RNA interference mediated inhibition of hepatitis B virus (HBV) using short interfering nucleic acid (siNA)

BACKGROUND OF THE INVENTION

[0002] The present invention concerns methods and reagents useful in modulating hepatitis B virus (HBV) gene expression and activity in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to short interfering nucleic acid (siNA) molecules capable of mediating RNA interference (RNAi) against HBV expression.

[0003] The following is a discussion of relevant art pertaining to RNAi. The discussion is provided only for understanding of the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.

[0004] RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.

[0005] The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about 21-23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

[0006] RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877) has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21-nucleotide siRNA duplexes are most active when containing 3′-terminal di-nucleotide overhangs. Furthermore, complete substitution of one or both siRNA strands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3′-terminal siRNA overhang nucleotides with 2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end of the siRNA guide sequence (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

[0007] Studies have shown that replacing the 3′-terminal nucleotide overhanging segments of a 21-mer siRNA duplex having two 2-nucleotide 3′-overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to 4 nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated whereas complete substitution with deoxyribonucleotides results in no RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877). In addition, Elbashir et al., supra, also report that substitution of siRNA with 2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al., International PCT Publication No. WO 00/44914, and Beach et al., International PCT Publication No. WO 01/68836, both suggest that siRNA “may include modifications to either the phosphate-sugar backbone or the nucleoside. . . to include at least one of a nitrogen or sulfur heteroatom”; however, neither application teaches to what extent these modifications are tolerated in siRNA molecules nor provides any examples of such modified siRNA. Kreutzer and Limmer, Canadian Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double-stranded-RNA-dependent protein kinase PKR, specifically 2′-amino or 2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-C methylene bridge. However, Kreutzer and Limmer similarly fail to show to what extent these modifications are tolerated in siRNA molecules nor do they provide any examples of such modified siRNA.

[0008] Parrish et al., 2000, Molecular Cell, 6, 1977-1087, tested certain chemical modifications targeting the unc-22 gene in C. elegans using long (>25 nt) siRNA transcripts. The authors describe the introduction of thiophosphate residues into these siRNA transcripts by incorporating thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and observed that “RNAs with two [phosphorothioate]-modified bases also had substantial decreases in effectiveness as RNAi triggers (data not shown); [phosphorothioate] modification of more than two residues greatly destabilized the RNAs in vitro and we were not able to assay interference activities.” Id. at 1081. The authors also tested certain modifications at the 2′-position of the nucleotide sugar in the long siRNA transcripts and observed that substituting deoxynucleotides for ribonucleotides “produced a substantial decrease in interference activity,” especially in the case of Uridine to Thymidine and/or Cytidine to deoxy-Cytidine substitutions. Id. In addition, the authors tested certain base modifications, including substituting, in sense and antisense strands of the siRNA, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine for guanosine. They found that whereas 4-thiouracil and 5-bromouracil were all well tolerated, inosine “produced a substantial decrease in interference activity” when incorporated in either strand. Incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resulted in substantial decrease in RNAi activity as well.

[0009] Beach et al., International PCT Publication No. WO 01/68836, describe specific methods for attenuating gene expression using endogenously-derived dsRNA. Tuschl et al., International PCT Publication No. WO 01/75164, describe a Drosophila in vitro RNAi system and the use of specific siRNA molecules for certain functional genomic and certain therapeutic applications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubts that RNAi can be used to cure genetic diseases or viral infection due “to the danger of activating interferon response.” Li et al., International PCT Publication No. WO 00/44914, describe the use of specific dsRNAs for use in attenuating the expression of certain target genes. Zernicka-Goetz et al., International PCT Publication No. WO 01/36646, describe certain methods for inhibiting the expression of particular genes in mammalian cells using certain dsRNA molecules. Fire et al., International PCT Publication No. WO 99/32619, describe particular methods for introducing certain dsRNA molecules into cells for use in inhibiting gene expression. Plaetinck et al., International PCT Publication No. WO 00/01846, describe certain methods for identifying specific genes responsible for conferring a particular phenotype in a cell using specific dsRNA molecules. Mello et al., International PCT Publication No. WO 01/29058, describe the identification of specific genes involved in dsRNA-mediated RNAi. Deschamps Depaillette et al., International PCT Publication No. WO 99/07409, describe specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents. Waterhouse et al., International PCT Publication No. 99/53050, describe certain methods for decreasing the phenotypic expression of a nucleic acid in plant cells. Driscoll et al., International PCT Publication No. WO 01/49844, describe specific DNA constructs for use in facilitating gene silencing in targeted organisms. Parrish et al., 2000, Molecular Cell, 6, 1977-1087, describe specific chemically-modified siRNA constructs targeting the unc-22 gene of C. elegans. Grossniklaus, International PCT Publication No. WO 01/38551, describes certain methods for regulating polycomb gene expression in plants. Churikov et al., International PCT Publication No. WO 01/42443, describes certain methods for modifying genetic characteristics of an organism. Cogoni et al., International PCT Publication No. WO 01/53475, describes certain methods for isolating a Neurospora silencing gene and uses thereof. Reed et al., International PCT Publication No. WO 01/68836, describes certain methods for gene silencing in plants. Honer et al., International PCT Publication No. WO 01/70944, describe certain methods of drug screening using transgenic nematodes as Parkinson's Disease models. Deak et al., International PCT Publication No. WO 01/72774, describe certain Drosophila-derived gene products. Arndt et al., International PCT Publication No. WO 01/92513, describe certain methods for mediating gene suppression by using factors that enhance RNAi. Tuschl et al., International PCT Publication No. WO 02/44321, describe certain synthetic siRNA constructs. Pachuk et al., International PCT Publication No. WO 00/63364, and Satishchandran et al., International PCT Publication No. WO 01/04313, describe certain methods and compositions for inhibiting the function of certain polynucleotide sequences. Echeverri et al., International PCT Publication No. WO 02/38805, describe certain C. elegans genes identified via RNAi. Kreutzer et al., International PCT Publications Nos. WO 02/055692 and WO 02/055693, describe certain methods for inhibiting gene expression using RNAi.

[0010] Chronic hepatitis B is caused by an enveloped virus, commonly known as the hepatitis B virus or HBV. HBV is transmitted via infected blood or other body fluids, especially saliva and semen, during delivery of a child, sexual activity, or sharing of needles contaminated by infected blood. Individuals can be “carriers” and transmit the infection to others without ever having experienced symptoms of the disease. Persons at highest risk are those with multiple sex partners, those with a history of sexually transmitted diseases, parenteral drug users, infants born to infected mothers, “close” contacts of or sexual partners of infected persons, and healthcare personnel or other service employees who have contact with blood. Transmission is also possible via tattooing, ear or body piercing, and acupuncture; the virus is also stable on razors, toothbrushes, baby bottles, eating utensils, and some hospital equipment such as respirators, scopes, and instruments. There is no evidence that HbsAg (an HBV surface antigen)-positive food handlers pose a health risk in an occupational setting; hence, they should not be excluded from the workplace. Hepatitis B has never been documented as being a food-borne disease. The average incubation period is 60 to 90 days, with a range of 45 to 180 days; the number of days appears to be related to the amount of virus to which the person was exposed. However, determining the length of incubation is difficult, since onset of symptoms is insidious. Approximately 50% of patients develop symptoms of acute hepatitis that last from 1 to 4 weeks. Two percent or less of these individuals develop fulminant hepatitis resulting in liver failure and death.

[0011] The determinants of severity include: (1) the size of the dose to which the person was exposed; (2) the person's age, with younger patients experiencing a milder form of the disease; (3) the status of the immune system, with those who are immunosuppressed experiencing milder cases; and (4) the presence or absence of co-infection with the Delta virus (hepatitis D), with more severe cases resulting from co-infection. In symptomatic cases, clinical signs include loss of appetite, nausea, vomiting, abdominal pain in the right upper quadrant, arthralgia, and tiredness/loss of energy. Jaundice is not experienced in all cases; however, jaundice is more likely to occur if the infection is due to transfusion or percutaneous serum transfer, and it is accompanied by mild pruritus in some patients. Bilirubin elevations are demonstrated in dark urine and clay-colored stools, and liver enlargement can occur accompanied by right upper-quadrant pain. The acute phase of the disease can be accompanied by severe depression, meningitis, Guillain-Barre syndrome, myelitis, encephalitis, agranulocytosis, and/or thrombocytopenia.

[0012] Hepatitis B is generally self-limiting and will resolve in approximately 6 months. Asymptomatic cases can be detected by serologic testing, since the presence of the virus leads to production of large amounts of HBsAg in the blood. This antigen is the first and most useful diagnostic marker for active infections. However, if HBsAg remains positive for 20 weeks or longer, the person is likely to remain positive indefinitely and is now a carrier. While only 10% of persons over age 6 who contract HBV become carriers, 90% of infants infected during the first year of life become carriers.

[0013] Hepatitis B virus (HBV) infects over 300 million people worldwide (Imperial, 1999, Gastroenterol. Hepatol., 14 (suppl), S1-5). In the United States, approximately 1.25 million individuals are chronic carriers of HBV as evidenced by the fact that they have measurable hepatitis B virus surface antigen HBsAg in their blood. The risk of becoming a chronic HBsAg carrier is dependent upon the mode of acquisition of infection as well as the age of the individual at the time of infection. For those individuals with high levels of viral replication, chronic active hepatitis with progression to cirrhosis, liver failure and hepatocellular carcinoma (HCC) is common, and liver transplantation is the only treatment option for patients with end-stage liver disease from HBV.

[0014] The natural progression of chronic HBV infection over a 10 to 20 year period leads to cirrhosis in 20-to-50% of patients and progression of HBV infection to hepatocellular carcinoma has been well documented. There have been no studies that have determined sub-populations that are most likely to progress to cirrhosis and/or hepatocellular carcinoma; thus all patients have equal risk of progression.

[0015] It is important to note that the survival for patients diagnosed with hepatocellular carcinoma is only 0.9 to 12.8 months from initial diagnosis (Takahashi et al., 1993, American Journal of Gastroenterology, 88, 240-243). Treatment of hepatocellular carcinoma with chemotherapeutic agents has not proven effective and only 10% of patients will benefit from surgery due to extensive tumor invasion of the liver (Trinchet et al., 1994, Presse Medicine, 23, 831-833). Given the aggressive nature of primary hepatocellular carcinoma, the only viable treatment alternative to surgery is liver transplantation (Pichlmayr et al., 1994, Hepatology., 20, 33S-40S).

[0016] Upon progression to cirrhosis, patients with chronic HBV infection present with clinical features that are common to clinical cirrhosis regardless of the initial cause (D'Amico et al., 1986, Digestive Diseases and Sciences, 31, 468-475). These clinical features can include: bleeding esophageal varices, ascites, jaundice, and encephalopathy (Zakim D, Boyer T D. Hepatology: A Textbook of Liver Disease, Second Edition, Volume 1, 1990, W. B. Saunders Company, Philadelphia). In the early stages of cirrhosis, patients are classified as compensated, meaning that although liver tissue damage has occurred, the patient's liver is still able to detoxify metabolites in the bloodstream. In addition, most patients with compensated liver disease are asymptomatic and the minority with symptoms report only minor symptoms such as dyspepsia and weakness. In the later stages of cirrhosis, patients are classified as decompensated meaning that their ability to detoxify metabolites in the bloodstream is diminished and it is at this stage that the clinical features described above will present.

[0017] In 1986, D'Amico et al. described the clinical manifestations and survival rates in 1155 patients with both alcoholic- and viral-associated cirrhosis (D'Amico, supra). Of the 1155 patients, 435 (37%) had compensated disease although 70% were asymptomatic at the beginning of the study. The remaining 720 patients (63%) had decompensated liver disease with 78% presenting with a history of ascites, 31% with jaundice, 17% with bleeding, and 16% with encephalopathy. Hepatocellular carcinoma was observed in 6 (0.5%) patients with compensated disease and in 30 (2.6%) patients with decompensated disease.

[0018] Over the course of six years, the patients with compensated cirrhosis developed clinical features of decompensated disease at a rate of 10% per year. In most cases, ascites was the first presentation of decompensation. In addition, hepatocellular carcinoma developed in 59 patients who initially presented with compensated disease by the end of the six-year study.

[0019] With respect to survival, the D'Amico study indicated that the five-year survival rate for all patients on the study was only 40%. The six-year survival rate for the patients who initially had compensated cirrhosis was 54% while the six-year survival rate for patients who initially presented with decompensated disease was only 21%. There were no significant differences in the survival rates between the patients who had alcoholic cirrhosis and the patients with viral related cirrhosis. The major causes of death for the patients in the D'Amico study were liver failure in 49%; hepatocellular carcinoma in 22%; and bleeding in 13% (D'Amico, supra).

[0020] Hepatitis B virus is a double-stranded circular DNA virus. It is a member of the Hepadnaviridae family. The virus is 42 nm in diameter, consisting of a central core that contains a core antigen (HBcAg) surrounded by an envelope containing a surface protein/surface antigen (HBsAg). It also contains an e antigen (HBeAg) that, along with HBcAg and HBsAg, is helpful in identifying this disease.

[0021] In HBV virions, the genome is found in an incomplete double-stranded form. HBV uses a reverse transcriptase to transcribe a positive-sense full-length RNA version of its genome back into DNA. This reverse transcriptase also contains DNA polymerase activity with which it begins replicating the newly-synthesized minus-sense DNA strand. However, it appears that the core protein encapsidates the reverse-transcriptase/polymerase before it completes replication.

[0022] From the free-floating form, the virus must first attach itself specifically to a host cell membrane. Viral attachment is one of the crucial steps that determine host and tissue specificity. Currently there are no in vitro cell lines that can be infected by HBV. There are, however, some cell lines, such as HepG2, which can support viral replication only upon transient or stable transfection using HBV DNA.

[0023] Cell Culture Models

[0024] As previously mentioned HBV does not infect cells in culture. However, transfection of HBV DNA (either as a head-to-tail dimer or as an “overlength” genome of >100%) into HuH7 or Hep G2 hepatocytes results in viral gene expression and production of HBV virions released into the media. Thus, HBV replication competent DNA could be co-transfected with ribozymes in cell culture. Such an approach has been used to report intracellular ribozyme activity against HBV (zu Putlitz, et al., 1999, J. Virol., 73, 5381-5387, and Kim et al., 1999, Biochem. Biophys. Res. Commun., 257, 759-765). In addition, stable hepatocyte cell lines have been generated that express HBV. In such cells, only the delivery of ribozymes would be required; however, a delivery screen would need to be performed.

[0025] Phenotypic Assays

[0026] Intracellular HBV gene expression can be assayed either by a Taqman® assay for HBV RNA or by ELISA for HBV protein. Extracellular virus can be assayed either by PCR for DNA or ELISA for protein. Antibodies are commercially available for HBV surface antigen and core protein. A secreted alkaline phosphatase expression plasmid can be used to normalize for differences in transfection efficiency and sample recovery.

[0027] Animal Models

[0028] There are several small animal models available to study HBV replication. One is the transplantation of HBV-infected liver tissue into irradiated mice. Viremia (as evidenced by measuring HBV DNA by PCR) is first detected 8 days after transplantation and peaks between 18- 25 days (Ilan et al., 1999, Hepatology, 29, 553-562).

[0029] Transgenic mice that express HBV have also been used as a model to evaluate potential anti-virals. HBV DNA is detectable in both liver and serum of the transgenic mice (Morrey et al., 1999, Antiviral Res., 42, 97-108).

[0030] An additional model is to establish subcutaneous tumors in nude mice with Hep G2 cells transfected with HBV. Tumors develop in about 2 weeks after inoculation and express HBV surface and core antigens. HBV DNA and surface antigen are also detected in the circulation of tumor-bearing mice (Yao et al., 1996, J. Viral Hepat., 3, 19-22).

[0031] Woodchuck hepatitis virus (WHV) is closely related to HBV in its virus structure, genetic organization, and mechanism of replication. As with HBV in humans, persistent WHV infection is common in natural woodchuck populations and is associated with chronic hepatitis and hepatocellular carcinoma (HCC). Experimental studies have established that WHV causes HCC in woodchucks and woodchucks chronically infected with WHV have been used as a model to test a number of anti-viral agents. For example, the nucleoside analogue 3T3 was observed to cause dose-dependent reduction in virus (50% reduction after two daily treatments at the highest dose) (Hurwitz et al., 1998. Antimicrob. Agents Chemother., 42, 2804-2809).

[0032] Therapeutic Approaches

[0033] Current therapeutic goals of treatment are three-fold: (1) to eliminate infectivity and transmission of HBV to others; (2) to arrest the progression of liver disease and improve the clinical prognosis; and (3) to prevent the development of hepatocellular carcinoma (HCC).

[0034] Interferon alpha (IFN-alpha) is the most common therapeutic for treating HBV infection; however, the FDA has recently approved Lamivudine (3TC®) as a therapeutic for treating chronic HBV infection. The standard duration of IFN-alpha therapy is 16 weeks; however, the optimal treatment length is still poorly defined. A complete response (where patients become both HBV DNA-negative and HbeAg-negative) occurs in approximately 25% of patients. Several factors have been identified that predict a favorable response to therapy, including: high ALT, low HBV DNA, being female, and heterosexual orientation.

[0035] There is also a risk of reactivation of the hepatitis B virus even after a successful response; this occurs in around 5% of responders and normally occurs within 1 year.

[0036] Side effects resulting from treatment with type 1 interferons can be divided into four general categories: (1) influenza-like symptoms, (2) neuropsychiatric side effects, (3) laboratory abnormalities, and (4) other miscellaneous side effects. Examples of influenza-like symptoms include, fatigue, fever, myalgia, malaise, appetite loss, tachycardia, rigors, headache and arthralgias. The influenza-like symptoms are usually short-lived and tend to abate after the first four weeks of dosing (Dusheiko et al., 1994, Journal of Viral Hepatitis, 1, 3-5). Neuropsychiatric side effects include irritability, apathy, mood changes, insomnia, cognitive changes, and depression. Laboratory abnormalities include the reduction of myeloid cells, including granulocytes, platelets, and, to a lesser extent, red blood cells. These changes in blood cell counts rarely lead to any significant clinical sequellae. In addition, increases in triglyceride concentrations and elevations in serum alaine and aspartate aminotransferase concentrations have been observed. Finally, thyroid abnormalities have been reported. These thyroid abnormalities are usually reversible after cessation of interferon therapy and can be controlled with appropriate medication during therapy. Miscellaneous side effects include nausea, diarrhea, abdominal and back pain, pruritus, alopecia, and rhinorrhea. In general, most side effects will abate after 4 to 8 weeks of therapy (Dushieko et al., supra).

