaggcggccg 6900

ggagaaggtt ggcgagaggg tcaccccctt ctatggccag ctcctcggct 6950

agccagctgt ccgctccatc tctcaaggca acttgcaccg ccaaccatga 7000

ctcccctgac gccgagctca tagaggctaa cctcctgtgg aggcaggaga 7050

tgggcggcaa catcaccagg gttgagtcag agaacaaagt ggtgattctg 7100

gactccttcg atccgcttgt ggcagaggag gatgagcggg aggtctccgt 7150

acccgcagaa attctgcgga agtctcggag attcgcccca gccctgcccg 7200

tctgggcgcg gccggactac aaccccctgc tagtagagac gtggaaaaag 7250

cctgactacg aaccacctgt ggtccatggc tgcccgctac cacctccacg 7300

gtcccctcct gtgcctccgc ctcggaaaaa gcgtacggtg gtcctcaccg 7350

aatcaaccct acctactgcc ttggccgagc ttgccaccaa aagttttggc 7400

agctcctcaa cttccggcat tacgggcgac aatacgacaa catcctctga 7450

gcccgcccct tctggctgcc cccccgactc cgacgttgag tcctattctt 7500

ccatgccccc cctggagggg gagcctgggg atccggatct cagcgacggg 7550

tcatggtcga cggtcagtag tggggccgac acggaagatg tcgtgtgctg 7600

ctcaatgtct tattcctgga caggcgcact cgtcaccccg tgcgctgcgg 7650

aggaacaaaa actgcccatc aacgcactga gcaactcgtt gctacgccat 7700

cacaatctgg tgtattccac cacttcacgc agtgcttgcc aaaggaagaa 7750

gaaagtcaca tttgacagac tgcaagttct ggacagccat taccaggacg 7800

tgctcaagga ggtcaaagca gcggcgtcaa aagtgaaggc taacttgcta 7850

tccgtagagg aagcttgcag cctggcgccc ccacattcag ccaaatccaa 7900

gtttggctat ggggcaaaag acgtccgttg ccatgccaga aaggccgtag 7950

cccacatcaa ctccgtgtgg aaagaccttc tggaagacag tgtaacacca 8000

atagacacta ccatcatggc caagaacgag gttttctgcg ttcagcctga 8050

gaaggggggt cgtaagccag ctcgtctcat cgtgttcccc gacctgggcg 8100

tgcgcgtgtg cgagaagatg gccctgtacg acgtggttag caagctcccc 8150

ttggccgtga tgggaagctc ctacggattc caatactcac caggacagcg 8200

ggttgaattc ctcgtgcaag cgtggaagtc caagaagacc ccgatggggc 8250

tctcgtatga tacccgctgt tttgactcca cagtcactga gagcgacatc 8300

cgtacggagg aggcaattta ccaatgttgt gacctggacc cccaagcccg 8350

cgtggccatc aagtccctca ctgagaggct ttatgttggg ggccctctta 8400

ctaattcaag gggggaaaac tgcggctacc gcaggtgccg cgcgagcaga 8450

gtactgacaa ctagctgtgg taacaccctc actcgctaca tcaaggcccg 8500

ggcagcctgt cgagccgcag ggctccagga ctgcaccatg ctcgtgtgtg 8550

gcgacgactt agtcgttatc tgtgaaagtg cgggggtcca ggaggacgcg 8600

gcgagcctga gagccttcac ggaggctatg accaggtact ccgccccccc 8650

cggggacccc ccacaaccag aatacgactt ggagcttata acatcatgct 8700

cctccaacgt gtcagtcgcc cacgacggcg ctggaaagag ggtctactac 8750

cttacccgtg accctacaac ccccctcgcg agagccgcgt gggagacagc 8800

aagacacact ccagtcaatt cctggctagg caacataatc atgtttgccc 8850

ccacactgtg ggcgaggatg atactgatga cccacttctt tagcgtcctc 8900