[0037] Lamivudine (3TC®) is a nucleoside analogue, which is a very potent and specific inhibitor of HBV DNA synthesis, and has recently been approved for the treatment of chronic hepatitis B. Unlike treatment with interferon, treatment with 3TC® does not eliminate the HBV from the patient. Rather, viral replication is controlled and chronic administration results in improvements in liver histology in over 50% of patients. Phase III studies with 3TC®, showed that treatment for one year was associated with reduced liver inflammation and a delay in scarring of the liver. In addition, patients treated with 3TC® (100 mg per day) had a 98% reduction in hepatitis B DNA and a significantly higher rate of seroconversion, suggesting disease improvements after completion of therapy. However, cessation of therapy resulted in a reactivation of HBV replication in most patients. In addition, recent reports have documented 3TC® resistance in approximately 30% of patients.

[0038] Therefore, current therapies for treating HBV infection, including interferon and nucleoside analogues, such as IFN-alpha and 3TC®, are only partially effective. In addition, drug resistance to nucleoside analogues is now emerging, making treatment of chronic hepatitis B more difficult. Thus, a need exists for effective treatment of this disease that utilizes antiviral inhibitors that work by mechanisms other than those currently utilized in the treatment of both acute and chronic hepatitis B infections.

SUMMARY OF THE INVENTION

[0039] This invention relates to compounds, compositions, and methods useful for modulating expression of genes, such as those genes associated with the development or maintenance of HBV infection, by RNA interference (RNAi) using short interfering nucleic acid (siNA). In particular, the instant invention features siNA molecules and methods to modulate the expression of HBV. A siNA of the invention can be unmodified or chemically modified. A siNA of the instant invention can be chemically synthesized, expressed from a vector, or enzymatically synthesized. The instant invention also features various chemically-modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating HBV gene expression/activity in cells by RNA inference (RNAi). The use of chemically-modified siNA is expected to improve various properties of native siNA molecules through increased resistance to nuclease degradation in vivo and/or improved cellular uptake. The siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering and pharmacogenomic applications.

[0040] In one embodiment, the invention features one or more siNA molecules and methods that independently or in combination modulate the expression of gene(s) encoding hepatitis B virus. Specifically, the present invention features siNA molecules that modulate the expression of HBV genes, for example genes encoding sequence referred to by Genbank Accession No. AB073834 or sequences referred to by Genbank Accession Nos. shown in Table I and/or homologous sequences thereof.

[0041] The description below of the various aspects and embodiments of the invention is provided with reference to the exemplary hepatitis B virus, including components or subunits thereof. However, the various aspects and embodiments are also directed to other genes that express other proteins associated with HBV infection, such as cellular proteins that are utilized in the HBV life-cycle. Those additional genes can be analyzed for target sites using the methods described for HBV herein. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.

[0042] In one embodiment, the invention features a siNA molecule which down-regulates expression of a HBV gene, for example, wherein the HBV gene comprises HBV encoding sequence.

[0043] In one embodiment, the invention features a siNA molecule having RNAi activity against HBV RNA, wherein the siNA molecule comprises a sequence complementary to any RNA having HBV encoding sequence, for example sequence referred to by Genbank Accession No. AB073834 or sequences referred to by Genbank Accession Nos. in Table I and/or homologous sequences thereof.

[0044] In another embodiment, the invention features a siNA molecule comprising sequences selected from the group consisting of SEQ ID NOs.: 1-1524. In another embodiment, the invention features a siNA molecule having an antisense region complementary to any sequence having SEQ ID NOs.: 1-646. In another embodiment, the invention features a siNA molecule having an antisense region having any of SEQ ID NOs.: 647-1292, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520, 1522, or 1524. In another embodiment, the invention features a siNA molecule having a sense region having any of SEQ ID NOs. 1-646, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519, 1521, or 1523. The sequences shown in SEQ ID NOs.: 1-1524 are not limiting. A siNA molecule of the invention can comprise any contiguous HBV sequence (e.g., wherein the sense region of the siNA comprises about 19 contiguous HBV nucleotides and the antisense region comprises sequence complementary to about 19 contiguous HBV nucleotides). In yet another embodiment, the invention features a siNA molecule comprising a sequence, for example the antisense sequence of the siNA construct, complementary to a sequence or portion of sequence comprising Genbank Accession No. AB073834 or Genbank Accession Nos. in Table I and/or homologous sequences thereof.

[0045] Due to the high sequence variability of the HBV genome, selection of siNA molecules for broad therapeutic applications would likely involve the conserved regions of the HBV genome. Specifically, the present invention describes siNA molecules that target the conserved regions of the HBV genome.

[0046] In one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by HBV genes, for example genes required for viral replication including genes required for HBV protein synthesis, such as the 5′-most 1500 nucleotides of the HBV pregenomic mRNA. This region controls the translational expression of the core protein (C), X protein (X), and DNA polymerase (P) genes, and plays a role in the replication of the viral DNA by serving as a template for reverse transcriptase. Disruption of this region in the RNA results in deficient protein synthesis as well as incomplete DNA synthesis (and inhibition of transcription from the defective genomes). Target sequences 5′ of the encapsidation site can result in the inclusion of the disrupted 3′ RNA within the core virion structure, and targeting sequences 3′ of the encapsidation site can result in the reduction in protein expression from both the 3′ and 5′ fragments. Alternative regions outside of the 5′-most 1500 nucleotides of the pregenomic mRNA also make suitable targets of siNA-mediated inhibition of HBV replication. Such targets include the mRNA regions that encode the viral S gene. Selection of particular target regions will depend upon the secondary structure of the pregenomic mRNA. Targets in the minor mRNAs can also be used, especially when folding or accessibility assays in these other RNAs reveal additional target sequences that are unavailable in the pregenomic mRNA species. A desirable target in the pregenomic RNA is a proposed bipartite stem-loop structure in the 3′-end of the pregenomic RNA that is believed to be critical for viral replication (Kidd and Kidd-Ljunggren, 1996. Nuc. Acid Res. 24:3295-3302). The 5′-end of the HBV pregenomic RNA carries a cis-acting encapsidation signal, which has inverted repeat sequences that are thought to form a bipartite stem-loop structure. Due to a terminal redundancy in the pregenomic RNA, the putative stem-loop also occurs at the 3′-end. While it is the 5′ copy that functions in polymerase binding and encapsidation, reverse transcription actually begins from the 3′ stem-loop. To start reverse transcription, a 4 nucleotide primer that is covalently attached to the polymerase is made, using a bulge in the 5′ encapsidation signal as template. This primer is then shifted, by an unknown mechanism, to the DRI primer binding site in the 3′ stem-loop structure, and reverse transcription proceeds from that point. The 3′ stem-loop, and especially the DRI primer binding site, appear to be highly effective targets for siNA mediated intervention. Sequences of the pregenomic RNA are shared by the mRNAs for surface, core, polymerase, and X proteins. Due to the overlapping nature of the HBV transcripts, all share a common 3′-end. Therefore, siNA targeting of this common 3′-end will thus disrupt the pregenomic RNA as well as all of the mRNAs for surface, core, polymerase and X proteins.

[0047] In one embodiment of the invention a siNA molecule is adapted for use to treat human hepatitis B virus infections, which include productive virus infection, latent or persistent virus infection, and HBV-induced hepatocyte transformation. The utility can be extended to other species of HBV that infect non-human animals where such infections are of veterinary importance. A siNA molecule can comprise a sense region and an antisense region, wherein said antisense region can comprise sequence complementary to an RNA sequence encoding HBV and the sense region can comprise sequence complementary to the antisense region. A siNA molecule can be assembled from two nucleic acid fragments wherein one fragment can comprise the sense region and the second fragment can comprise the antisense region of said siNA molecule. The sense region and antisense region can be covalently connected via a linker molecule. The linker molecule can be a polynucleotide or non-nucleotide linker. The sense region of a siNA molecule of the invention can comprise a 3′-terminal overhang and the antisense region can comprise a 3′-terminal overhang. The 3′-terminal overhangs each can comprise about 2 nucleotides. The antisense region 3′-terminal nucleotide overhang can be complementary to RNA encoding HBV. The sense region can comprise a terminal cap moiety at the 3′-end, 5′-end, and/or both the 3′ and the 5′-ends of the sense region. The antisense region can also comprise a terminal cap moiety at the 3′-end, 5′-end, and/or both the 3′ and the 5′-ends of the antisense region.

[0048] In one embodiment, the invention features one or more siNA molecules and methods that independently or in combination modulate the expression of genes representing cellular targets for HBV infection, such as cellular receptors, cell surface molecules, cellular enzymes, cellular transcription factors, and/or cytokines, second messengers, and cellular accessory molecules including but not limited to interferon regulatory factors (IRFs such as Genbank Accession No. AF082503.1), cellular PKR protein kinase (such as Genbank Accession No. XM—002661.7), human eukaryotic initiation factors 2B (elF2Bgamma, such as Genbank Accession No. AF256223 and/or elF2gamma, such as Genbank Accession No. NM—006874.1), human DEAD Box protein DDX3 (such as Genbank Accession No. XM—018021.2), and cellular proteins that are essential for the maintenance of persistent infection of hepatocytes, such as proteins that interact with the HBV-encoded HBx regulatory protein.

[0049] In one embodiment, nucleic acid molecules of the invention that act as mediators of the RNA interference gene silencing response are double-stranded nucleic acid molecules. In another embodiment, the siNA molecules of the invention consist of duplexes containing about 19 base pairs between oligonucleotides comprising about 19 to about 25 nucleotides. In yet another embodiment, siNA molecules of the invention comprise duplexes with overhanging ends of about 1-3 (e.g., about 1, 2, or 3) nucleotides, for example about 21-nucleotide duplexes with about 19 base pairs and a 3′-terminal mononucleotide, dinucleotide, or trinucleotide overhang.

[0050] In one embodiment, the invention features one or more chemically-modified siNA constructs having specificity for HBV expressing nucleic acid molecules. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incorporation. These chemical modifications, when used in various siNA constructs, are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Furthermore, contrary to the data published by Parrish et al., supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions are well-tolerated and confer substantial increases in serum stability for modified siNA constructs.

[0051] The antisense region of a siNA molecule of the invention can comprise a phosphorothioate internucleotide linkage at the 3′-end of said antisense region. The antisense region can comprise between about one and about five phosphorothioate internucleotide linkages at the 5′-end of said antisense region. The 3′-terminal nucleotide overhangs of a siNA molecule of the invention can comprise ribonucleotides or deoxyribonucleotides that are chemically modified at a nucleic acid sugar, base, or backbone. The 3′-terminal nucleotide overhangs can comprise one or more universal base ribonucleotides. The 3′-terminal nucleotide overhangs can comprise one or more acyclic nucleotides.

[0052] In a non-limiting example, the introduction of chemically-modified nucleotides into nucleic acid molecules will provide a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules that are delivered exogenously. For example, the use of chemically-modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically-modified nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically-modified nucleic acid molecule is reduced as compared to a native nucleic acid molecule, for example when compared to an all RNA nucleic acid molecule, the overall activity of the modified nucleic acid molecule can be greater than the native molecule due to improved stability and/or delivery of the molecule. Unlike native unmodified siNA, chemically-modified siNA can also minimize the possibility of activating interferon activity in humans.

[0053] One embodiment of the invention provides an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention in a manner that allows expression of the nucleic acid molecule. Another embodiment of the invention provides a mammalian cell comprising such an expression vector. The mammalian cell can be a human cell. The siNA molecule of the expression vector can comprise a sense region and an antisense region and the antisense region can comprise sequence complementary to a RNA sequence encoding HBV and the sense region can comprise sequence complementary to the antisense region. The siNA molecule can comprise two distinct strands having complementary sense and antisense regions. The siNA molecule can comprise a single strand having complementary sense and antisense regions.

[0054] In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising a backbone modified internucleotide linkage having Formula I:

[0055] wherein each R1 and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be naturally occurring or chemically modified, each X and Y is independently O, S, N, alkyl, or substituted alkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, or aralkyl, and wherein W, X, Y, and Z are optionally not all O.

[0056] The chemically-modified internucleotide linkages having Formula I, for example wherein any Z, W, X, and/or Y independently comprises a sulphur atom, can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modified internucleotide linkages having Formula I at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise between about 1 and about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified internucleotide linkages having Formula I at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In another embodiment, a siNA molecule of the invention having internucleotide linkage(s) of Formula I also comprises a chemically-modified nucleotide or non-nucleotide having any of Formulae I-VII.

[0057] In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula II:

[0058] wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA.

[0059] The chemically-modified nucleotide or non-nucleotide of Formula II can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotide or non-nucleotide of Formula II at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise between about 1 and about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide or non-nucleotide of Formula II at the 5′-end of the sense strand, the antisense strand, or both strands. In anther non-limiting example, an exemplary siNA molecule of the invention can comprise between about 1 and about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide or non-nucleotide of Formula II at the 3′-end of the sense strand, the antisense strand, or both strands.

[0060] In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula III:

[0061] wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be employed to be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA.

[0062] The chemically-modified nucleotide or non-nucleotide of Formula III can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotide or non-nucleotide of Formula III at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise between about 1 and about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide or non-nucleotide of Formula III at the 5′-end of the sense strand, the antisense strand, or both strands. In anther non-limiting example, an exemplary siNA molecule of the invention can comprise between about 1 and about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide or non-nucleotide of Formula III at the 3′-end of the sense strand, the antisense strand, or both strands.

[0063] In another embodiment, a siNA molecule of the invention comprises a nucleotide having Formula II or III, wherein the nucleotide having Formula II or III is in an inverted configuration. For example, the nucleotide having Formula II or III is connected to the siNA construct in a 3′,3′; 3′-2′, 2′-3′; or 5′,5′ configuration, such as at the 3′-end, 5′-end, or both 3′ and 5′-ends of one or both siNA strands.

[0064] In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a 5′-terminal phosphate group having Formula IV:

[0065] wherein each X and Y is independently O, S, N, alkyl, substituted alkyl, or alkylhalo; each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or alkylhalo; and wherein W, X, Y and Z are not all O.

[0066] In one embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand, for example a strand complementary to a target RNA, wherein the siNA molecule comprises an all RNA siNA molecule. In another embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand wherein the siNA molecule also comprises 1-3 (e.g., 1, 2, or 3) nucleotide 3′-terminal nucleotide overhangs having between about 1 and about 4 (e.g., about 1, 2, 3, or 4) deoxyribonucleotides on the 3′-end of one or both strands. In another embodiment, a 5′-terminal phosphate group having Formula IV is present on the target-complementary strand of a siNA molecule of the invention, for example a siNA molecule having chemical modifications having any of Formulae I-VII.

[0067] In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more phosphorothioate internucleotide linkages. For example, in a non-limiting example, the invention features a chemically-modified short interfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one siNA strand. In yet another embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both siNA strands. The phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more phosphorothioate internucleotide linkages at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise between about 1 and about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate internucleotide linkages at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands.

[0068] In one embodiment, the invention features a siNA molecule, wherein the sense strand comprises one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5 or more) universal base-modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises any of between 1 and 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5 or more) universal base-modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, pyrimidine nucleotides of the sense and/or antisense siNA stand are chemically modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

[0069] In another embodiment, the invention features a siNA molecule, wherein the sense strand comprises between about 1 and about 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base-modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises any of between about 1 and about 5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5 or more) universal base-modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA stand are chemically modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without between about 1 and about 5 or more, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

[0070] In one embodiment, the invention features a siNA molecule, wherein the antisense strand comprises one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or between one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base-modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises any of between about 1 and about 10, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base-modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA stand are chemically modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

[0071] In another embodiment, the invention features a siNA molecule, wherein the antisense strand comprises between about 1 and about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5 or more) universal base-modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises any of between about 1 and about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base-modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA stand are chemically modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without between about 1 and about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

[0072] In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule having between about 1 and about 5, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages in each strand of the siNA molecule.

[0073] In another embodiment, the invention features a siNA molecule comprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) can be at 3′-end the 5′-end, the 3′-end, or both of the 5′- and 3′-ends of one or both siNA sequence strands. In addition, the 2′-5′ internucleotide linkage(s) can be present at various other positions within one or both siNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a pyrimidine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a purine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage.

[0074] In another embodiment, a chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically modified, wherein each strand is between about 18 and about 27 (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides in length, wherein the duplex has between about 18 and about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the chemical modification comprises a structure having any of Formulae I-VII. For example, an exemplary chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically modified with a chemical modification having any of Formulae I-VII, wherein each strand consists of about 21 nucleotides, each having two 2-nucleotide 3′-terminal nucleotide overhangs, and wherein the duplex has 19 base pairs.

[0075] In another embodiment, a siNA molecule of the invention comprises a single-stranded hairpin structure, wherein the siNA is between about 36 and about 70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having between about 18 and about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the siNA can include a chemical modification comprising a structure having any of Formulae I-VII. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having between about 42 and about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically modified with a chemical modification having any of Formulae I-VII, wherein the linear oligonucleotide forms a hairpin structure having 19 base pairs and a 2 nucleotide 3′-terminal nucleotide overhang.

[0076] In another embodiment, a linear hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. For example, a linear hairpin siNA molecule of the invention is designed such that degradation of the loop portion of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

[0077] In another embodiment, a siNA molecule of the invention comprises a circular nucleic acid molecule, wherein the siNA is between about 38 and about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having between about 18 and about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the siNA can include a chemical modification, which comprises a structure having any of Formulae I-VII. For example, an exemplary chemically-modified siNA molecule of the invention comprises a circular oligonucleotide having between about 42 and about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically modified with a chemical modification having any of Formulae I-VII, wherein the circular oligonucleotide forms a dumbbell-shaped structure having 19 base pairs and 2 loops.

[0078] In another embodiment, a circular siNA molecule of the invention contains two loop motifs, wherein one or both loop portions of the siNA molecule is biodegradable. For example, a circular siNA molecule of the invention is designed such that degradation of the loop portions of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

[0079] In one embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety, for example a compound having Formula V:

[0080] wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S═O, CHF, or CF2.

[0081] In one embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasic moiety, for example a compound having Formula VI:

[0082] wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I; R9 is O, S, CH2, S═O, CHF, or CF2, and either R2, R3, R8 or R13 serve as points of attachment to the siNA molecule of the invention.