atagccaggg atcagcttga acaggctctc aactgcgaga tctacggagc 8950

ctgctactcc atagaaccac tggatctacc tccaatcatt caaagactcc 9000

atggcctcag cgcattttca ctccacagtt actctccagg tgaaattaat 9050

agggtggccg catgcctcag aaaacttggg gtcccgccct tgcgagcttg 9100

gagacaccgg gcctggagcg tccgcgctag gcttctggcc agaggaggca 9150

›Example 6 · 5 of 6

aggctgccat atgtggcaag tacctcttca actgggcagt aagaacaaag 9200

ctcaaactca ctccgataac ggccgctggc cggctggact tgtccggctg 9250

gttcacggct ggctacagcg ggggagacat ttatcacagc gtgtctcatg 9300

cccggccccg ctggttctgg ttttgcctac tcctgcttgc tgcaggggta 9350

ggcatctacc tcctccccaa ccgatgaaga ttgggctaac cactccaggc 9400

caataggcca ttccct 9416

cgattggggg cgacactcca ccatagatca ctcccctgtg aggaactact 50

gtcttcacgc agaaagcgtc tagccatggc gttagtatga gtgtcgtgca 100

gcctccagga ccccccctcc cgggagagcc atagtggtct gcggaaccgg 150

tgagtacacc ggaattgcca ggacgaccgg gtcctttctt ggatcaaccc 200

gctcaatgcc tggagatttg ggcgtgcccc cgcgagactg ctagccgagt 250

agtgttgggt cgcgaaaggc cttgtggtac tgcctgatag ggtgcttgcg 300

agtgccccgg gaggtctcgt agaccgtgca ccatgagcac gaatcctaaa 350

cctcaaagaa aaaccaaacg taacaccaac cgccgcccac aggacgtcaa 400

gttcccgggc ggtggtcaga tcgttggtgg agtttacctg ttgccgcgca 450

ggggccccag gttgggtgtg cgcgcgccca ggaagacttc cgagcggtcg 500

caacctcgtg gaaggcgaca acctatcccc aaggctcgcc ggcccgaggg 550

caggacctgg gctcagcccg ggtacccttg gcctctctat ggcaatgagg 600

gcttagggtg ggcaggatgg ctcctgtcac cccgcggctc ccggcctagt 650

tggggcccca cggacccccg gcgtaggtcg cgtaatttgg gtaaggtcat 700

cgataccctc acatgcggct tcgccgatct catggggtac attccgctcg 750

tcggcgcccc cctggggggc gctgccaggg ccctggcaca tggtgtccgg 800

gttctggagg acggcgtgaa ctatgcaaca gggaatctgc ccggttgctc 850

tttttctatc ttcctcttgg ctctgctgtc ctgcctgacc accccagctt 900

ccgcttacga agtgcacaac gtgtccggga tatatcatgt cacgaacgac 950

tgctccaacg caagcattgt gtatgaggca gcggacttga tcatgcatac 1000

tcctgggtgc gtgccctgcg ttcgggaagg caactcctcc cgctgctggg 1050

tagcgctcac tcccacgctc gcagccagga acgtcaccat ccccaccacg 1100

acgatacgac gccacgtcga tctgctcgtt ggggcggctg ctttctgttc 1150

cgctatgtac gtgggggacc tctgcggatc tgttttcctc gtctctcagc 1200

tgttcacctt ctcgcctcgc cggcatgtga cattacagga ctgtaactgc 1250

tcaatttatc ccggccatgt gtcgggtcac cgtatggctt gggacatgat 1300

gatgaactgg tcgcccacaa cagccctagt ggtgtcgcag ttactccgga 1350

tcccacaagc cgtcgtggac atggtggcgg gggcccactg gggagtcctg 1400

gcgggccttg cctactattc catggcgggg aactgggcta aggttctgat 1450

tgtgatgcta ctttttgctg gcgttgacgg ggatacccac gtgacagggg 1500

gggcgcaagc caaaaccacc aacaggctcg tgtccatgtt cgcaagtggg 1550

ccgtctcaga aaatccagct tataaacacc aatgggagtt ggcacatcaa 1600

caggactgcc ctgaactgca atgactctct ccagactggg tttcttgccg 1650

cgctgttcta cacacatagt ttcaactcgt ccgggtgccc agagcgcatg 1700

gcccagtgcc gcaccattga caagttcgac cagggatggg gtcccattac 1750

ttatgctgag tctagcagat cagaccagag gccatattgc tggcactacc 1800

cacctccaca atgtaccatc gtacctgcgt cggaggtgtg cggcccagtg 1850

tactgcttca ccccaagccc tgtcgtcgtg gggacgaccg atcgtttcgg 1900

tgtccctacg tatagatggg gggagaacga gactgacgtg ctgctgctca 1950

acaacacgcg gccgccgcaa ggcaactggt tcggctgcac atggatgaat 2000

agcaccgggt tcaccaagac atgtgggggg cccccgtgta acatcggggg 2050

ggtcggcaac aacaccctga cctgccccac ggactgcttc cggaagcacc 2100

ccgaggctac ctacacaaaa tgtggttcgg ggccttggct gacacctagg 2150

tgcatggttg actatccata caggctctgg cattacccct gcactgttaa 2200

ctttaccatc ttcaaggtta ggatgtatgt ggggggggtg gagcacaggc 2250

tcaatgctgc atgcaattgg acccgaggag agcgttgtga cttggaggac 2300

agggataggc cggagctcag cccgctgctg ctgtctacaa cagagtggca 2350

ggtactgccc tgttccttca ccaccctacc agctctgtcc actggcttga 2400

ttcacctcca tcagaacatc gtggacgtgc aatacctata cggtataggg 2450

tcagcggttg tctcctttgc aatcaaatgg gagtatgtcc tgttgctttt 2500

ccttctccta gcggacgcac gtgtctgtgc ctgcttgtgg atgatgctgc 2550

tgatagccca ggccgaggcc gccttggaga acctggtggt cctcaattcg 2600

gcgtctgtgg ccggcgcaca tggcatcctc tccttccttg tgttcttctg 2650

tgccgcctgg tacatcaaag gcaggctggt ccctggggcg acatatgctc 2700

tttatggcgt gtggccgctg ctcctgctct tgctggcatt accaccgcga 2750

gcttacgcca tggaccggga gatggctgca tcgtgcggag gcgcggtttt 2800

tgtgggtctg gtactcctga ctttgtcacc atactacaag gtgttcctcg 2850

ctaggctcat atggtggtta caatatttta ccaccagagc cgaggcggac 2900

ttacatgtgt ggatcccccc cctcaacgct cggggaggcc gcgatgccat 2950

catcctcctc atgtgcgcag tccatccaga gctaatcttt gacatcacca 3000

aacttctaat tgccatactc ggtccgctca tggtgctcca agctggcata 3050

accagagtgc cgtacttcgt gcgcgctcaa gggctcattc atgcatgcat 3100

gttagtgcgg aaggtcgctg ggggtcatta tgtccaaatg gccttcatga 3150

agctgggcgc gctgacaggc acgtacattt acaaccatct taccccgcta 3200

cgggattggc cacgcgcggg cctacgagac cttgcggtgg cagtggagcc 3250

cgtcgtcttc tccgacatgg agaccaagat catcacctgg ggagcagaca 3300

ccgcggcgtg tggggacatc atcttgggtc tgcccgtctc cgcccgaagg 3350

ggaaaggaga tactcctggg cccggccgat agtcttgaag ggcgggggtt 3400

gcgactcctc gcgcccatca cggcctactc ccaacagacg cggggcctac 3450

ttggttgcat catcactagc cttacaggcc gggacaagaa ccaggtcgag 3500