[0083] In another embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) substituted polyalkyl moieties, for example a compound having Formula VII:

[0084] wherein each n is independently an integer from 1 to 12, R1, R2 and R3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I, and either R1, R2 or R3 serve as points of attachment to the siNA molecule of the invention.

[0085] In another embodiment, the invention features a compound having Formula VII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises O and is the point of attachment to the 3′-end, 5-end, or both 3′ and 5′-ends of one or both strands of a double-stranded siNA molecule of the invention or to a single-stranded siNA molecule of the invention. This modification is referred to herein as “glyceryl” (for example modification 6 in FIG. 10).

[0086] In another embodiment, a moiety having any of Formula V, VI or VII of the invention is at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of a siNA molecule of the invention. For example, a moiety having Formula V, VI or VII can be present at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand, the sense strand, or both the antisense and sense strands of the siNA molecule. In addition, a moiety having Formula VII can be present at the 3′-end or the 5′-end of a hairpin siNA molecule as described herein.

[0087] In another embodiment, a siNA molecule of the invention comprises an abasic residue having Formula V or VI, wherein the abasic residue having Formula V or VI is connected to the siNA construct in a 3′-3′, 3′-2′, 2′-3′, or 5′-5′ configuration, such as at the 3′-end, 5′-end, or both 3′ and ′5′-ends of one or both siNA strands.

[0088] In one embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acid (LNA) nucleotides, for example at the 5′-end, 3′-end, 5′ and 3′-end, or any combination thereof, of the siNA molecule.

[0089] In another embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides, for example at the 5′-end, 3′-end, 5′ and 3′-end, or any combination thereof, of the siNA molecule.

[0090] In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein the chemically-modified siNA comprises a sense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides).

[0091] In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein the chemically-modified siNA comprises a sense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said sense region are 2′-deoxy nucleotides.

[0092] In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein the chemically-modified siNA comprises an antisense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

[0093] In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein the chemically-modified siNA comprises an antisense region, where any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said antisense region are 2′-deoxy nucleotides.

[0094] In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein the chemically-modified siNA comprises a sense region, where one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where one or more purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), and inverted deoxy abasic modifications that are optionally present at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense region, the sense region optionally further comprising a 3′-terminal nucleotide overhang having between about 1 and about 4 (e.g, about 1, 2, 3, or 4) 2′-deoxyribonucleotides; and wherein the chemically-modified short interfering nucleic acid molecule comprises an antisense region, where one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein one or more purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense sequence, the antisense region optionally further comprising a 3′-terminal overhang having between about 1 and about 4 (e.g, about 1, 2, 3, or 4) 2′-deoxynucleotides, wherein the overhang nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4 ) phosphorothioate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown in FIGS. 4 and 5 (SEQ ID NOs.: 396/397 and 406/407) herein.

[0095] In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein the siNA comprises a sense region, where one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and where one or more purine nucleotides present in the sense region are purine ribonucleotides (e.g., wherein all purine nucleotides are purine ribonucleotides or alternately a plurality of purine nucleotides are purine ribonucleotides), and inverted deoxy abasic modifications that are optionally present at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense region, the sense region optionally further comprising a 3′-terminal nucleotide overhang having between about 1 and about 4 (e.g, about 1, 2, 3, or 4) 2′-deoxyribonucleotides; and wherein the siNA comprises an antisense region, where one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense sequence, the antisense region optionally further comprising a 3′-terminal nucleotide overhang having between about 1 and about 4 (e.g, about 1, 2, 3, or 4) 2′-deoxynucleotides, wherein the overhang nucleotides can further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown in FIGS. 4 and 5 (SEQ ID NOs.: 398/397 and 408/407) herein.

[0096] In one embodiment, the invention features a chemically-modified short interfering nucleic acid molecule (siNA) capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a conjugate covalently attached to the chemically-modified siNA molecule. In another embodiment, the conjugate is covalently attached to the chemically-modified siNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In another embodiment, the conjugate molecule is attached at the 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In yet another embodiment, the conjugate molecule is attached to both the 3′-end and the 5′-end of the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule, or any combination thereof. In one embodiment, a conjugate molecule of the invention comprises a molecule that facilitates delivery of a chemically-modified siNA molecule molecule into a biological system such as a cell. In another embodiment, the conjugate molecule attached to the chemically-modified siNA molecule is a poly ethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to chemically-modified siNA molecules are described in Vargeese et al., U.S. Ser. No. 60/311,865, incorporated by reference herein.

[0097] In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein one or both strands of the siNA molecule that are assembled from two separate oligonucleotides comprise ribonucleotides at positions within the siNA that are critical for siNA mediated RNAi in a cell. All other positions within the siNA can include chemically-modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having any of Formulae I-VII or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.

[0098] In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein neither of the strands of the siNA molecule that are assembled from two separate oligonucleotides comprise ribonucleotides that are critical for siNA mediated RNAi in a cell. For example, all the positions within the siNA molecule can include chemically-modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having any of Formulae I-VII or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.

[0099] In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein the antisense region and/or the sense region of the siNA molecule comprise ribonucleotides at positions within the siNA that are critical for siNA mediated RNAi in a cell. All other positions within the siNA can include chemically-modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having any of Formulae I-VII or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.

[0100] In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against HBV inside a cell or reconstituted in vitro system, wherein wherein the antisense region and/or the sense region of the siNA molecule that are assembled from two separate oligonucleotides comprise ribonucleotides that are critical for siNA mediated RNAi in a cell. For example, all the positions within the siNA molecule can include chemically-modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having any of Formulae I-VII or any combination thereof to the extent that the ability of the siNA molecule molecule to support RNAi activity in a cell is maintained.

[0101] In one embodiment, the invention features a method for modulating the expression of a HBV gene within a cell, comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically modified, wherein one of the siNA strands includes a sequence complementary to RNA of the HBV gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the HBV gene in the cell.

[0102] In one embodiment, the invention features a method for modulating the expression of a HBV gene within a cell, comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically modified, wherein one of the siNA strands includes a sequence complementary to RNA of the HBV gene and wherein the sense strand sequence of the siNA is identical to the complementary sequence of the target RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the HBV gene in the cell.

[0103] In another embodiment, the invention features a method for modulating the expression of more than one HBV gene within a cell, comprising: (a) synthesizing siNA molecules of the invention, which can be chemically modified, wherein one of the siNA strands includes a sequence complementary to RNA of the HBV genes; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate the expression of the HBV genes in the cell.

[0104] In another embodiment, the invention features a method for modulating the expression of more than one HBV gene within a cell, comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically modified, wherein one of the siNA strands includes a sequence complementary to RNA of the HBV gene and wherein the sense strand sequence of the siNA is identical to the complementary sequence of the target RNA; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate the expression of the HBV genes in the cell.

[0105] In one embodiment, the invention features a method of modulating the expression of a HBV gene in a tissue explant, comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically modified, wherein one of the siNA strands includes a sequence complementary to RNA of the HBV gene; (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the HBV gene in the tissue explant, and (c) optionally introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the HBV gene in that organism.

[0106] In one embodiment, the invention features a method of modulating the expression of a HBV gene in a tissue explant, comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically modified, wherein one of the siNA strands includes a sequence complementary to RNA of the HBV gene and wherein the sense strand sequence of the siNA is identical to the complementary sequence of the target RNA; (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the HBV gene in the tissue explant, and (c) optionally introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the HBV gene in that organism.

[0107] In another embodiment, the invention features a method of modulating the expression of more than one HBV gene in a tissue explant, comprising: (a) synthesizing siNA molecules of the invention, which can be chemically modified, wherein one of the siNA strands includes a sequence complementary to RNA of the HBV genes; (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the HBV genes in the tissue explant, and (c) optionally introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the HBV genes in that organism.

[0108] In one embodiment, the invention features a method of modulating the expression of a HBV gene in an organism, comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically modified, wherein one of the siNA strands includes a sequence complementary to RNA of the HBV gene; and (b) introducing the siNA molecule into the organism under conditions suitable to modulate the expression of the HBV gene in the organism.

[0109] In another embodiment, the invention features a method of modulating the expression of more than one HBV gene in an organism, comprising: (a) synthesizing siNA molecules of the invention, which can be chemically modified, wherein one of the siNA strands includes a sequence complementary to RNA of the HBV genes; and (b) introducing the siNA molecules into the organism under conditions suitable to modulate the expression of the HBV genes in the organism.

[0110] The siNA molecules of the invention can be designed to inhibit target (HBV) gene expression through RNAi targeting of a variety of RNA molecules. In one embodiment, the siNA molecules of the invention are used to target various RNAs corresponding to a target gene. Non-limiting examples of such RNAs include messenger RNA (mRNA), alternate RNA splice variants of target gene(s), post-transcriptionally modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA templates. If alternate splicing produces a family of transcipts that are distinguished by usage of appropriate exons, the instant invention can be used to inhibit gene expression through the appropriate exons to specifically inhibit or to distinguish among the functions of gene family members. For example, a protein that contains an alternatively spliced transmembrane domain can be expressed in both membrane bound and secreted forms. Use of the invention to target the exon containing the transmembrane domain can be used to determine the functional consequences of pharmaceutical targeting of membrane bound as opposed to the secreted form of the protein. Non-limiting examples of applications of the invention relating to targeting these RNA molecules include therapeutic pharmaceutical applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example using single nucleotide polymorphism mapping with siNA molecules of the invention. Such applications can be implemented using known gene sequences or from partial sequences available from an expressed sequence tag (EST).

[0111] In another embodiment, the siNA molecules of the invention are used to target conserved sequences corresponding to a gene family or gene families such as HBV genes. As such, siNA molecules targeting multiple HBV targets can provide increased therapeutic effect. In addition, siNA can be used to characterize pathways of gene function in a variety of applications. For example, the present invention can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis. The invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The invention can be used to understand pathways of gene expression involved in, for example, HBV infection.

[0112] In one embodiment, siNA molecule(s) and/or methods of the invention are used to inhibit the expression of gene(s) that encode RNA referred to by Genbank Accession, for example HBV genes such genes encoding RNA sequence(s) referred to herein by Genbank Accession number, for example Genbank Accession No. AB073834 or Accession numbers shown in Table I. Such sequences are readily obtained using these Genbank Accession numbers.

[0113] In one embodiment, the invention features a method comprising: (a) generating a library of siNA constructs having a predetermined complexity; and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target RNA sequence. In another embodiment, the siNA molecules of (a) have strands of a fixed length, for example about 23 nucleotides in length. In yet another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In yet another embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target target RNA sequence. In another embodiment, the target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

[0114] In one embodiment, the invention features a method comprising: (a) generating a randomized library of siNA constructs having a predetermined complexity, such as of 4N, where N represents the number of base paired nucleotides in each of the siNA construct strands (eg. for a siNA construct having 21-nucleotide sense and antisense strands with 19 base pairs, the complexity would be 419); and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target HBV RNA sequence. In another embodiment, the siNA molecules of (a) have strands of a fixed length, for example about 23 nucleotides in length. In yet another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In yet another embodiment, the assay can comprise a reconstituted in vitro siNA assay as described in Example 7 herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of HBV RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target HBV RNA sequence. In another embodiment, the target HBV RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

[0115] In another embodiment, the invention features a method comprising: (a) analyzing the sequence of a RNA target encoded by a target gene; (b) synthesizing one or more sets of siNA molecules having sequence complementary to one or more regions of the RNA of (a); and (c) assaying the siNA molecules of (b) under conditions suitable to determine RNAi targets within the target RNA sequence. In another embodiment, the siNA molecules of (b) have strands of a fixed length, for example about 23 nucleotides in length. In yet another embodiment, the siNA molecules of (b) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In yet another embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. Fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by expression in in vivo systems.

[0116] By “target site” is meant a sequence within a target RNA that is “targeted” for cleavage mediated by a siNA construct which contains sequences within its antisense region that are complementary to the target sequence.

[0117] By “detectable level of cleavage” is meant cleavage of target RNA (and formation of cleaved product RNAs) to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation of the target RNA. Production of cleavage products from 1-5% of the target RNA is sufficient to detect above the background for most methods of detection.

[0118] In one embodiment, the invention features a composition comprising a siNA molecule of the invention, which can be chemically modified, in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a pharmaceutical composition comprising siNA molecules of the invention, which can be chemically modified, targeting one or more genes in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a method for treating or preventing a disease or condition in a patient, comprising administering to the patient a composition of the invention under conditions suitable for the treatment or prevention of the disease or condition in the patient, alone or in conjunction with one or more other therapeutic compounds. In yet another embodiment, the invention features a method for reducing or preventing tissue rejection in a patient comprising administering to the patient a composition of the invention under conditions suitable for the reduction or prevention of tissue rejection in the patient.

[0119] In another embodiment, the invention features a method for validating a HBV gene target, comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically modified, wherein one of the siNA strands includes a sequence complementary to RNA of a HBV target gene; (b) introducing the siNA molecule into a cell, tissue, or organism under conditions suitable for modulating expression of the HBV target gene in the cell, tissue, or organism; and (c) determining the function of the gene by assaying for any phenotypic change in the cell, tissue, or organism.

[0120] By “phenotypic change” is meant any detectable change to a cell that occurs in response to contact or treatment with a nucleic acid molecule of the invention (e.g., siNA). Such detectable changes include but are not limited to changes in shape, size, proliferation, protein expression or RNA expression or detection of viral antigens as can be assayed by methods known in the art. The detectable change can also include expression of reporter genes/molecules such as Green Florescent Protein (GFP) or various tags that are used to identify an expressed protein or any other cellular component that can be assayed.

[0121] In one embodiment, the invention features a kit containing a siNA molecule of the invention, which can be chemically modified, that can be used to modulate the expression of a HBV target gene in a cell, tissue, or organism. In another embodiment, the invention features a kit containing more than one siNA molecule of the invention, which can be chemically modified, that can be used to modulate the expression of more than one HBV target gene in a cell, tissue, or organism.

[0122] In one embodiment, the invention features a cell containing one or more siNA molecules of the invention, which can be chemically modified. In another embodiment, the cell containing a siNA molecule of the invention is a mammalian cell. In yet another embodiment, the cell containing a siNA molecule of the invention is a human cell.

[0123] In one embodiment, the synthesis of a siNA molecule of the invention, which can be chemically modified, comprises: (a) synthesis of two complementary strands of the siNA molecule; (b) annealing the two complementary strands together under conditions suitable to obtain a double-stranded siNA molecule. In another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase tandem oligonucleotide synthesis.

[0124] In one embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing a first oligonucleotide sequence strand of the siNA molecule, wherein the first oligonucleotide sequence strand comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of the second oligonucleotide sequence strand of the siNA; (b) synthesizing the second oligonucleotide sequence strand of siNA on the scaffold of the first oligonucleotide sequence strand, wherein the second oligonucleotide sequence strand further comprises a chemical moiety than can be used to purify the siNA duplex; (c) cleaving the linker molecule of (a) under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex; and (d) purifying the siNA duplex utilizing the chemical moiety of the second oligonucleotide sequence strand. In another embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example under hydrolysis conditions using an alkylamine base such as methylamine. In another embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place concomitantly. In another embodiment, the chemical moiety of (b) that can used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group, which can be employed in a trityl-on synthesis strategy as described herein. In yet another embodiment, the chemical moiety, such as a dimethoxytrityl group, is removed during purification, for example using acidic conditions.

[0125] In a further embodiment, the method for siNA synthesis is a solution phase synthesis or hybrid phase synthesis wherein both strands of the siNA duplex are synthesized in tandem using a cleavable linker attached to the first sequence which acts a scaffold for synthesis of the second sequence. Cleavage of the linker under conditions suitable for hybridization of the separate siNA sequence strands results in formation of the double-stranded siNA molecule.

[0126] In another embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing one oligonucleotide sequence strand of the siNA molecule, wherein the sequence comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of another oligonucleotide sequence; (b) synthesizing a second oligonucleotide sequence having complementarity to the first sequence strand on the scaffold of (a), wherein the second sequence comprises the other strand of the double-stranded siNA molecule and wherein the second sequence further comprises a chemical moiety than can be used to isolate the attached oligonucleotide sequence; (c) purifying the product of (b) utilizing the chemical moiety of the second oligonucleotide sequence strand under conditions suitable for isolating the full-length sequence comprising both siNA oligonucleotide strands connected by the cleavable linker; and (d) under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex. In another embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example under hydrolysis conditions. In another embodiment, cleavage of the linker molecule in (c) above takes place after deprotection of the oligonucleotide. In another embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity or differing reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place either concomitantly or sequentially. In another embodiment, the chemical moiety of (b) that can used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group.

[0127] In another embodiment, the invention features a method for making a double-stranded siNA molecule in a single synthetic process, comprising: (a) synthesizing an oligonucleotide having a first and a second sequence, wherein the first sequence is complementary to the second sequence, and the first oligonucleotide sequence is linked to the second sequence via a cleavable linker, and wherein a terminal 5′-protecting group, for example a 5′-O-dimethoxytrityl group (5′-O-DMT) remains on the oligonucleotide having the second sequence; (b) deprotecting the oligonucleotide whereby the deprotection results in the cleavage of the linker joining the two oligonucleotide sequences; and (c) purifying the product of (b) under conditions suitable for isolating the double-stranded siNA molecule, for example using a trityl-on synthesis strategy as described herein.

[0128] In one embodiment, the invention features siNA constructs that mediate RNAi against HBV, wherein the siNA construct comprises one or more chemical modifications, for example one or more chemical modifications having any of Formulae I-VII or any combination thereof that increases the nuclease resistance of the siNA construct.

[0129] In another embodiment, the invention features a method for generating siNA molecules with increased nuclease resistance comprising (a) introducing nucleotides having any of Formula I-VII into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased nuclease resistance.

[0130] In one embodiment, the invention features siNA constructs that mediate RNAi against HBV, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the sense and antisense strands of the siNA construct.

[0131] In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the sense and antisense strands of the siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the sense and antisense strands of the siNA molecule.

[0132] In one embodiment, the invention features siNA constructs that mediate RNAi against HBV, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target RNA sequence within a cell.

[0133] In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence, comprising (a) introducing nucleotides having any of Formula I-VII into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence.

[0134] In one embodiment, the invention features siNA constructs that mediate RNAi against HBV, wherein the siNA construct comprises one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA construct.

[0135] In another embodiment, the invention features a method for generating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA molecule.

[0136] In one embodiment, the invention features chemically-modified siNA constructs that mediate RNAi against HBV in a cell, wherein the chemical modifications do not significantly effect the interaction of siNA with a target RNA molecule and/or proteins or other factors that are essential for RNAi in a manner that would decrease the efficacy of RNAi mediated by such siNA constructs.