ggagaggttc aggtggtttc caccgcaaca caatccttcc tggcgacctg 3550

cgtcaacggc gtgtgttgga ccgtttacca tggtgctggc tcaaagacct 3600

tagccgcgcc aaaggggcca atcacccaga tgtacactaa tgtggaccag 3650

gacctcgtcg gctggcccaa gccccccggg gcgcgttcct tgacaccatg 3700

cacctgtggc agctcagacc tttacttggt cacgagacat gctgacgtca 3750

ttccggtgcg ccggcggggc gacagtaggg ggagcctgct ctcccccagg 3800

cctgtctcct acttgaaggg ctcttcgggt ggtccactgc tctgcccctt 3850

cgggcacgct gtgggcatct tccgggctgc cgtatgcacc cggggggttg 3900

cgaaggcggt ggactttgtg cccgtagagt ccatggaaac tactatgcgg 3950

tctccggtct tcacggacaa ctcatccccc ccggccgtac cgcagtcatt 4000

tcaagtggcc cacctacacg ctcccactgg cagcggcaag agtactaaag 4050

tgccggctgc atatgcagcc caagggtaca aggtgctcgt cctcaatccg 4100

tccgttgccg ctaccttagg gtttggggcg tatatgtcta aggcacacgg 4150

tattgacccc aacatcagaa ctggggtaag gaccattacc acaggcgccc 4200

ccgtcacata ctctacctat ggcaagtttc ttgccgatgg tggttgctct 4250

gggggcgctt atgacatcat aatatgtgat gagtgccatt caactgactc 4300

gactacaatc ttgggcatcg gcacagtcct ggaccaagcg gagacggctg 4350

gagcgcggct tgtcgtgctc gccaccgcta cgcctccggg atcggtcacc 4400

gtgccacacc caaacatcga ggaggtggcc ctgtctaata ctggagagat 4450

ccccttctat ggcaaagcca tccccattga agccatcagg gggggaaggc 4500

atctcatttt ctgtcattcc aagaagaagt gcgacgagct cgccgcaaag 4550

ctgtcaggcc tcggaatcaa cgctgtggcg tattaccggg ggctcgatgt 4600

gtccgtcata ccaactatcg gagacgtcgt tgtcgtggca acagacgctc 4650

tgatgacggg ctatacgggc gactttgact cagtgatcga ctgtaacaca 4700

tgtgtcaccc agacagtcga cttcagcttg gatcccacct tcaccattga 4750

gacgacgacc gtgcctcaag acgcagtgtc gcgctcgcag cggcggggta 4800

ggactggcag gggtaggaga ggcatctaca ggtttgtgac tccgggagaa 4850

›Example 6 · 6 of 6

cggccctcgg gcatgttcga ttcctcggtc ctgtgtgagt gctatgacgc 4900

gggctgtgct tggtacgagc tcaccccggc cgagacctcg gttaggttgc 4950

gggcctacct gaacacacca gggttgcccg tttgccagga ccacctggag 5000

ttctgggaga gtgtcttcac aggcctcacc catatagatg cacacttctt 5050

gtcccagacc aagcaggcag gagacaactt cccctacctg gtagcatacc 5100

aagccacggt gtgcgccagg gctcaggccc cacctccatc atgggatcaa 5150

atgtggaagt gtctcatacg gctgaaacct acgctgcacg ggccaacacc 5200

cttgctgtac aggctgggag ccgtccagaa tgaggtcacc ctcacccacc 5250

ccataaccaa atacatcatg gcatgcatgt cggctgacct ggaggtcgtc 5300

actagcacct gggtgctggt gggcggagtc cttgcagctc tggccgcgta 5350

ttgcctgaca acaggcagtg tggtcattgt gggtaggatt atcttgtccg 5400

ggaggccggc cattgttccc gacagggagc ttctctacca ggagttcgat 5450

gaaatggaag agtgcgcctc gcacctccct tacatcgagc agggaatgca 5500

gctcgccgag caattcaagc agaaagcgct cgggttactg caaacagcca 5550

ccaaacaagc ggaggctgct gctcccgtgg tggagtccaa gtggcgagcc 5600

cttgagacat tctgggcgaa gcacatgtgg aatttcatca gcgggataca 5650

gtacttagca ggcttatcca ctctgcctgg gaaccccgca atagcatcat 5700

tgatggcatt cacagcctct atcaccagcc cgctcaccac ccaaagtacc 5750

ctcctgttta acatcttggg ggggtgggtg gctgcccaac tcgccccccc 5800

cagcgccgct tcggctttcg tgggcgccgg catcgccggt gcggctgttg 5850

gcagcatagg ccttgggaag gtgcttgtgg acattctggc gggttatgga 5900

gcaggagtgg ccggcgcgct cgtggccttt aaggtcatga gcggcgagat 5950

gccctccacc gaggacctgg tcaatctact tcctgccatc ctctctcctg 6000

gcgccctggt cgtcggggtc gtgtgtgcag caatactgcg tcgacacgtg 6050

ggtccgggag agggggctgt gcagtggatg aaccggctga tagcgttcgc 6100

ctcgcggggt aatcatgttt cccccacgca ctatgtgcct gagagcgacg 6150

ccgcagcgcg tgttactcag atcctctcca gccttaccat cactcagctg 6200

ctgaaaaggc tccaccagtg gattaatgaa gactgctcca caccgtgttc 6250

cggctcgtgg ctaagggatg tttgggactg gatatgcacg gtgttgactg 6300

acttcaagac ctggctccag tccaagctcc tgccgcagct acctggagtc 6350

ccttttttct cgtgccaacg cgggtacaag ggagtctggc ggggagacgg 6400

catcatgcaa accacctgcc catgtggagc acagatcacc ggacatgtca 6450

aaaacggttc catgaggatc gtcgggccta agacctgcag caacacgtgg 6500

catggaacat tccccatcaa cgcatacacc acgggcccct gcacaccctc 6550

tccagcgcca aactattcta gggcgctgtg gcgggtggcc gctgaggagt 6600

acgtggaggt cacgcgggtg ggggatttcc actacgtgac gggcatgacc 6650

actgacaacg taaagtgccc atgccaggtt ccggctcctg aattcttctc 6700

ggaggtggac ggagtgcggt tgcacaggta cgctccggcg tgcaggcctc 6750

tcctacggga ggaggttaca ttccaggtcg ggctcaacca atacctggtt 6800

gggtcacagc taccatgcga gcccgaaccg gatgtagcag tgctcacttc 6850

catgctcacc gacccctccc acatcacagc agaaacggct aagcgtaggt 6900

tggccagggg gtctcccccc tccttggcca gctcttcagc tagccagttg 6950

tctgcgcctt ccttgaaggc gacatgcact acccaccatg tctctccgga 7000

cgctgacctc atcgaggcca acctcctgtg gcggcaggag atgggcggga 7050

acatcacccg cgtggagtcg gagaacaagg tggtagtcct ggactctttc 7100

gacccgcttc gagcggagga ggatgagagg gaagtatccg ttccggcgga 7150

gatcctgcgg aaatccaaga agttccccgc agcgatgccc atctgggcgc 7200

gcccggatta caaccctcca ctgttagagt cctggaagga cccggactac 7250

gtccctccgg tggtgcacgg gtgcccgttg ccacctatca aggcccctcc 7300

aataccacct ccacggagaa agaggacggt tgtcctaaca gagtcctccg 7350

tgtcttctgc cttagcggag ctcgctacta agaccttcgg cagctccgaa 7400

tcatcggccg tcgacagcgg cacggcgacc gcccttcctg accaggcctc 7450

cgacgacggt gacaaaggat ccgacgttga gtcgtactcc tccatgcccc 7500

cccttgaggg ggaaccgggg gaccccgatc tcagtgacgg gtcttggtct 7550