[0137] In another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against HBV, comprising (a) introducing nucleotides having any of Formula I-VII into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity.

[0138] In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against an HBV target RNA, comprising (a) introducing nucleotides having any of Formula I-VII into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the target RNA.

[0139] In one embodiment, the invention features siNA constructs that mediate RNAi against HBV, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the cellular uptake of the siNA construct.

[0140] In another embodiment, the invention features a method for generating siNA molecules against HBV with improved cellular uptake, comprising (a) introducing nucleotides having any of Formula I-VII into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved cellular uptake.

[0141] In one embodiment, the invention features siNA constructs that mediate RNAi against HBV, wherein the siNA construct comprises one or more chemical modifications described herein that increases the bioavailability of the siNA construct, for example by attaching polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the siNA construct, or by attaching conjugates that target specific tissue types or cell types in vivo. Non-limiting examples of such conjugates are described in Vargeese et al., U.S. Ser. No. 60/311,865 incorporated by reference herein.

[0142] In one embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability, comprising (a) introducing a conjugate into the structure of a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such conjugates can include ligands for cellular receptors such as peptides derived from naturally occurring protein ligands, protein localization sequences including cellular ZIP code sequences, antibodies, nucleic acid aptamers, vitamins and other co-factors such as folate and N-acetylgalactosamine, polymers such as polyethyleneglycol (PEG), phospholipids, polyamines such as spermine or spermidine, and others.

[0143] In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability, comprising (a) introducing an excipient formulation to a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such excipients include polymers such as cyclodextrines, lipids, cationic lipids, polyamines, phospholipids, and others.

[0144] In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability, comprising (a) introducing nucleotides having any of Formula I-VII into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability.

[0145] In another embodiment, polyethylene glycol (PEG) can be covalently attached to siNA compounds of the present invention. The attached PEG can be any molecular weight, preferably from about 2,000 to about 50,000 daltons (Da).

[0146] The present invention can be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples and/or subjects. For example, preferred components of the kit include the siNA and a vehicle that promotes introduction of the siNA. Such a kit can also include instructions to allow a user of the kit to practice the invention.

[0147] The term “short interfering nucleic acid”, “siNA”, “short interfering RNA”, “siRNA”, “short interfering nucleic acid molecule”, “short interfering oligonucleotide molecule”, or “chemically-modified short interfering nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of mediating RNA interference (“RNAi”) or gene silencing; see for example Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914. Non-limiting examples of siRNA molecules of the invention are shown in FIG. 10. For example the siNA can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises complementarity to a target nucleic acid molecule. The siNA can be a single-stranded hairpin polynucleotide having self-complementary sense and antisense regions, wherein the antisense region comprises complementarity to a target nucleic acid molecule. The siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises complementarity to a target nucleic acid molecule, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA capable of mediating RNAi. As used herein, siNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules of the invention lack 2′-hydroxy (2′-OH) containing nucleotides. Applicant describes in certain embodiments short interfering nucleic acids that do not require the presence of nucleotides having a 2′-hydroxy group for mediating RNAi and as such, short interfering nucleic acid molecules of the invention optionally do not contain any ribonucleotides (e.g., nucleotides having a 2′-OH group). The modified short interfering nucleic acid molecules of the invention can also be referred to as short interfering modified oligonucleotides “siMON.” As used herein, the term siNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA, short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post-transciptional gene silencing.

[0148] By “modulate” is meant that the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up-regulated or down-regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator. For example, the term “modulate” can mean “inhibit” but the use of the word “modulate” is not limited to this definition.

[0149] By “inhibit” it is meant that the activity of a gene expression product or level of RNAs or equivalent RNAs encoding one or more gene products is reduced below that observed in the absence of the nucleic acid molecule of the invention. In one embodiment, inhibition with a siNA molecule preferably is below that level observed in the presence of an inactive or attenuated molecule that is unable to mediate an RNAi response. In another embodiment, inhibition of gene expression with the siNA molecule of the instant invention is greater in the presence of the siNA molecule than in its absence.

[0150] By “gene” or “target gene” is meant, a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide. The target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. The cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrates or invertebrates. Non-limiting examples of fungi include molds or yeasts.

[0151] By “HBV proteins” is meant, a peptide or protein comprising a component of HBV and/or encoded by a HBV gene.

[0152] By “highly conserved sequence region” is meant, a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.

[0153] By “complementarity” is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.

[0154] The siNA molecules of the invention represent a novel therapeutic approach to treat a variety of pathologic indications, such as HBV infection, liver failure, cirrhosis, hepatocellular carcinoma, and any other diseases or conditions that are related to or will respond to the levels of HBV in a cell or tissue, alone or in combination with other therapies. The reduction of HBV expression (specifically HBV gene RNA levels) and thus reduction in the level of the respective protein relieves, to some extent, the symptoms of the disease or condition.

[0155] In one embodiment of the present invention, each sequence of a siNA molecule of the invention is independently 18 to 24 nucleotides in length, in specific embodiments about 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. In another embodiment, the siNA duplexes of the invention independently comprise between 17 and 23 base pairs. In yet another embodiment, siNA molecules of the invention comprising hairpin or circular structures are 35 to 55 nucleotides in length, or 38-44 nucleotides in length and comprising 16-22 base pairs. Exemplary siNA molecules of the invention are shown in Table II. Exemplary synthetic siNA molecules of the invention are shown in Table III and/or FIGS. 4-5.

[0156] As used herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.

[0157] The siNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers. In particular embodiments, the nucleic acid molecules of the invention comprise sequences shown in Tables II-III and/or FIGS. 4-5. Examples of such nucleic acid molecules consist essentially of sequences defined in these tables and figures.

[0158] In another aspect, the invention provides mammalian cells containing one or more siNA molecules of this invention. The one or more siNA molecules can independently be targeted to the same or different sites.

[0159] By “RNA” is meant a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribo-furanose moiety. The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

[0160] By “subject” is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Subject” also refers to an organism to which the nucleic acid molecules of the invention can be administered. In one embodiment, a subject is a mammal or mammalian cells. In another embodiment, a subject is a human or human cells.

[0161] The term “phosphorothioate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise a sulfur atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.

[0162] The term “universal base” as used herein refers to nucleotide base analogs that form base pairs with each of the natural DNA/RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0163] The term “acyclic nucleotide” as used herein refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (C1, C2, C3, C4, or C5), are independently or in combination absent from the nucleotide.

[0164] The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed herein. For example, to treat a particular disease or condition, the siNA molecules can be administered to a patient or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.

[0165] In a further embodiment, the siNA molecules can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described molecules could be used in combination with one or more known therapeutic agents to treat a disease or condition. Non-limiting examples of other therapeutic agents that can be readily combined with a siNA molecule of the invention are enzymatic nucleic acid molecules, allosteric nucleic acid molecules, antisense, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules, and other organic and/or inorganic compounds including metals, salts and ions.

[0166] In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention, in a manner that allows expression of the siNA molecule. For example, the vector can contain sequence(s) encoding both strands of a siNA molecule comprising a duplex. The vector can also contain sequence(s) encoding a single nucleic acid molecule that is self-complementary and thus forms a siNA molecule. Non-limiting examples of such expression vectors are described in Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi:10.1038/nm725.

[0167] In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.

[0168] In yet another embodiment, the expression vector of the invention comprises a sequence for a siNA molecule having complementarity to a RNA molecule referred to by a Genbank Accession number, for example Genbank Accession No. AB073834 or Genbank Accession Nos. shown in Table I.

[0169] In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more siNA molecules, which can be the same or different.

[0170] In another aspect of the invention, siNA molecules that interact with target RNA molecules and down-regulate gene encoding target RNA molecules (for example target RNA molecules referred to by Genbank Accession numbers herein) are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecules bind and down-regulate gene function or expression via RNA interference (RNAi). Delivery of siNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell.

[0171] By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

[0172] By “comprising” is meant including, but not limited to, whatever follows the word “comprising.” Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0173] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0174] First the drawings will be described briefly.

[0175] Drawings

[0176]
FIG. 1 shows a non-limiting example of a scheme for the synthesis of siNA molecules. The complementary siNA sequence strands, strand 1 and strand 2, are synthesized in tandem and are connected by a cleavable linkage, such as a nucleotide succinate or abasic succinate, which can be the same or different from the cleavable linker used for solid phase synthesis on a solid support. The synthesis can be either solid phase or solution phase, in the example shown, the synthesis is a solid phase synthesis. The synthesis is performed such that a protecting group, such as a dimethoxytrityl group, remains intact on the terminal nucleotide of the tandem oligonucleotide. Upon cleavage and deprotection of the oligonucleotide, the two siNA strands spontaneously hybridize to form a siNA duplex, which allows the purification of the duplex by utilizing the properties of the terminal protecting group, for example by applying a trityl on purification method wherein only duplexes/oligonucleotides with the terminal protecting group are isolated.

[0177]
FIG. 2 shows a MALDI-TOV mass spectrum of a purified siNA duplex synthesized by a method of the invention. The two peaks shown correspond to the predicted mass of the separate siNA sequence strands. This result demonstrates that the siNA duplex generated from tandem synthesis can be purified as a single entity using a simple trityl-on purification methodology.

[0178]
FIG. 3 shows a non-limiting proposed mechanistic representation of target RNA degradation involved in RNAi. Double stranded RNA (dsRNA), which is generated by RNA-dependent RNA polymerase (RdRP) from foreign single-stranded RNA, for example viral, transposon, or other exogenous RNA, activates the DICER enzyme that in turn generates siNA duplexes. Alternately, synthetic or expressed siNA can be introduced directely into a cell by appropriate means. An active siNA complex forms which recognizes a target RNA, resulting in degradation of the target RNA by the RISC endonuclease complex or in the synthesis of additional RNA by RNA-dependent RNA polymerase (RdRP), which can activate DICER and result in additional siNA molecules, thereby amplifying the RNAi response.

[0179] FIGS. 4A-F shows non-limiting examples of chemically-modified siNA constructs of the present invention. In the figure, N stands for any nucleotide (adenosine, guanosine, cytosine, uridine, or optionally thymidine, for example thymidine can be substituted in the overhanging regions designated by parenthesis (N N). Various modifications are shown for the sense and antisense strands of the siNA constructs.

[0180]
FIG. 4A: The sense strand comprises 21 nucleotides having four phosphorothioate 5′ and 3′-terminal internucleotide linkages, wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methy or 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-tenninal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and having one 3′-terminal phosphorothioate internucleotide linkage and four 5′-terminal phosphorothioate internucleotide linkages and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.

[0181]
FIG. 4B: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.

[0182]
FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and having one 3′-terminal phosphorothioate internucleotide linkage and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.

[0183]
FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and having one 3′-terminal phosphorothioate internucleotide linkage and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.

[0184]
FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein.

[0185]
FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and having one 3′-terminal phosphorothioate internucleotide linkage and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand of constructs A-F comprise sequence complementary to target RNA sequence of the invention.

[0186] FIGS. 5A-F shows non-limiting examples of specific chemically-modified siNA sequences of the invention. FIGS. 5A-F applies the chemical modifications described in FIGS. 4A-F to an HBV siNA sequence.

[0187]
FIG. 6 shows non-limiting examples of different siNA constructs of the invention. The examples shown (constructs 1, 2, and 3) have 19 representative base pairs; however, different embodiments of the invention include any number of base pairs described herein. Bracketed regions represent nucleotide overhangs, for example comprising between about 1, 2, 3, or 4 nucleotides in length, preferably about 2 nucleotides. Constructs 1 and 2 can be used independently for RNAi activity. Construct 2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker. In one embodiment, the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of construct 1 in vivo and/or in vitro. In another example, construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siNA construct 2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siNA construct 1 in vivo and/or in vitro. As such, the stability and/or activity of the siNA constructs can be modulated based on the design of the siNA construct for use in vivo or in vitro and/or in vitro.

[0188] FIGS. 7A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate siNA hairpin constructs.

[0189]
FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1) sequence followed by a region having sequence identical (sense region of siNA) to a predetermined HBV target seqeunce, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, which is followed by a loop sequence of defined sequence (X), comprising, for example, between about 3 and 10 nucleotides.

[0190]
FIG. 7B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence that will result in a siNA transcript having specificity for an HBV target sequence and having self-complementary sense and antisense regions.

[0191]
FIG. 7C: The construct is heated (for example to about 95° C.) to linearize the sequence, thus allowing extension of a complementary second DNA strand using a primer to the 3′-restriction sequence of the first strand. The double-stranded DNA is then inserted into an appropriate vector for expression in cells. The construct can be designed such that a 3′-terminal nucleotide overhang results from the transcription, for example by engineering restriction sites and/or utilizing a poly-U termination region as described in Paul et al., 2002, Nature Biotechnology, 29, 505-508.

[0192] FIGS. 8A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate double-stranded siNA constructs.

[0193]
FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) site sequence followed by a region having sequence identical (sense region of siNA) to a predetermined HBV target seqeunce, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, and which is followed by a 3′-restriction site (R2) which is adjacent to a loop sequence of defined sequence (X).

[0194]
FIG. 8B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence.

[0195]
FIG. 8C: The construct is processed by restriction enzymes specific to R1 and R2 to generate a double-stranded DNA which is then inserted into an appropriate vector for expression in cells. The transcription cassette is designed such that a U6 promoter region flanks each side of the dsDNA which generates the separate sense and antisense strands of the siNA. Poly T termination sequences can be added to the constructs to generate U overhangs in the resulting transcript.

[0196] FIGS. 9A-E is a diagrammatic representation of a method used to determine target sites for siNA mediated RNAi within a particular target nucleic acid sequence, such as messenger RNA.

[0197]
FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein the antisense region of the siNA constructs has complementarity to target sites across the target nucleic acid sequence, and wherein the sense region comprises sequence complementary to the antisense region of the siNA.

[0198] FIGS. 9B & C: (FIG. 9B) The sequences are pooled and are inserted into vectors such that (FIG. 9C) transfection of a vector into cells results in the expression of the siNA.

[0199]
FIG. 9D: Cells are sorted based on phenotypic change that is associated with modulation of the target nucleic acid sequence.

[0200]
FIG. 9E The siNA is isolated from the sorted cells and is sequenced to identify efficacious target sites within the target nucleic acid sequence.

[0201]
FIG. 10 shows non-limiting examples of different stabilization chemistries (1-10) that can be used, for example, to stabilize the 3′-end of siNA sequences of the invention, including (1) [3-3′]-inverted deoxyribose; (2) deoxyribonucleotide; (3) [5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5) [5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7) [3-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9) [5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. In addition to modified and unmodified backbone chemistries indicated in the figure, these chemistries can be combined with different backbone modifications as described herein, for example, backbone modifications having Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to the terminal modifications shown can be another modified or unmodified nucleotide or non-nucleotide described herein, for example modifications having Formulae I-VII.

[0202]
FIG. 11 shows a graphical representation of siNA mediated inhibition of HBV in a cell culture experiment. Results are shown with reference to the siRNA construct used (sense strand SEQ ID NO: 1338/antisense strand SEQ ID NO: 1342) at different lipid concentrations (2.5, 5.0, 7.5, 10.0 and 12.5 ug/ml). Inverted sequence duplexes were used as negative controls (sense strand SEQ ID NO: 1358/antisense strand SEQ ID NO: 1350). Levels of secreted HBV surface antigen (HBsAg) were analyzed by ELISA.

MECHANISM OF ACTION OF NUCLEIC ACID MOLECULES OF THE INVENTION

[0203] The discussion that follows discusses the proposed mechanism of RNA interference mediated by short interfering RNA as is presently known, and is not meant to be limiting and is not an admission of prior art. Applicant demonstrates herein that chemically-modified short interfering nucleic acids possess similar or improved capacity to mediate RNAi as do siRNA molecules and are expected to possess improved stability and activity in vivo; therefore, this discussion is not meant to be limiting only to siRNA and can be applied to siNA as a whole. By “improved capacity to mediate RNAi” is meant to include RNAi activity measured in vitro and/or in vivo where the RNAi activity is a reflection of both the ability of the siNA to mediate RNAi and the stability of the siRNAs of the invention. In this invention, the product of these activities can be increased in vitro and/or in vivo compared to an all RNA siRNA or an siNA containing a plurality of ribonucleotides. In some cases, the activity or stability of the siNA molecule can be decreased (i.e., less than ten-fold), but the overall activity of the siNA molecule is enhanced, in vitro and/or in vivo RNA interference refers to the process of sequence specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′, 5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.

[0204] The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as Dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from Dicer activity are typically about 21-23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

[0205] RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21-nucleotide siRNA duplexes are most active when containing two 2-nucleotide 3′-terminal nucleotide overhangs. Furthermore, substitution of one or both siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of 3′-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated. Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309); however siRNA molecules lacking a 5′-phosphate are active when introduced exogenously, suggesting that 5′-phosphorylation of siRNA constructs may occur in vivo.

[0206] Synthesis of Nucleic Acid Molecules

[0207] Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (“small” refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.

[0208] Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. All of these references are incorporated herein by reference. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45 sec coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoro nucleotides. Table IV outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-fold excess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by calorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM I2, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

[0209] Deprotection of the DNA-based oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.

[0210] The method of synthesis used for RNA including certain siNA molecules of the invention follows the procedure as described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2′-O-methylated nucleotides. Table IV outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I2, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide0.05 M in acetonitrile) is used.

[0211] Deprotection of the RNA is performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEA•3HF to provide a 1.4 M HF concentration) and heated to 65° C. After 1.5 h, the oligomer is quenched with 1.5 M NH4HCO3.

[0212] Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65° C. for 15 min. The vial is brought to r.t. TEA•3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 min. The sample is cooled at −20° C. and then quenched with 1.5 M NH4HCO3.

[0213] For purification of the trityl-on oligomers, the quenched NH4HCO3 solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 min. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.

[0214] The average stepwise coupling yields are typically >98% (Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96-well format.

[0215] Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT Publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.

[0216] The siNA molecules of the invention can also be synthesized via a tandem synthesis methodology as described in Example 1 herein, wherein both siNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siNA fragments or strands that hybridize and permit purification of the siNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siNA as described herein can be readily adapted to both multiwell/multiplate synthesis platforms such as 96 well or similarly larger multi-well platforms. The tandem synthesis of siNA as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns and the like.

[0217] A siNA molecule can also be assembled from two distinct nucleic acid strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the RNA molecule.