accgtgagcg aggaagctag tgaggatgtc gtctgctgct caatgtccta 7600

cacatggaca ggcgccttga tcacgccatg cgctgcggag gaaagcaagc 7650

tgcccatcaa cgcgttgagc aactctttgc tgcgccacca taacatggtt 7700

tatgccacaa catctcgcag cgcaggcctg cggcagaaga aggtcacctt 7750

tgacagactg caagtcctgg acgaccacta ccgggacgtg ctcaaggaga 7800

tgaaggcgaa ggcgtccaca gttaaggcta aactcctatc cgtagaggaa 7850

gcctgcaagc tgacgccccc acattcggcc aaatccaagt ttggctatgg 7900

ggcaaaggac gtccggaacc tatccagcaa ggccgttaac cacatccact 7950

ccgtgtggaa ggacttgctg gaagacactg tgacaccaat tgacaccacc 8000

atcatggcaa aaaatgaggt tttctgtgtc caaccagaga aaggaggccg 8050

taagccagcc cgccttatcg tattcccaga tctgggagtc cgtgtatgcg 8100

agaagatggc cctctatgat gtggtctcca cccttcctca ggtcgtgatg 8150

ggctcctcat acggattcca gtactctcct gggcagcgag tcgagttcct 8200

ggtgaatacc tggaaatcaa agaaaaaccc catgggcttt tcatatgaca 8250

ctcgctgttt cgactcaacg gtcaccgaga acgacatccg tgttgaggag 8300

tcaatttacc aatgttgtga cttggccccc gaagccagac aggccataaa 8350

atcgctcaca gagcggcttt atatcggggg tcctctgact aattcaaaag 8400

ggcagaactg cggttatcgc cggtgccgcg cgagcggcgt gctgacgact 8450

agctgcggta acaccctcac atgttacttg aaggcctctg cagcctgtcg 8500

agctgcgaag ctccaggact gcacgatgct cgtgaacgga gacgacctcg 8550

tcgttatctg tgaaagcgcg ggaacccaag aggacgcggc gagcctacga 8600

gtcttcacgg aggctatgac taggtactcc gccccccccg gggacccgcc 8650

ccaaccagaa tacgacttgg agctgataac atcatgttcc tccaatgtgt 8700

cggtcgccca cgatgcatca ggcaaaaggg tgtactacct cacccgtgat 8750

cccaccaccc ccctagcacg ggctgcgtgg gagacagcta gacacactcc 8800

agttaactcc tggctaggca acattattat gtatgcgccc actttgtggg 8850

caaggatgat tctgatgact cacttcttct ccatccttct agcgcaggag 8900

caacttgaaa aagccctgga ctgccagatc tacggggcct gttactccat 8950

tgagccactt gacctacctc agatcattga acgactccat ggccttagcg 9000

cattttcact ccatagttac tctccaggtg agatcaatag ggtggcttca 9050

tgcctcagga aacttggggt accacccttg cgagtctgga gacatcgggc 9100

caggagcgtc cgcgctaggc tactgtccca gggagggagg gccgccactt 9150

gtggcaaata cctcttcaac tgggcagtaa aaaccaaact taaactcact 9200

ccaatcccgg ctgcgtcccg gctggacttg tccggctggt tcgttgctgg 9250

ttacagcggg ggagacatat atcacagcct gtctcgtgcc cgaccccgtt 9300

ggttcatgct gtgcctactc ctactttctg taggggtagg catctacctg 9350

ctccccaacc gatgaacggg gagataaaca ctccaggcca ataggccatc 9400

cccctttttt tttttt 9416

›Tables in the description — 28

TABLE 1 — RNA SEQUENCE TARGETS AND ANTISENSE OLIGONUCLEOTIDES FOR HCV [Sequences are from HCV-1 (US) and HCV-J (Japan)]