[0218] The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is hereby incorporated herein by reference) and re-suspended in water.

[0219] In another aspect of the invention, siNA molecules of the invention are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules.

[0220] Optimizing Activity of the Nucleic Aacid Molecule of the Invention

[0221] Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al., supra; all of which are incorporated by reference herein). All of the above references describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.

[0222] There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-O-allyl, and/or 2′-H nucleotide base modifications (for a review, see Usman and Cedergren, 1992, TIBS, 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication, PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Picken et al., Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al., International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711; Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404 (filed on Apr. 20, 1998); Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the siNA nucleic acid molecules of the instant invention so long as the ability of siNA to promote RNAi is cells is not significantly inhibited.

[0223] While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorothioate, and/or 5′-methylphosphonate linkages improves stability, excessive modifications can cause some toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity, resulting in increased efficacy and higher specificity of these molecules.

[0224] Short interfering nucleic acid (siNA) molecules having chemical modifications that maintain or enhance activity are provided. Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid. Accordingly, the in vitro and/or in vivo activity should not be significantly lowered. In cases in which modulation is the goal, therapeutic nucleic acid molecules delivered exogenously should optimally be stable within cells until translation of the target RNA has been modulated long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211,3-19 (incorporated by reference herein)) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability, as described above.

[0225] In one embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in nucleic acid molecules of the invention results in both enhanced affinity and specificity to nucleic acid targets, complementary sequences, or template strands. In another embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleic acid” nucleotides such as a 2′, 4′-C mythylene bicyclo nucleotide (see for example Wengel et al., International PCT Publication No. WO 00/66604 and WO 99/14226).

[0226] In another embodiment, the invention features conjugates and/or complexes of siNA molecules of the invention. Such conjugates and/or complexes can be used to facilitate delivery of siNA molecules into a biological system, such as a cell. The conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.

[0227] The term “biodegradable linker” as used herein, refers to a nucleic acid or non-nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a siNA molecule of the invention or the sense and antisense strands of a siNA molecule of the invention. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base-modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.

[0228] The term “biodegradable” as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.

[0229] The term “biologically active molecule” as used herein, refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active siNA molecules either alone or in combination with othe molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs thereof. Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.

[0230] The term “phospholipid” as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.

[0231] Therapeutic nucleic acid molecules (e.g., siNA molecules) delivered exogenously optimally are stable within cells until reverse trascription of the RNA has been modulated long enough to reduce the levels of the RNA transcript. The nucleic acid molecules are resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.

[0232] In yet another embodiment, siNA molecules having chemical modifications that maintain or enhance enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and/or in vivo the activity should not be significantly lowered.

[0233] Use of the nucleic acid-based molecules of the invention will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules). The treatment of subjects with siNA molecules can also include combinations of different types of nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes), allozymes, antisense molecules, 2,5-A oligoadenylate, decoys, and aptamers.

[0234] In another aspect a siNA molecule of the invention comprises one or more 5′- and/or a 3′-cap structure, for example on only the sense siNA strand, antisense siNA strand, or both siNA strands.

[0235] By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5′-terminus (5′-cap) or at the 3′-terminal (3′-cap) or may be present on both termini. In non-limiting examples: the 5′-cap is selected from the group comprising glyceryl, inverted deoxy abasic residue (moiety); 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety.

[0236] In yet another embodiment, the 3′-cap is selected from a group comprising glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).

[0237] By the term “non-nucleotide” is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, and therefore lacks a base at the 1′-position.

[0238] An “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO2 or N(CH3)2, amino, or SH. The term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably, it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO2, halogen, N(CH3)2, amino, or SH. The term “alkyl” also includes alkynyl groups that have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO2 or N(CH3)2, amino or SH.

[0239] Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylaryl or hydrogen.

[0240] By “nucleotide” as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1′ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents.

[0241] In one embodiment, the invention features modified siNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39.

[0242] By “abasic” is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1′ position, see for example Adamic et al., U.S. Pat. No. 5,998,203.

[0243] By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, or uracil joined to the 1′ carbon of β-D-ribo-furanose.

[0244] By “modified nucleoside” is meant any nucleotide base that contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.

[0245] In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′-NH2 or 2′-O- NH2, which may be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878, which are both incorporated by reference in their entireties.

[0246] Various modifications to nucleic acid siNA structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.

[0247] Administration of Nucleic Acid Molecules

[0248] A siNA molecule of the invention can be adapted for use to treat, for example, HBV infection, liver failure cirrhosis, hepatocellular carcinoma and any other indications that can respond to the level of HBV in a cell or tissue, alone or in combination with other therapies. For example, a siNA molecule can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, all of which are incorporated herein by reference. Beigelman et al., U.S. Pat. No. 6,395,713, and Sullivan et al., PCT WO 94/02595, further describe the general methods for delivery of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al., 1999, Clin. Cancer Res., 5, 2330-2337 and Barry et al., International PCT Publication No. WO 99/31262. The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, modulate the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a patient.

[0249] Thus, the invention features a pharmaceutical composition comprising one or more nucleic acid(s) of the invention in an acceptable carrier, such as a stabilizer, buffer, and the like. The polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions, suspensions for injectable administration, and the other compositions known in the art.

[0250] The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

[0251] A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or patient, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.

[0252] By “systemic administration” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes that lead to systemic absorption include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes the siNA molecules of the invention to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells.

[0253] By “pharmaceutically acceptable formulation” is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Nonlimiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85), which can enhance entry of drugs into the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, D F et al., 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge, Mass.); and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

[0254] The invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al.,1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

[0255] The present invention also includes compositions prepared for storage or administration, which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985) hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.

[0256] The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985), hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.

[0257] A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.

[0258] The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.

[0259] Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.

[0260] Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

[0261] Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0262] Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.

[0263] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.

[0264] Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.

[0265] Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0266] The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

[0267] Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.

[0268] Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.

[0269] It is understood that the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

[0270] For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.

[0271] The nucleic acid molecules of the present invention can also be administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication canincrease the beneficial effects while reducing the presence of side effects.

[0272] In one embodiment, the invention comprises compositions suitable for administering nucleic acid molecules of the invention to specific cell types, such as hepatocytes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). Binding of such glycoproteins or synthetic glycoconjugates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose and galactosamine based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to the treatment of liver disease such as HBV infection or hepatocellular carcinoma. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavialability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of nucleic acid bioconjugates of the invention.

[0273] Alternatively, certain siNA molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990, Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

[0274] In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another embodiment, pol III based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pat. Nos. 5,902,880 and 6,146,886). The recombinant vectors capable of expressing the siNA molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from a patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510).

[0275] In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the instant invention. The expression vector can encode one or both strands of a siNA duplex, or a single self complementary strand that self hybridizes into a siNA duplex. The nucleic acid sequences encoding the siNA molecules of the instant invention can be operably linked in a manner that allows expression of the siNA molecule (see for example Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi:10.1038/nm725).

[0276] In another aspect, the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siNA molecules of the instant invention; wherein said sequence is operably linked to said initiation region and said termination region, in a manner that allows expression and/or delivery of the siNA molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the siNA of the invention; and/or an intron (intervening sequences).

[0277] Transcription of the siNA molecule sequences can be driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang, 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that nucleic acid molecules expressed from such promoters can function in mammalian cells (e.g., Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. U S A, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U. S. A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as siNA in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al., International PCT Publication No. WO 96/18736). The above siNA transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).

[0278] In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the siNA molecules of the invention, in a manner that allows expression of that siNA molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; and c) a nucleic acid sequence encoding at least one strand of the siNA molecule; wherein the sequence is operably linked to the initiation region and the termination region, in a manner that allows expression and/or delivery of the siNA molecule.

[0279] In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; and d) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame; and wherein the sequence is operably linked to the initiation region, the open reading frame and the termination region, in a manner that allows expression and/or delivery of the siNA molecule. In yet another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; and d) a nucleic acid sequence encoding at least one siNA molecule; wherein the sequence is operably linked to the initiation region, the intron and the termination region, in a manner which allows expression and/or delivery of the nucleic acid molecule.

[0280] In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; and e) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame; and wherein the sequence is operably linked to the initiation region, the intron, the open reading frame and the termination region, in a manner which allows expression and/or delivery of the siNA molecule.

EXAMPLES

[0281] The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.

Example 1

Tandem Synthesis of siNA Constructs

[0282] Exemplary siNA molecules of the invention are synthesized in tandem using a cleavable linker, for example a succinyl-based linker. Tandem synthesis as described herein is followed by a one-step purification process that provides RNAi molecules in high yield. This approach is highly amenable to siNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.

[0283] After completing a tandem synthesis of an siNA oligo and its compliment in which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact (trityl on synthesis), the oligonucleotides are deprotected as described above. Following deprotection, the siNA sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5′-O-DMT group while the complementary strand comprises a terminal 5′-hydroxyl. The newly formed duplex behaves as a single molecule during routine solid-phase extraction purification (Trityl-On purification) even though only one molecule has a dimethoxytrityl group. Because the strands form a stable duplex, this dimethoxytrityl group (or an equivalent group, such as other trityl groups or other hydrophobic moieties) is all that is required to purify the pair of oligos, for example by using a C18 cartridge.

[0284] Standard phosphoramidite synthesis chemistry is used up to point of introducing a tandem linker, such as an inverted deoxy abasic succinate or glyceryl succinate linker (see FIG. 1) or an equivalent cleavable linker. A non-limiting example of linker coupling conditions that can be used includes a hindered base such as diisopropylethylamine (DIPA) and/or DMAP in the presence of an activator reagent such as Bromotripyrrolidinophosphoniumhexaflurorophosphate (PyBrOP). After the linker is coupled, standard synthesis chemistry is utilized to complete synthesis of the second sequence leaving the terminal the 5′-O-DMT intact. Following synthesis, the resulting oligonucleotide is deprotected according to the procedures described herein and quenched with a suitable buffer, for example with 50 mM NaOAc or 1.5M NH4H2CO3.

[0285] Purification of the siNA duplex can be readily accomplished using solid phase extraction, for example using a Waters C18 SepPak 1 g cartridge conditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2 CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50 mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with 50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with 1 CV H2O followed by on-column detritylation, for example by passing 1 CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then adding a second CV of 1% aqueous TFA to the column and allowing to stand for approx. 10 minutes. The remaining TFA solution is removed and the column washed with H2O followed by 1 CV 1M NaCl and additional H2O. The siNA duplex product is then eluted, for example using 1 CV 20% aqueous CAN.

[0286]
FIG. 2 provides an example of MALDI-TOV mass spectrometry analysis of a purified siNA construct in which each peak corresponds to the calculated mass of an individual siNA strand of the siNA duplex. The same purified siNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably corresponding to the duplex siNA, and two peaks presumably corresponding to the separate siNA sequence strands. Ion exchange HPLC analysis of the same siNA contract only shows a single peak. Testing of the purified siNA construct using a luciferase reporter assay described below demonstrated the same RNAi activity compared to siNA constructs generated from separately synthesized oligonucleotide sequence strands.

Example 2

Identification of Potential siNA Target Sites in any RNA Sequence

[0287] The sequence of an RNA target of interest, such as a viral or human mRNA transcript, is screened for target sites, for example by using a computer folding algorithm. In a non-limiting example, the sequence of a gene or RNA gene transcript derived from a database, such as Genbank, is used to generate siNA targets having complimentarily to the target. Such sequences can be obtained from a database, or can be determined experimentally as known in the art. Target sites that are known, for example, those target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease or condition such as those sites containing mutations or deletions, can be used to design siNA molecules targeting those sites as well. Various parameters can be used to determine which sites are the most suitable target sites within the target RNA sequence. These parameters include but are not limited to secondary or tertiary RNA structure, the nucleotide base composition of the target sequence, the degree of homology between various regions of the target sequence, or the relative position of the target sequence within the RNA transcript. Based on these determinations, any number of target sites within the RNA transcript can be chosen to screen siNA molecules for efficacy, for example by using in vitro RNA cleavage assays, cell culture, or animal models. In a non-limiting example, anywhere from 1 to 1000 target sites are chosen within the transcript based on the size of the siNA contruct to be used. High throughput screening assays can be developed for screening siNA molecules using methods known in the art, such as with multi-well or multi-plate assays to determine efficient reduction in target gene expression.

Example 3

Selection of siNA Molecule Target Sites in a RNA

[0288] The following non-limiting steps can be used to carry out the selection of siNAs targeting a given gene sequence or transcipt.

[0289] 1. The target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, contained within the target sequence. This step is typically carried out using a custom Perl script, but commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package can be employed as well.

[0290] 2. In some instances the siNAs correspond to more than one target sequence; such would be the case for example in targeting many different strains of a viral sequence, for targeting different transcipts of the same gene, targeting different transcipts of more than one gene, or for targeting both the human gene and an animal homolog. In this case, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find matching sequences in each list. The subsequences are then ranked according to the number of target sequences that contain the given subsequence; the goal is to find subsequences that are present in most or all of the target sequences. Alternately, the ranking can indentify subsequences that are unique to a target sequence, such as a mutant target sequence. Such an approach would enable the use of siNA to target specifically the mutant sequence and not effect the expression of the normal sequence.

[0291] 3. In some instances the siNA subsequences are absent in one or more sequences while present in the desired target sequence; such would be the case if the siNA targets a gene with a paralogous family member that is to remain untargeted. As in case 2 above, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find sequences that are present in the target gene but are absent in the untargeted paralog.

[0292] 4. The ranked siNA subsequences can be further analyzed and ranked according to GC content. A preference can be given to sites containing 30-70% GC, with a further preference to sites containing 40-60% GC.

[0293] 5. The ranked siNA subsequences can be further analyzed and ranked according to self-folding and internal hairpins. Weaker internal folds are preferred; strong hairpin structures are to be avoided.

[0294] 6. The ranked siNA subsequences can be further analyzed and ranked according to whether they have runs of GGG or CCC in the sequence. GGG (or even more Gs) in either strand can make oligonucleotide synthesis problematic, so it is avoided whenever better sequences are available. CCC is searched in the target strand because that will place GGG in the antisense strand.

[0295] 7. The ranked siNA subsequences can be further analyzed and ranked according to whether they have the dinucleotide UU (uridine dinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end of the sequence (to yield 3′ UU on the antisense sequence). These sequences allow one to design siNA molecules with terminal TT thymidine dinucleotides.

[0296] 8. Four or five target sites are chosen from the ranked list of subsequences as described above. For example, in subsequences having 23 nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the upper (sense) strand of the siNA duplex, while the reverse complement of the left 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the lower (antisense) strand of the siNA duplex. If terminal TT residues are desired for the sequence (as described in paragraph 7), then the two 3′ terminal nucleotides of both the sense and antisense strands are replaced by TT prior to synthesizing the oligos.

[0297] 9. The siNA molecules are screened in an in vitro, cell culture or animal model system to identify the most active siNA molecule or the most preferred target site within the target RNA sequence.

[0298] In an alternate approach, a pool of siNA constructs specific to an HBV target sequence is used to screen for target sites in cells expressing HBV RNA. The general strategy used in this approach is shown in FIG. 9. Cells expressing HBV (e.g., HEPG2) are transfected with the pool of siNA constructs and cells that demonstrate a phenotype associated with HBV inhibition are sorted. The pool of siNA constructs can be expressed from transcription cassettes inserted into appropriate vectors (see for example FIG. 7 and FIG. 8). Cells in which HBV expression is decreased due to siNA treatment demonstrate a phenotypic change, for example decreased production of HBV RNA or protein(s) compared to untreated cells or cells treated with a control siNA. The siNA from cells demonstrating a positive phenotypic change (e.g., decreased HBV RNA or protein), are sequenced to determine the most suitable target site(s) within the target RNA sequence.

Example 4

HBV Targeted siNA Design

[0299] siNA target sites were chosen by analyzing sequences of the HBV RNA target and generating a consensus HBV sequence based on a minimun 65% homology for sequences referred to by Genbank Accession Numbers in Table I. This way, conserved sequences encoding HBV are targeted by siNA molecules of the invention. Alternately, target sequences are chosen using the methodology described in Example 3. siNA molecules were designed that could bind each target and are optionally individually analyzed by computer folding to assess whether the siNA molecule can interact with the target sequence. Varying the length of the siNA molecules can be chosen to optimize activity. Generally, a sufficient number of complementary nucleotide bases are chosen to bind to, or otherwise interact with, the target RNA, but the degree of complementarity can be modulated to accommodate siNA duplexes or varying length or base composition. By using such methodologies, siNA molecules can be designed to target sites within any known RNA sequence, for example those RNA sequences corresponding to the any gene transcript.

Example 5

Chemical Synthesis and Purification of siNA

[0300] siNA molecules can be designed to interact with various sites in the RNA message, for example target sequences within the RNA sequences described herein. The sequence of one strand of the siNA molecule(s) is complementary to the target site sequences described above. The siNA molecules can be chemically synthesized using methods described herein. Inactive siNA molecules that are used as control sequences can be synthesized by scrambling the sequence of the siNA molecules such that it is not complementary to the target sequence.

Example 6

Models Used to Evaluate the Down-Regulation of HBV Gene Expression

[0301] Nucleic acid molecules targeted to the human HBV RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the procedures described below. A variety of endpoints have been used in cell culture models to evaluate HBV-mediated effects after treatment with anti-HBV agents. Phenotypic endpoints include inhibition of cell proliferation, apoptosis assays and reduction of HBV RNA/protein expression. There are several methods by which these endpoints can be measured. For example, a nucleic acid-mediated decrease in the level of HBV RNA and/or HBV protein expression can be evaluated using methods known in the art, such as RT-PCR, Northern blot, ELISA, Western blot, and immunoprecipitation analyses, to name a few techniques.

[0302] Phenotypic Assays

[0303] Intracellular HBV gene expression can be assayed either by a Taqman® assay for HBV RNA or by ELISA for HBV protein. Extracellular virus can be assayed either by PCR for DNA or ELISA for protein. Antibodies are commercially available for HBV surface antigen and core protein. A secreted alkaline phosphatase expression plasmid can be used to normalize for differences in transfection efficiency and sample recovery. The method consists of coating a micro-titer plate with an antibody such as anti-HBsAg Mab (for example, Biostride B88-95-31ad,ay) at 0.1 to 10 μg/ml in a buffer (for example, carbonate buffer, such as Na2CO3 15 mM, NaHCO3 35 mM, pH 9.5) at 4° C. overnight. The microtiter wells are then washed with PBST or the equivalent thereof, (for example, PBS, 0.05% Tween 20) and blocked for 0.1-24 hr at 37° C. with PBST, 1% BSA or the equivalent thereof. Following washing as above, the wells are dried (for example, at 37° C. for 30 min). Biotinylated goat anti-HBsAg or an equivalent antibody (for example, Accurate YVS1807) is diluted (for example at 1:1000) in PBST and incubated in the wells (for example, 1 hr. at 37° C.). The wells are washed with PBST (for example, 4×). A conjugate, (for example, Streptavidin/Alkaline Phosphatase Conjugate, Pierce 21324) is diluted to 10-10,000 ng/ml in PBST, and incubated in the wells (for example, 1 hr. at 37° C.). After washing as above, a substrate (for example, p-nitrophenyl phosphate substrate, Pierce 37620) is added to the wells, which are then incubated (for example, 1 hr. at 37° C.). The optical density is then determined (for example, at 405 nm). SEAP levels are then assayed, for example, using the Great EscAPe® Detection Kit (Clontech K2041-1), as per the manufacturer's instructions. In the above example, incubation times and reagent concentrations can be varied to achieve optimum results.

[0304] Cell Culture Models

[0305] HBV does not infect cells in culture. However, transfection of HBV DNA (either as a head-to-tail dimer or as an “overlength” genome of >100%) into HuH7 or Hep G2 hepatocytes results in viral gene expression and production of HBV virions released into the media. Thus, HBV replication competent DNA would be co-transfected with siNA molecules in cell culture. Such an approach has been used to report intracellular enzymatic nucleic acid molecule activity against HBV (zu Putlitz, et al., 1999, J. Virol., 73, 5381-5387, and Kim et al., 1999, Biochem. Biophys. Res. Commun., 257, 759-765). In addition, stable hepatocyte cell lines have been generated that express HBV.

[0306] Animal Models

[0307] The development of new antiviral agents for the treatment of chronic Hepatitis B has been aided by the use of animal models that are permissive to replication of related Hepadnaviridae such as Woodchuck Hepatitis Virus (WHV) and Duck Hepatitis Virus (DHV). In addition the use of transgenic mice has also been employed. Macejak et al., U.S. Ser. No. 60/335,059 (incorporated by reference herein in its entirety), describe a model in which the human hepatoblastoma cell line, HepG2.2.15, implanted as a subcutaneous (SC) tumor, was evaluated in terms of its usefulness in producing Hepatitis B viremia in mice. This model is useful for evaluating new HBV therapies such as siNA molecules described herein. The study showed that in mice bearing HepG2.2.15 SC tumors, HBV viremia was present. HBV DNA was detected in serum beginning on Day 35. Maximum serum viral levels reached 1.9×105 copies/mL by day 49. This study also determined that the minimum tumor volume associated with viremia was 300 mm3. Therefore, the HepG2.2.15 cell line grown as a SC tumor produces a useful model of HBV viremia in mice. This model is suitable for evaluating siNA molecules of the invention targeting HBV RNA.

[0308] There are several other small animal models to study HBV replication. One is the transplantation of HBV-infected liver tissue into irradiated mice. Viremia (as evidenced by measuring HBV DNA by PCR) is first detected 8 days after transplantation and peaks between 18-25 days (Ilan et al., 1999, Hepatology, 29, 553-562). Transgenic mice that express HBV have also been used as a model to evaluate potential anti-virals. HBV DNA is detectable in both liver and serum (Morrey et al., 1999, Antiviral Res., 42, 97-108). An additional model is to establish subcutaneous tumors in nude mice with Hep G2 cells transfected with HBV. Tumors develop in about 2 weeks after inoculation and express HBV surface and core antigens. HBV DNA and surface antigen is also detected in the circulation of tumor-bearing mice (Yao et al., 1996, J. Viral Hepat., 3, 19-22). Woodchuck hepatitis virus (WHV) is closely related to HBV in its virus structure, genetic organization, and mechanism of replication. As with HBV in humans, persistent WHV infection is common in natural woodchuck populations and is associated with chronic hepatitis and hepatocellular carcinoma (HCC). Experimental studies have established that WHV causes HCC in woodchucks and woodchucks chronically infected with WHV have been used as a model to test a number of anti-viral agents. For example, the nucleoside analogue 3T3 was observed to cause dose dependent reduction in virus (50% reduction after two daily treatments at the highest dose) (Hurwitz et al., 1998. Antimicrob. Agents Chemother., 42, 2804-2809).

Example 7

Inhibition of HBV Using siNA Molecules of the Invention

[0309] Transfection of HepG2 Cells with psHB V-1 and siNA

[0310] The human hepatocellular carcinoma cell line Hep G2 was grown in Dulbecco's modified Eagle media supplemented with 10% fetal calf serum, 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 25 mM Hepes, 100 units penicillin, and 100 μg/ml streptomycin. To generate a replication competent cDNA, prior to transfection the HBV genomic sequences are excised from the bacterial plasmid sequence contained in the psHBV-1 vector (Those skilled in the art understand that other methods can be used to generate a replication competent cDNA). This was done with an EcoRi and Hind III restriction digest. Following completion of the digest, a ligation was performed under dilute conditions (20 μg/ml) to favor intermolecular ligation. The total ligation mixture was then concentrated using Qiagen spin columns.

[0311] Transfection of the human hepatocellular carcinoma cell line, Hep G2, with replication-competent HBV DNA results in the expression of HBV proteins and the production of virions. To test the efficacy of siNAs targeted against HBV RNA, the siNA duplex (sense strand SEQ ID NO: 1338/antisense strand SEQ ID NO: 1342) was co-transfected with HBV genomic DNA twice at 25 nM, the first time with siNA and lipid 12.5 ug/ml and the second time with siNA and lipid at 2.5 ug/ml, 5.0 ug/ml, 7.5 ug/ml and 10 ug/ml, into Hep G2 cells, and the subsequent levels of secreted HBV surface antigen (HBsAg) were analyzed by ELISA. Inverted sequence duplexes were used as negative controls (sense strand SEQ ID NO: 1358/antisense strand SEQ ID NO: 1350). Alternately, the siNA duplex (sense strand SEQ ID NO: 1338/antisense strand SEQ ID NO: 1342) and (two right side colums in FIG. 11) was co-transfected with HBV genomic DNA once at 25 nM with lipid at 12.5 ug/ml into Hep G2 cells, and the subsequent levels of secreted HBV surface antigen (HBsAg) were analyzed by ELISA.

[0312] Analysis of HBsAg Levels Following siNA Treatment

[0313] Immulon 4 (Dynax) microtiter wells were coated overnight at 4° C. with anti-HBsAg Mab (Biostride B88-95-31ad,ay) at 1 μg/ml in Carbonate Buffer (Na2CO3 15 mM, NaHCO3 35 mM, pH 9.5). The wells were then washed 4× with PBST (PBS, 0.05% Tween® 20) and blocked for 1 hr at 37° C. with PBST, 1% BSA. Following washing as above, the wells were dried at 37° C. for 30 min. Biotinylated goat ant-HBsAg (Accurate YVS1807) was diluted 1:1000 in PBST and incubated in the wells for 1 hr. at 37° C. The wells were washed 4× with PBST. Streptavidin/Alkaline Phosphatase Conjugate (Pierce 21324) was diluted to 250 ng/ml in PBST, and incubated in the wells for 1 hr. at 37° C. After washing as above, p-nitrophenyl phosphate substrate (Pierce 37620) was added to the wells, which were then incubated for 1 hr. at 37° C. The optical density at 405 nm was then determined. Results of this study are summarized in FIG. 11, where the siNA duplex (sense strand SEQ ID NO: 1338/antisense strand SEQ ID NO: 1342) and inverted control siNA duplex (sense strand SEQ ID NO: 1358/antisense strand SEQ ID NO: 1350) were tested at differing lipid concentrations as indicated in the figure. As shown in FIG. 11, the siRNA construct targeting site 413 of HBV RNA provides significant inhibition of viral replication/activity when compared to an inverted siRNA control. This effect is seen consistently at differing concentrations of lipid transfection agent.

Example 8

RNAi In Vitro Assay to Assess siNA Activity

[0314] An in vitro assay that recapitulates RNAi in a cell free system is used to evaluate siNA constructs targeting HBV RNA targets. The assay comprises the system described by Tuschl et al., 1999, Genes and Development, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33 adapted for use with HBV target RNA. A Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro. Target RNA is generated via in vitro transcription from an appropriate HBV expressing plasmid using T7 RNA polymerase or via chemical synthesis as described herein. Sense and antisense siNA strands (for example 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 min. at 90° C. followed by 1 hour at 37° C., then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide. The Drosophila lysate is prepared using zero- to two-hour-old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated. The assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10% [vol/vol] lysis buffer containing siNA (10 nM final concentration). The reaction mixture also contains 10 mM creatine phosphate, 10 ug.ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reactions are pre-assembled on ice and preincubated at 25° C. for 10 minutes before adding RNA, then incubated at 25° C. for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25× Passive Lysis Buffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which siNA is omitted from the reaction.

[0315] Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [a-32P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5′-32P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing intact control RNA or RNA from control reactions without siNA and the cleavage products generated by the assay.

[0316] In one embodiment, this assay is used to determine target sites the HBV RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA constructs are screened for RNAi mediated cleavage of the HBV RNA target, for example by analyzing the assay reaction by electrophoresis of labeled target RNA, or by northern blotting, as well as by other methodologies well known in the art.

Example 9

Diagnostic Uses

[0317] The siNA molecules of the invention can be used in a variety of diagnostic applications, such as in identifying molecular targets such as RNA in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings. Such diagnostic use of siNA molecules involves utilizing reconstituted RNAi systems, for example using cellular lysates or partially purified cellular lysates. siNA molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous, for example viral, RNA in a cell. The close relationship between siNA activity and the structure of the target RNA allows the detection of mutations in any region of the molecule, which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple siNA molecules described in this invention, one can map nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with siNA molecules can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease or infection. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes, siNA molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations siNA molecules and/or other chemical or biological molecules). Other in vitro uses of siNA molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with a disease, infection, or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a siNA using standard methodologies, for example fluorescence resonance emission transfer (FRET).

[0318] In a specific example, siNA molecules that can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first siNA molecules (i.e., those that can cleave only wild-type foms of target RNA) are used to identify wild-type RNA present in the sample and the second siNA molecules (i.e., those that can cleave only mutant forms of target RNA) will be used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA will be cleaved by both siNA molecules to demonstrate the relative siNA efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis will require two siNA molecules, two substrates, and one unknown sample, which will be combined into six reactions. The presence of cleavage products will be determined using an RNase protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., disease related or infection related) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

[0319] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

[0320] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

[0321] It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims.