SEQ ID	Antisense oligo	Target	Target
NO:	sequence:	description:	strand:
1	5′-ATG GTG GAG TGT CGC CCC GTC-3′	5′ end hairpin	+
2	5′-GGA GTG ATC TAT GGT GGA GTG-3′	5′ end 6-bp repeat	+
3	5′-GAT TCG TGC TCA TGG TGC ACG-3′	Polyprotein AUG	+
4	5′-TCC AGG CAT TGA GCG GGT TGA-3′	ORF 3 AUG	+
5	5′-TGG CCT GGA GTG TTT ATC TCC-3′	3′-untranslated	+
6	5′-GGG GTA GGC ATC TAC CTG CTC-3′	3′ palindrome	−
7	5′-CGC CCC CAT CAG GGG GCT GGC-3′	5′ end hairpin	+
8	5′-TTC ATG GTG GAG TGT CGC CCC-3′	5′ end hairpin	+
9	5′-GTT CCT CAC AGG GGA GTG ATT-3′	5′ untranslated	+
10	5′-TAC TAA CGC CAT GGC TAG ACG-3′	5′ untranslated	+
11	5′-CTA TGG CTC TCC CGG GAG GGG-3′	5′ untranslated	+
12	5′-CCA CTA TGG CTC TCC CGG GAG-3′	5′ untranslated	+
13	5′-CGG TGT ACT CAC CGG TTC CGC-3′	5′ untranslated	+
14	5′-CTG GCA ATT CCG GTG TAC TCA-3′	5′ untranslated	+
15	5′-GGG GCA CGC CCA AAT CTC CAG-3′	5′ untranslated	+
16	5′-CCT TTC GCG ACC CAA CAC TAC-3′	5′ untranslated	+
17	5′-CCC TAT CAG GCA GTA CCA CAA-3′	5′ untranslated	+
18	5′-CTC CCG GGG CAC TCG CAA GCA-3′	5′ untranstated	+
19	5′-CAT GGT GCA CGG TCT ACG AGA-3′	Polyprotein AUG	+
20	5′-GAT TCG TGC TCA TGG TGC ACG-3′	Polyprotein AUG	+
21	5′-TTT AGG ATT CGT GCT CAT GGT-3′	Polyprotein AUG	+
22	5′-GAG TGG TTA GCC CAA TCT TCA-3′	3′ untranslated	+
23	5′-TAT TGG CCT GGA GTG GTT AGC-3′	R2	+
24	5′-AGG GAA TGG CCT ATT GGC CTG-3′	R2/3′ hairpin	+

21	Nucleic	Single	Linear
Yes	1

21	Nucleic	Single	Linear
Yes	2

21	Nucleic	Single	Linear
Yes	3

21	Nucleic	Single	Linear
Yes	4

21	Nucleic	Single	Linear
Yes	5

21	Nucleic	Single	Linear
Yes	6

21	Nucleic	Single	Linear
Yes	7

21	Nucleic	Single	Linear
Yes	8

21	Nucleic	Single	Linear
Yes	9

21	Nucleic	Single	Linear
Yes	10

21	Nucleic	Single	Linear
Yes	11

21	Nucleic	Single	Linear
Yes	12

21	Nucleic	Single	Linear
Yes	13

21	Nucleic	Single	Linear
Yes	14

21	Nucleic	Single	Linear
Yes	15

21	Nucleic	Single	Linear
Yes	16

21	Nucleic	Single	Linear
Yes	17

21	Nucleic	Single	Linear
Yes	18

21	Nucleic	Single	Linear
Yes	19

21	Nucleic	Single	Linear
Yes	20

21	Nucleic	Single	Linear
Yes	21

21	Nucleic	Single	Linear
Yes	22

21	Nucleic	Single	Linear
Yes	23

21	Nucleic	Single	Linear
Yes	24

9401	Nucleic	Single	Linear
NO	25

9416	Nucleic	Single	Linear
NO	26

9416	Nucleic	Single	Linear
NO	27

1 of 19 part labels are ours — the grant heads the rest

Claims as granted

1 claim

Classifications

6 codes

IPC · International Patent Classification

Section C — Chemistry; metallurgy

C12Q1/70
C07K14/18
C07H21/00
C12N15/113

USPC · US Patent Classification

536/24.5536/23.1

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Pendency

5.4 y

1,975 days filing → grant

Office actions

non-final + final

Responses

no RCE

Examiner

Andrew Wang

art unit 1635 · TC 1600

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Citations: 22 back · 52 forward

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