[0322] The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

[0323] In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

1TABLE IHBV SequencesAccessionSeq NameTotal ScoreNo.LOCUSgi|5019947|gb|AF143301.1|AF143301230559AF143301.1AF143301gi|6063432|dbj|AB033552.1|AB033552292977AB033552.1AB033552gi|12060433|dbj|AB049609.1|267942AB049609.1gi|6692481|gb|AF121242.1|AF121242228783AF121242.1AF121242gi|21280241|dbj|AB073828.1|307127AB073828.1gi|1107590|emb|X80925.1|HBVP6PCXX208895X80925.1HBVP6PCXXgi|13491148|gb|AF330110.1|AF330110295703AF330110.1AF330110gi|21280285|dbj|AB073850.1|250338AB073850.1gi|21326584|ref|NC_003977.1|292733NC_003977.1gi|19568072|gb|AY077736.1|159292AY077736.1gi|1914688|emb|X98073.1|HBVCGINCX272903X98073.1HBVCGINCXgi|9454475|gb|AF282918.1|AF282918296123AF282918.1AF282918gi|21280269|dbj|AB073842.1|261966AB073842.1gi|329628|gb|M54923.1|HPBADWZ251770M54923.1HPBADWZgi|6009770|dbj|AB026813.1|AB026813302607AB026813.1AB026813gi|19568077|gb|AY077735.1|169063AY077735.1gi|21280295|dbj|AB073855.1|245594AB073855.1gi|452637|emb|X75658.1|HHVBFFOU170005X75658.1HHVBFFOUgi|11191867|dbj|AB036909.1|AB036909184492AB036909.1AB036909gi|15425688|dbj|AB056513.1|185259AB056513.1gi|6692485|gb|AF121246.1|AF121246300222AF121246.1AF121246gi|21280279|dbj|AB073847.1|251950AB073847.1gi|5019984|gb|AF143308.1|AF143308225170AF143308.1AF143308gi|10441111|gb|AF182804.1|AF182804262414AF182804.1AF182804gi|5019979|gb|AF143307.1 |AF143307227324AF143307.1AF143307gi|10441106|gb|AF182803.1|AF182803264971AF182803.1AF182803gi|10443814|gb|AF241408.1|AF241408248822AF241408.1AF241408gi|15419852|gb|AF297624.1|AF297624248555AF297624.1AF297624gi|6063467|dbj|AB033559.1|AB033559220175AB033559.1AB033559gi|15419847|gb|AF297623.1|AF297623218711AF297623.1AF297623gi|11071965|dbj|A8042283.1|AB042283289018AB042283.1AB042283gi|5114069|gb|AF090839.1|AF090839250372AF090839.1AF090839gi|5019943|gb|AF143300.1|AF143300223920AF143300.1AF143300gi|6063422|dbj|AB033550.1|AB033550298540AB033550.1AB033550gi|1914693|emb|X98072.1|HBVCGINSC274513X98072.1HBVCGINSCgi|12060438|dbj|AB049610.1|255954AB049610.1gi|21280227|dbj|AB073821.1|286641AB073821.1gi|11191835|dbj|AB036905.1|AB036905184492AB036905.1AB036905gi|11191851|dbj|AB036907.1|AB036907183325AB036907.1AB036907gi|15778318|gb|AF411408.1|AF411408286382AF411408.1AF411408gi|21280253|dbj|AB073834.1|309461AB073834.1gi|560062|dbj|D23678.1|HPBA2HYS2261916D23678.1HPBA2HYS2gi|10443830|gb|AF241410.1|AF241410244968AF241410.1AF241410gi|6009760|dbj|AB026811.1|AB026811303731AB026811.1AB026811gi|21280237|dbj|AB073826.1|306003AB073826.1gi|1914710|emb|X98075.1|HBVDEFVP2282323X98075.1HBVDEFVP2gi|1914704|emb|X98074.1|HBVDEFVP1259015X98074.1HBVDEFVP1gi|1914699|emb|X98077.1|HBVCGWITY285934X98077.1HBVCGWITYgi|18621111|emb|AJ344116.1|HEB344116218625AJ344116.1HEB344116gi|21280263|dbj|AB073839.1|274633AB073839.1gi|2182117|gb|U95551.1|U95551222999U95551.1U95551gi|18845081|gb|AF473543.1|275269AF473543.1gi|11191939|dbj|AB036918.1|AB036918180426AB036918.1AB036918gi|18146697|dbj|AB064316.1|164182AB064316.1gi|221497|dbj|D00329.1|HPBADW1269342D00329.1HPBADW1gi|6063457|dbj|AB033557.1|AB033557276860AB033557.1AB033557gi|807711|dbj|D50489.1|HPBA11A271115D50489.1HPBA11Agi|6692478|gb|AF121239.1|AF121239222516AF121239.1AF121239gi|12246985|gb|AF223958.1|AF223958277556AF223958.1AF223958gi|4140292|emb|AJ131956.1|HBV131956223626AJ131956.1HBV131956gi|13991863|gb|AF363961.1|AF363961287052AF363961.1AF363961gi|5019931|gb|AF143298.1|AF143298234175AF143298.1AF143298gi|12585611|gb|M57663.2|HPBADWZCG213646M57663.2HPBADWZCGgi|1814218|gb|U46935.1|HBU46935170791U46935.1HBU46935gi|4323201|gb|AF100309.1|291448AF100309.1gi|15778322|gb|AF411409.1|AF411409274184AF411409.1AF411409gi|11191907|dbj|AB036914.1|AB036914184305AB036914.1AB036914gi|9454470|gb|AF282917.1|AF282917278685AF282917.1AF282917gi|12247041|gb|AF223965.1|AF223965174200AF223965.1AF223965gi|13365550|dbj|AB048705.1|AB048705221176AB048705.1AB048705gi|11191923|dbj|AB036916.1|AB036916184242AB036916.1AB036916gi|21280301|dbj|AB073858.1|246498AB073858.1gi|21280265|dbj|AB073840.1|293680AB073840.1gi|16751309|gb|AY057948.1|265183AY057948.1gi|6692479|gb|AF121240.1|AF121240237011AF121240.1AF121240gi|6692482|gb|AF121243.1|AF121243300260AF121243.1AF121243gi|21280291|dbj||AB073853.1|217945AB073853.1gi|21280249|dbj|AB073832.1|255818AB073832.1gi|21624227|dbj|AB074755.1|271043AB074755.1gi|21280275|dbj|AB073845.1|257829AB073845.1gi|20800457|gb|U87746.3|220314U87746.3gi|5019974|gb|AF143306.1|AF143306220769AF143306.1AF143306gi|18146685|dbj|AB064314.1|247361AB064314.1gi|21280259|dbj|AB073837.1|290908AB073837.1gi|10441101|gb|AF182802.1|AF182802255610AF182802.1AF182802gi|1155012|emb|X51970.1|HVHEPB230814X51970.1HVHEPBgi|6063447|dbj|AB033555.1|AB033555258566AB033555.1AB033555gi|4490403|emb|Y18857.1|HBV18857274668Y18857.1HBV18857gi|4468847|emb|AJ131133.1|HBV131133273880AJ131133.1HBV131133gi|15419842|gb|AF297622.1|AF297622232545AF297622.1AF297622gi|21431678|gb|U87747.3|273379U87747.3gi|6692486|gb|AF121247.1|AF121247284619AF121247.1AF121247gi|15419837|gb|AF297621.1|AF297621221472AF297621.1AF297621gi|329667|gb|M32138.1|HPBHBVAA215731M32138.1HPBHBVAAgi|5114064|gb|AF090838.1|AF090838222057AF090838.1AF090838gi|12247001|gb|AF223960.1|AF223960271086AF223960.1AF223960gi|18389985|gb|AF462041.1|250341AF462041.1gi|560087|dbj|D23683.1|HPBC5HKO2247233D23683.1HPBC5HKO2gi|14334406|gb|AY034878.1|214004AY034878.1gi|11935071|gb|AF305327.1|AF305327181882AF305327.1AF305327gi|59455|emb|X70185.1|HBVXCPS255514X70185.1HBVXCPSgi|11191875|dbj|AB036910.1|AB036910179530AB036910.1AB036910gi|18252538|gb|AF458665.1|AF458665256655AF458665.1AF458665gi|12247017|gb|AF223962.1|AF223962171583AF223962.1AF223962gi|11191891|dbj|AB036912.1|AB036912184598AB036912.1AB036912gi|21280233|dbj|AB073824.1|273493AB073824.1gi|15425700|dbj|AB056516.1|172653AB056516.1gi|12060183|dbj|AB037927.1|AB037927161772AB037927.1AB037927gi|6692490|gb|AF121251.1|AF121251281174AF121251.1AF121251gi|13365546|dbj|AB048703.1|AB048703221017AB048703.1AB048703gi|18252589|gb|AF461043.1|AF461043276940AF461043.1AF461043gi|451966|gb|L27106.1|HPBMUT199839L27106.1HPBMUTgi|21280243|dbj|AB073829.1|304269AB073829.1gi|18146673|dbj|AB064312.1|181001AB064312.1gi|15211885|emb|AJ309369.1|HEB309369233378AJ309369.1HEB309369gi|6063437|dbj|AB033553.1|AB033553293149AB033553.1AB033553gi|21280287|dbj|AB073851.1|256764AB073851.1gi|19849032|gb|AF405706.1|186584AF405706.1gi|5019968|gb|AF143305.1|AF143305219149AF143305.1AF143305gi|21280297|dbj|AB073856.1|253274AB073856.1gi|474959|gb|M12906.1|HPBADRA283060M12906.1HPBADRAgi|6009775|dbj|AB026814.1|AB026814303971AB026814.1AB026814gi|4490393|emb|Y18855.1|HBV18855274699Y18855.1HBV18855gi|10443806|gb|AF241407.1|AF241407248499AF241407.1AF241407gi|4490408|emb|Y18858.1|HBV18858275693Y18858.1HBV18858gi|527440|emb|Z35717.1|HBVGEN2253405Z35717.1HBVGEN2gi|5211892|emb|AJ309370.1|HEB309370235466AJ309370.1HEB309370gi|5257487|gb|AF151735.1|AF151735227289AF151735.1AF151735gi|329649|gb|M38636.1|HPBCGADR261795M38636.1HPBCGADRgi|11071967|dbj|AB042284.1|AB042284281386AB042284.1AB042284gi|16751304|gb|AY057947.1|292195AY057947.1gi|560067|dbj|D23679.1|HPBA3HMS2270945D23679.1HPBA3HMS2gi|21280245|dbj|AB073830.1|271145AB073830.1gi|18146661|dbj|AB064310.1|179324AB064310.1gi|560077|dbj|D23681.1|HPBC4HST2258955D23681.1HPBC4HST2gi|21280271|dbj|AB073843.1|264504AB073843.1gi|21280229|dbj|AB073822.1|295616AB073822.1gi|1359675|emb|X97848.1|HBVP2CSX226618X97848.1HBVP2CSXgi|6063427|dbj|AB033551.1|AB033551291777AB033551.1AB033551gi|21280255|dbj|AB073835.1|264943AB073835.1gi|6692483|gb|AF121244.1|AF121244301914AF121244.1AF121244gi|21280281|dbj|AB073848.1|279357AB073848.1gi|21280239|dbj|AB073827.1|307559AB073827.1gi|15425692|dbj|AB056514.1|184194AB056514.1gi|12246961|gb|AF223955.1|AF223955282408AF223955.1AF223955gi|10934053|dbj|AB050018.1|AB050018298098AB050018.1AB050018gi|18032031|gb|AY066028.1|285293AY066028.1gi|19224211|gb|AF479684.1|293124AF479684.1gi|6009765|dbj|AB026812.1|AB026812302802AB026812.1AB026812gi|11191859|dbj|AB036908.1|AB036908179271AB036908.1AB036908gi|1514493|emb|Y07587.1|HBVAYWGEN234664Y07587.1HBVAYWGENgi|10443838|gb|AF241411.1|AF241411247518AF241411.1AF241411gi|18621118|emb|AJ344117.1|HEB344117222960AJ344117.1HEB344117gi|6692487|gb|AF121248.1|AF121248286004AF121248.1AF121248gi|12246977|gb|AF223957.1|AF223957285866AF223957.1AF223957gi|18252533|gb|AF458664.1|AF458664299135AF458664.1AF458664gi|527435|emb|Z35716.1|HBVGEN1231786Z35716.1HBVGEN1gi|4490398|emb|Y18856.1|HBV18856269371Y18856.1HBV18856gi|4323196|gb|AF100308.1|AF100308295195AF100308.1AF100308gi|13365542|dbj|AB048701.1|AB048701203430AB048701.1AB048701gi|4206634|gb|AF068756.1|AF068756278450AF068756.1AF068756gi|12247033|gb|AF223964.1|AF223964165757AF223964.1AF223964gi|11191843|dbj|AB036906.1|AB036906183325AB036906.1AB036906gi|11877208|emb|AJ132335.1|HBV132335201764AJ132335.1HBV132335gi|1107583|emb|X80926.1|HBVP5PCXX219450X80926.1HBVP5PCXXgi|5019963|gb|AF143304.1|AF143304241689AF143304.1AF143304gi|21280267|dbj|AB073841.1|277079AB073841.1gi|2288869|dbj|D28880.1|D28880255723D28880.1D28880gi|5019958|gb|AF143303.1|AF143303220264AF143303.1AF143303gi|18621103|emb|AJ344115.1|HEB344115201900AJ344115.1HEB344115gi|21280293|dbj|AB073854.1|241071AB073854.1gi|18031713|gb|AY033073.1|267088AY033073.1gi|14485224|gb|AF384372.1|AF384372276619AF384372.1AF384372gi|5114084|gb|AF090842.1|AF090842214699AF090842.1AF090842gi|21280277|dbj|AB073846.1|266691AB073846.1gi|5114079|gb|AF090841.1|AF090841232925AF090841.1AF090841gi|18031708|gb|AY033072.1|270012AY033072.1gi|11191947|dbj|AB036919.1|AB036919184078AB036919.1AB036919gi|221505|dbj|D00220.1|HPBVCG178041D00220.1HPBVCGgi|6063462|dbj|AB033558.1|AB033558197912AB033558.1AB033558gi|541655|dbj|D16665.1|HPBADRM253402D16665.1HPBADRMgi|11071963|dbj|AB042282.1|AB042282283035AB042282.1AB042282gi|10443822|gb|AF241409.1|AF241409226800AF241409.1AF241409gi|15778336|gb|AF411412.1|AF411412263874AF411412.1AF411412gi|1359690|emb|X97850.1|HBVP4CSX253482X97850.1HBVP4CSXgi|313780|emb|X59795.1|HBVAYWMCG214427X59795.1HBVAYWMCGgi|12247009|gb|AF223961.1|AF223961274267AF223961.1AF223961gi|6692480|gb|AF121241.1|AF121241236801AF121241.1AF121241gi|21280251|dbj|AB073833.1|294823AB073833.1gi|11191915|dbj|AB036915.1|AB036915183839AB036915.1AB036915gi|21280235|dbj|AB073825.1|266911AB073825.1gi|11191931|dbj|AB036917.1|AB036917184242AB036917.1AB036917gi|1107576|emb|X80924.1|HBVP4PCXX218080X80924.1HBVP4PCXXgi|221500|dbj|D12980.1|HPBCG289726D12980.1HPBCGgi|21280261|dbj|AB073838.1|248223AB073838.1gi|13365548|dbj|AB048704.1|AB048704218626AB048704.1AB048704gi|21624234|dbj|AB074756.1|270134AB074756.1gi|11191899|dbj|AB036913.1|AB036913177808AB036913.1AB036913gi|14290239|gb|AF384371.1|AF384371285891AF384371.1AF384371gi|18146691|dbj|AB064315.1|121870AB064315.1gi|15419830|gb|AF297620.1|AF297620237532AF297620.1AF297620gi|6983934|gb|AF160501.1|AF160501182351AF160501.1AF160501gi|21388705|dbj|AB074047.1|244069AB074047.1gi|6063452|dbj|AB033556.1|AB033556290959AB033556.1AB033556gi|6692484|gb|AF121245.1|AF121245300260AF121245.1AF121245gi|21280289|dbj|AB073852.1|246774AB073852.1gi|18146679|dbj|AB064313.1|175470AB064313.1gi|221499|dbj|D00331.1|HPBADW3252986D00331.1HPBADW3gi|15211900|emb|AJ309371.1|HEB309371239614AJ309371.1HEB309371gi|560082|dbj|D23682.1|HPBB5HKO1255559D23682.1HPBB5HKO1gi|21280299|dbj|AB073857.1|247755AB073857.1gi|15778332|gb|AF411411.1|AF411411286382AF411411.1AF411411gi|15778327|gb|AF411410.1|AF411410267992AF411410.1AF411410gi|12246993|gb|AF223959.1|AF223959279509AF223959.1AF223959gi|2829154|gb|AF043594.1|AF043594223502AF043594.1AF043594gi|6692488|gb|AF121249.1|AF121249305912AF121249.1AF121249gi|10441116|gb|AF182805.1 51 AF182805265419AF182805.1AF182805gi|15419857|gb|AF297625.1|AF297625216580AF297625.1AF297625gi|11191883|dbj|AB036911.1|AB036911184393AB036911.1AB036911gi|5019937|gb|AF143299.1|AF143299235942AF143299.1AF143299gi|11071969|dbj|AB042285.1|AB042285257613AB042285.1AB042285gi|12060190|dbj|AB037928.1|AB037928168481AB037928.1AB037928gi|21280247|dbj|AB073831.1|287868AB073831.1gi|1359682|emb|X97849.1|HBVP3CSX218388X97849.1HBVP3CSXgi|329616|gb|M38454.1|HPBADR1CG272026M38454.1HPBADR1CGgi|21280273|dbj|AB073844.1|261056AB073844.1gi|1359698|emb|X97851.1|HBVP6CSX295810X97851.1HBVP6CSXgi|6063442|dbj|AB033554.1|AB033554257344AB033554.1AB033554gi|5114074|gb|AF090840.1|AF090840247925AF090840.1AF090840gi|6692489|gb|AF121250.1|AF121250294273AF121250.1AF121250gi|18146667|dbj|AB064311.1|179163AB064311.1gi|21280257|dbj|AB073836.1|264148AB073836.1gi|12246953|gb|AF223954.1|AF223954285309AF223954.1AF223954gi|9082083|gb|AF233236.1|AF233236282508AF233236.1AF233236gi|288927|emb|X72702.1|HBVORFS225307X72702.1HBVORFSgi|221498|dbj|D00330.1|HPBADW2299926D00330.1HPBADW2gi|21280283|dbj|AB073849.1|230392AB073849.1gi|1914716|emb|X98076.1|HBVDEFVP3228252X98076.1HBVDEFVP3gi|15425696|dbj|AB056515.1|180333AB056515.1gi|560057|dbj|D23677.1|HPBA1HKK2269146D23677.1HPBA1HKK2gi|6009780|dbj|AB026815.1|AB026815294151AB026815.1AB026815gi|12246969|gb|AF223956.1|AF223956278063AF223956.1AF223956gi|15072539|gb|AY040627.1|287659AY040627.1gi|2829148|gb|AF043593.1|AF043593226770AF043593.1AF043593gi|560072|dbj|D23680.1|HPBB4HST1279791D23680.1HPBB4HST1gi|13991873|gb|AF363963.1|AF363963279252AF363963.1AF363963gi|560092|dbj|D23684.1|HPBC6T588291474D23684.1HPBC6T588gi|21280231|dbj|AB073823.1|279375AB073823.1gi|11191955|dbj|AB036920.1|AB036920183088AB036920.1AB036920gi|15419825|gb|AF297619.1|AF297619231156AF297619.1AF297619gi|13991868|gb|AF363962.1|AF363962259635AF363962.1AF363962gi|20151226|gb|U87742.3|203103U87742.3gi|13365544|dbj|AB048702.1|AB048702219013AB048702.1AB048702gi|5019952|gb|AF143302.1|AF143302229555AF143302.1AF143302gi|12247025|gb|AF223963.1|AF223963161165AF223963.1AF223963

[0324]

2

TABLE II

HBV siRNA and Target Sequences

Sequence
Seq ID
Upper seq
Seq ID
Lower seq
Seq ID

GAUCCUGCUGCUAUGCCUC
1
CAUCCUGCUGCUAUGCCUC
1
GAGGCAUAGCAGCAGGAUG
647

AUCCUGCUGCUAUGCCUCA
2
AUCCUGCUGCUAUGCCUCA
2
UGAGGCAUAGCAGCAGGAU
648

GGCUGUAGGCAUAAAUUGG
3
GGCUGUAGGCAUAAAUUGG
3
CCAAUUUAUGCCUACAGCC
649

GCUGUAGGCAUAAAUUGGU
4
GCUGUAGGCAUAAAUUGGU
4
ACCAAUUUAUGCCUACAGC
650

GCUGCUAUGCCUCAUCUUC
5
GCUGCUAUGCCUCAUCUUC
5
GAAGAUGAGGCAUAGCAGC
651

UGCUAUGCCUCAUCUUCUU
6
UGCUAUGCCUCAUCUUCUU
6
AAGAAGAUGAGGCAUAGCA
652

UGCUGCUAUGCCUCAUCUU
7
UGCUGCUAUGCCUCAUCUU
7
AAGAUGAGGCAUAGCAGCA
653

CUGCUAUGCCUCAUCUUCU
8
CUGCUAUGCCUCAUCUUCU
8
AGAAGAUGAGGCAUAGCAG
654

CUGCUGCUAUGCCUCAUCU
9
CUGCUGCUAUGCCUGAUCU
9
AGAUGAGGCAUAGCAGCAG
655

UCCUGCUGCUAUGCCUCAU
10
UCCUGCUGCUAUGCCUCAU
10
AUGAGGCAUAGCAGCAGGA
656

CCUGCUGCUAUGCCUCAUC
11
CCUGCUGCUAUGCCUCAUC
11
GAUGAGGCAUAGCAGCAGG
657

GAAGAAGAACUCCCUCGCC
12
GAAGAAGAACUCCCUCGCC
12
GGCGAGGGAGUUCUUCUUC
658

GAGGCUGUAGGCAUAAAUU
13
GAGGCUGUAGGCAUAAAUU
13
AAUUUAUGCCUACAGCCUC
659

AGGCUGUAGGCAUAAAUUG
14
AGGCUGUAGGCAUAAAUUG
14
CAAUUUAUGCCUACAGCCU
660

GGAGGCUGUAGGCAUAAAU
15
GGAGGCUGUAGGCAUAAAU
15
AUUUAUGCCUACAGCCUCC
661

AAGCCUCCAAGCUGUGCCU
16
AAGCCUCCAAGCUGUGCCU
16
AGGCACAGCUUGGAGGCUU
662

CCUCCAAGCUGUGCCUUGG
17
CCUCCAAGCUGUGCCUUGG
17
CCAAGGCACAGCUUGGAGG
663

GAAGAACUCCCUCGCCUCG
18
GAAGAACUCCCUCGCCUCG
18
CGAGGCGAGGGAGUUCUUC
664

UCAAGCCUCCAAGCUGUGC
19
UCAAGCCUCCAAGCUGUGC
19
GCACAGCUUGGAGGCUUGA
665

UCGUGGUGGACUUCUCUCA
20
UCGUGGUGGACUUCUCUCA
20
UGAGAGAAGUCCACCACGA
666

CUCGUGGUGGACUUCUCUC
21
CUCGUGGUGGACUUCUCUC
21
GAGAGAAGUCCACCACGAG
667

AAGAACUCCCUCGCCUCGC
22
AAGAACUCCCUCGCCUCGC
22
GCGAGGCGAGGGAGUUCUU
668

UUCAAGCCUCCAAGCUGUG
23
UUCAAGCCUCCAAGCUGUG
23
CACAGCUUGGAGGCUUGAA
669

GCCUCCAAGCUGUGCCUUG
24
GCCUCCAAGCUGUGCCUUG
24
CAAGGCACAGCUUGGAGGC
670

AAGAAGAACUCCCUCGCCU
25
AAGAAGAACUCCCUCGCCU
25
AGGCGAGGGAGUUCUUCUU
671

CAAGCCUCCAAGCUGUGCC
26
CAAGCCUCCAAGCUGUGCC
26
GGCACAGCUUGGAGGCUUG
672

AGAAGAACUCCCUCGCCUC
27
AGAAGAACUCCCUCGCCUC
27
GAGGCGAGGGAGUUCUUCU
673

AGCCUCCAAGCUGUGCCUU
28
AGCCUCCAAGCUGUGCGUU
28
AAGGCACAGCUUGGAGGCU
674

ACUCGUGGUGGACUUCUCU
29
ACUCGUGGUGGACUUCUCU
29
AGAGAAGUCCACCACGAGU
675

UGUGCACUUCGCUUCACCU
30
UGUGCACUUCGCUUCACCU
30
AGGUGAAGCGAAGUGCACA
676

CCGUGUGCACUUCGCUUCA
31
CCGUGUGCACUUCGCUUCA
31
UGAAGCGAAGUGCACACGG
677

CGUGUGCACUUCGCUUCAC
32
CGUGUGCACUUCGCUUCAC
32
GUGAAGCGAAGUGCACACG
678

UCGCUUCACCUCUGCACGU
33
UCGCUUCACCUCUGCACGU
33
ACGUGCAGAGGUGAAGCGA
679

GUGUGCACUUCGCUUCACC
34
GUGUGCACUUCGCUUCACC
34
GGUGAAGCGAAGUGCACAC
680

UUCGCUUCACCUCUGCACG
35
UUCGCUUCACCUCUGCACG
35
CGUGCAGAGGUGAAGCGAA
681

ACUUCGCUUCACCUCUGCA
36
ACUUCGCUUCACCUCUGCA
36
UGCAGAGGUGAAGCGAAGU
682

CUUCGCUUCACCUCUGCAC
37
CUUCGCUUCACCUCUGCAC
37
GUGCAGAGGUGAAGCGAAG
683

CACUUCGCUUCACCUCUGC
38
CACUUCGCUUCACCUCUGC
38
GCAGAGGUGAAGCGAAGUG
684

CCUAUGGGAGUGGGCCUCA
39
CCUAUGGGAGUGGGCCUCA
39
UGAGGCCCACUCCCAUAGG
685

CGCACCUCUCUUUACGCGG
40
CGCACCUCUCUUUACGCGG
40
CCGCGUAAAGAGAGGUGCG
686

GUGCACUUCGCUUCACCUC
41
GUGCACUUCGCUUCACCUC
41
GAGGUGAAGCGAAGUGCAC
687

GACUCGUGGUGGACUUCUC
42
GACUCGUGGUGGACUUCUC
42
GAGAAGUCCACCACGAGUC
688

UCUAGACUCGUGGUGGACU
43
UCUAGACUCGUGGUGGACU
43
AGUCCACCACGAGUCUAGA
689

CUAGACUCGUGGUGGACUU
44
CUAGACUCGUGGUGGACUU
44
AAGUCCACCACGAGUCUAG
690

GCACUUCGCUUCACCUCUG
45
GCACUUCGCUUCACGUCUG
45
CAGAGGUGAAGCGAAGUGC
691

AGACUCGUGGUGGACUUCU
46
AGACUCGUGGUGGACUUCU
46
AGAAGUCCACCACGAGUCU
692

UGCACUUCGCUUCACCUCU
47
UGCACUUCGCUUCACCUCU
47
AGAGGUGAAGCGAAGUGCA
693

UAGACUCGUGGUGGACUUC
48
UAGACUCGUGGUGGACUUC
48
GAAGUCCACCACGAGUCUA
694

AGUCUAGACUGGUGGUGGA
49
AGUCUAGACUCGUGGUGGA
49
UCCACCACGAGUCUAGACU
695

GAGUCUAGACUCGUGGUGG
50
GAGUCUAGACUCGUGGUGG
50
CCACCACGAGUCUAGACUC
696

GUCUAGACUCGUGGUGGAC
51
GUCUAGACUCGUGGUGGAC
51
GUCCACCACGAGUCUAGAC
697

GUUCAAGCCUCCAAGCUGU
52
GUUCAAGCCUCCAAGCUGU
52
ACAGCUUGGAGGCUUGAAC
698

AAGCUGUGCCUUGGGUGGC
53
AAGCUGUGCCUUGGGUGGC
53
GCCACCCAAGGCACAGCUU
699

CUGUGCCUUGGGUGGCUUU
54
CUGUGCCUUGGGUGGCUUU
54
AAAGCCACCCAAGGCACAG
700

UGUUCAAGCCUCCAAGCUG
55
UGUUCAAGCCUCCAAGCUG
55
CAGCUUGGAGGCUUGAACA
701

CAAGCUGUGCCUUGGGUGG
56
CAAGCUGUGCCUUGGGUGG
56
CCACCCAAGGCACAGCUUG
702

CUCCAAGCUGUGCCUUGGG
57
CUCCAAGCUGUGCCUUGGG
57
CCCAAGGCACAGCUUGGAG
703

CCAAGCUGUGCCUUGGGUG
58
CCAAGCUGUGCCUUGGGUG
58
CACCCAAGGCACAGCUUGG
704

UCCAAGCUGUGCCUUGGGU
59
UCCAAGCUGUGCCUUGGGU
59
ACCCAAGGCACAGCUUGGA
705

CUAUGGGAGUGGGCCUCAG
60
CUAUGGGAGUGGGCCUCAG
60
CUGAGGCCCACUCCCAUAG
706

AGCUGUGCCUUGGGUGGCU
61
AGCUGUGCCUUGGGUGGCU
61
AGCCACCCAAGGCACAGCU
707

ACUGUUCAAGCCUCCAAGC
62
ACUGUUCAAGCCUCCAAGC
62
GCUUGGAGGCUUGAACAGU
708

AGAGUCUAGACUCGUGGUG
63
AGAGUCUAGACUCGUGGUG
63
CACCACGAGUCUAGACUCU
709

GCUGUGCCUUGGGUGGCUU
64
GCUGUGCCUUGGGUGGCUU
64
AAGCCACCCAAGGCACAGC
710

CUGUUCAAGCCUCCAAGCU
65
CUGUUCAAGCCUCCAAGCU
65
AGCUUGGAGGCUUGAACAG
711

GGUAUGUUGCCCGUUUGUC
66
GGUAUGUUGCCCGUUUGUC
66
GACAAACGGGCAACAUACC
712

UGGAUGUGUCUGCGGCGUU
67
UGGAUGUGUCUGCGGCGUU
67
AACGCCGCAGACACAUCCA
713

CUGCUGGUGGCUCCAGUUC
68
CUGCUGGUGGCUCCAGUUC
68
GAACUGGAGCCACCAGCAG
714

CCUGCUGGUGGCUCCAGUU
69
CCUGCUGGUGGCUCCAGUU
69
AACUGGAGCCACCAGCAGG
715

GUAUGUUGCCCGUUUGUCC
70
GUAUGUUGCCCGUUUGUCC
70
GGACAAACGGGCAACAUAC
716

UGGCUCAGUUUACUAGUGC
71
UGGCUCAGUUUACUAGUGC
71
GCACUAGUAAACUGAGCCA
717

CCGAUCCAUACUGCGGAAC
72
CCGAUCCAUACUGCGGAAC
72
GUUCCGCAGUAUGGAUCGG
718

CGAUCCAUACUGCGGAACU
73
CGAUCCAUACUGCGGAACU
73
AGUUCCGCAGUAUGGAUCG
719

AGGUAUGUUGCCCGUUUGU
74
AGGUAUGUUGCCCGUUUGU
74
ACAAACGGGCAACAUACCU
720

CAGAGUCUAGACUCGUGGU
75
CAGAGUCUAGACUCGUGGU
75
ACCACGAGUCUAGACUCUG
721

UGGACUUCUCUCAAUUUUC
76
UGGACUUCUCUCAAUUUUC
76
GAAAAUUGAGAGAAGUCCA
722

GGACUUCUCUCAAUUUUCU
77
GGACUUCUCUCAAUUUUCU
77
AGAAAAUUGAGAGAAGUCC
723

UGGUGGACUUCUCUCAAUU
78
UGGUGGACUUCUCUCAAUU
78
AAUUGAGAGAAGUCCACCA
724

AUGUUGCCCGUUUGUCCUC
79
AUGUUGCCCGUUUGUCCUC
79
GAGGACAAACGGGCAACAU
725

GACUUCUCUCAAUUUUCUA
80
GACUUCUCUCAAUUUUCUA
80
UAGAAAAUUGAGAGAAGUC
726

CUCCUCUGCCGAUCCAUAC
81
CUCCUCUGCCGAUCCAUAC
81
GUAUGGAUCGGCAGAGGAG
727

GUGGUGGACUUCUCUCAAU
82
GUGGUGGACUUCUCUCAAU
82
AUUGAGAGAAGUCCACCAC
728

UAUGUUGCCCGUUUGUCCU
83
UAUGUUGCCCGUUUGUCCU
83
AGGACAAACGGGCAACAUA
729

GUGGACUUCUCUCAAUUUU
84
GUGGACUUCUCUCAAUUUU
84
AAAAUUGAGAGAAGUCCAC
730

AACUUUUUCACCUCUGCCU
85
AACUUUUUCACCUCUGCCU
85
AGGCAGAGGUGAAAAAGUU
731

GAGUGUGGAUUCGCACUCC
86
GAGUGUGGAUUCGCACUCC
86
GGAGUGCGAAUCCACACUC
732

AUGUGUCUGCGGCGUUUUA
87
AUGUGUCUGCGGCGUUUUA
87
UAAAACGCCGCAGACACAU
733

GAUGUGUCUGCGGCGUUUU
88
GAUGUGUCUGCGGCGUUUU
88
AAAACGCCGCAGACACAUC
734

GGUGGACUUCUCUCAAUUU
89
GGUGGACUUCUCUCAAUUU
89
AAAUUGAGAGAAGUCCACC
735

GUGUCUGCGGCGUUUUAUC
90
GUGUCUGCGGCGUUUUAUC
90
GAUAAAACGCCGCAGACAC
736

UAGAAGAAGAACUCCCUCG
91
UAGAAGAAGAACUCCCUCG
91
CGAGGGAGUUCUUCUUCUA
737

AGAAGAAGAACUCCCUCGC
92
AGAAGAAGAACUCCCUCGC
92
GCGAGGGAGUUCUUCUUCU
738

UGUCUGCGGCGUUUUAUCA
93
UGUCUGCGGCGUUUUAUCA
93
UGAUAAAACGCCGCAGACA
739

ACUUUUUCACCUCUGCCUA
94
ACUUUUUCACCUCUGCCUA
94
UAGGCAGAGGUGAAAAAGU
740

CCUGCUCGUGUUACAGGCG
95
CCUGCUCGUGUUACAGGCG
95
CGCCUGUAACACGAGCAGG
741

GUCUGCGGCGUUUUAUCAU
96
GUCUGCGGCGUUUUAUCAU
96
AUGAUAAAACGCCGCAGAC
742

ACUUCUCUCAAUUUUCUAG
97
ACUUCUCUCAAUUUUCUAG
97
CUAGAAAAUUGAGAGAAGU
743

CCUAGAAGAAGAACUCCCU
98
CCUAGAAGAAGAACUCCCU
98
AGGGAGUUCUUCUUCUAGG
744

GGAGUGUGGAUUCGCACUC
99
GGAGUGUGGAUUCGCACUC
99
GAGUGCGAAUCCACACUCC
745

UGUGUCUGCGGCGUUUUAU
100
UGUGUCUGCGGCGUUUUAU
100
AUAAAACGCCGCAGACACA
746

CUAGAAGAAGAACUCCCUC
101
CUAGAAGAAGAACUCCCUC
101
GAGGGAGUUCUUCUUCUAG
747

CCCUAGAAGAAGAACUCCC
102
CCCUAGAAGAAGAACUCCC
102
GGGAGUUCUUCUUCUAGGG
748

CUGCUCGUGUUACAGGCGG
103
CUGCUCGUGUUACAGGCGG
103
CCGCCUGUAACACGAGCAG
749

GGAUGUGUCUGCGGCGUUU
104
GGAUGUGUCUGCGGCGUUU
104
AAACGCCGCAGACACAUCC
750

CCCCUAGAAGAAGAACUCC
105
CCCCUAGAAGAAGAACUCC
105
GGAGUUCUUCUUCUAGGGG
751

CGUGGUGGACUUCUCUCAA
106
CGUGGUGGACUUCUCUCAA
106
UUGAGAGAAGUCCACCACG
752

GGACCCCUGCUCGUGUUAC
107
GGACCCCUGCUCGUGUUAC
107
GUAACACGAGCAGGGGUCC
753

UGUUGCCCGUUUGUCCUCU
108
UGUUGCCCGUUUGUCCUCU
108
AGAGGACAAACGGGCAACA
754

CCCUGCUCGUGUUACAGGC
109
CCCUGCUCGUGUUACAGGC
109
GCCUGUAACACGAGCAGGG
755

GACCCCUGCUCGUGUUACA
110
GACCCCUGCUCGUGUUACA
110
UGUAACACGAGCAGGGGUC
756

CCCCUGCUCGUGUUACAGG
111
CCCCUGCUCGUGUUACAGG
111
CCUGUAACACGAGCAGGGG
757

UUCUCUCAAUUUUCUAGGG
112
UUCUCUCAAUUUUCUAGGG
112
CCCUAGAAAAUUGAGAGAA
758

ACCCCUGCUCGUGUUACAG
113
ACCCCUGCUCGUGUUACAG
113
CUGUAACACGAGCAGGGGU
759

CUUCUCUCAAUUUUCUAGG
114
CUUCUCUCAAUUUUCUAGG
114
CCUAGAAAAUUGAGAGAAG
760

AAGGUAUGUUGCCCGUUUG
115
AAGGUAUGUUGCCCGUUUG
115
CAAACGGGCAACAUACCUU
761

GCCGAUCCAUACUGCGGAA
116
GCCGAUCCAUACUGCGGAA
116
UUCCGCAGUAUGGAUCGGC
762

GAUCCAUACUGCGGAACUC
117
GAUCCAUACUGCGGAACUC
117
GAGUUCCGCAGUAUGGAUC
763

UCCAUACUGCGGAACUCCU
118
UCCAUACUGCGGAACUCCU
118
AGGAGUUCCGCAGUAUGGA
764

UCUCUCAAUUUUCUAGGGG
119
UCUCUCAAUUUUCUAGGGG
119
CCCCUAGAAAAUUGAGAGA
765

AUCCAUACUGCGGAACUCC
120
AUCCAUACUGCGGAACUCC
120
GGAGUUCCGCAGUAUGGAU
766

UGCCGAUCCAUACUGCGGA
121
UGCCGAUCCAUACUGCGGA
121
UCCGCAGUAUGGAUCGGCA
767

AACUCCCUCGCCUCGCAGA
122
AACUCCCUCGCCUCGCAGA
122
UCUGCGAGGCGAGGGAGUU
768

CGUCGCAGAAGAUCUCAAU
123
CGUCGCAGAAGAUCUCAAU
123
AUUGAGAUCUUCUGCGACG
769

CUGCCGAUCCAUACUGCGG
124
CUGCCGAUCCAUACUGCGG
124
CCGCAGUAUGGAUCGGCAG
770

GAACUCCCUCGCCUCGCAG
125
GAACUCCCUCGCCUCGCAG
125
CUGCGAGGCGAGGGAGUUC
771

GUCGCAGAAGAUCUCAAUC
126
GUCGCAGAAGAUCUCAAUC
126
GAUUGAGAUCUUCUGCGAC
772

AGGACCCCUGCUCGUGUUA
127
AGGACCCCUGCUCGUGUUA
127
UAACACGAGCAGGGGUCCU
773

UCGCAGAAGAUCUCAAUCU
128
UCGCAGAAGAUCUCAAUCU
128
AGAUUGAGAUCUUCUGCGA
774

AGAACUCCCUCGCCUCGCA
129
AGAACUCCCUCGCCUCGCA
129
UGCGAGGCGAGGGAGUUCU
775

UCUGCCGAUCCAUACUGCG
130
UCUGCCGAUCCAUACUGCG
130
CGCAGUAUGGAUCGGCAGA
776

CGCGUCGCAGAAGAUCUCA
131
CGCGUCGCAGAAGAUCUCA
131
UGAGAUCUUCUGCGACGCG
777

CCUCUGCCGAUCCAUACUG
132
CCUCUGCCGAUCCAUACUG
132
CAGUAUGGAUCGGCAGAGG
778

GCGUCGCAGAAGAUCUCAA
133
GCGUCGCAGAAGAUCUCAA
133
UUGAGAUCUUCUGCGACGC
779

CUCUGCCGAUCCAUACUGC
134
CUCUGCCGAUCCAUACUGC
134
GCAGUAUGGAUCGGCAGAG
780

CGCAGAAGAUCUCAAUCUC
135
CGCAGAAGAUCUCAAUCUC
135
GAGAUUGAGAUCUUCUGCG
781

UCCUCUGCCGAUCCAUACU
136
UCCUCUGCCGAUCCAUACU
136
AGUAUGGAUCGGCAGAGGA
782

UCCCCUAGAAGAAGAACUC
137
UCCCCUAGAAGAAGAACUC
137
GAGUUCUUCUUCUAGGGGA
783

CCGCGUCGCAGAAGAUCUC
138
CCGCGUCGCAGAAGAUCUC
138
GAGAUCUUCUGCGACGCGG
784

CCAAGUGUUUGCUGACGCA
139
CCAAGUGUUUGCUGACGCA
139
UGCGUCAGCAAACACUUGG
785

UGCCAAGUGUUUGCUGACG
140
UGCCAAGUGUUUGCUGACG
140
CGUCAGCAAACACUUGGCA
786

AGGAGGCUGUAGGCAUAAA
141
AGGAGGCUGUAGGCAUAAA
141
UUUAUGCCUACAGCCUCCU
787

UAGGAGGCUGUAGGCAUAA
142
UAGGAGGCUGUAGGCAUAA
142
UUAUGCCUACAGCCUCCUA
788

GCCAAGUGUUUGCUGACGC
143
GCCAAGUGUUUGCUGACGC
143
GCGUCAGCAAACACUUGGC
789

CUCCCUCGCCUCGCAGACG
144
CUCCCUCGCCUCGCAGACG
144
CGUCUGCGAGGCGAGGGAG
790

UUGCUGACGCAACCCCCAC
145
UUGCUGACGCAACCCCCAC
145
GUGGGGGUUGCGUCAGCAA
791

UCCCGUCGGCGCUGAAUCC
146
UCCCGUCGGCGCUGAAUCC
146
GGAUUCAGCGCCGACGGGA
792

GCAACUUUUUCACCUCUGC
147
GCAACUUUUUCACCUCUGC
147
GCAGAGGUGAAAAAGUUGC
793

GGUCCCCUAGAAGAAGAAC
148
GGUCCCCUAGAAGAAGAAC
148
GUUCUUCUUCUAGGGGACC
794

GCAGGUCCCCUAGAAGAAG
149
GCAGGUCCCCUAGAAGAAG
149
CUUCUUCUAGGGGACCUGC
795

ACUCCCUCGCCUCGCAGAC
150
ACUCCCUCGCCUCGGAGAC
150
GUCUGCGAGGCGAGGGAGU
796

CGUCCCGUCGGCGCUGAAU
151
CGUCCCGUCGGCGCUGAAU
151
AUUCAGCGCCGACGGGACG
797

CAGGUCCCCUAGAAGAAGA
152
CAGGUCCCCUAGAAGAAGA
152
UCUUCUUCUAGGGGACCUG
798

AGGUCCCCUAGAAGAAGAA
153
AGGUCCCCUAGAAGAAGAA
153
UUCUUCUUCUAGGGGACCU
799

UACGUCCCGUCGGCGCUGA
154
UACGUCCCGUCGGCGCUGA
154
UCAGCGCCGACGGGACGUA
800

CAAGUGUUUGCUGACGCAA
155
CAAGUGUUUGCUGACGCAA
155
UUGCGUCAGCAAACACUUG
801

ACGUCCCGUCGGCGCUGAA
156
ACGUCCCGUCGGCGCUGAA
156
UUCAGCGCCGACGGGACGU
802

CAAGGUAUGUUGCCCGUUU
157
CAAGGUAUGUUGCCCGUUU
157
AAACGGGCAACAUACCUUG
803

UUUGCUGACGCAACCCCCA
158
UUUGCUGACGCAACCCCCA
158
UGGGGGUUGCGUCAGCAAA
804

CAACUUUUUCACCUCUGCC
159
CAACUUUUUCACCUCUGCC
159
GGCAGAGGUGAAAAAGUUG
805

UCCUAGGACCCCUGCUCGU
160
UCCUAGGACCCCUGCUCGU
160
ACGAGCAGGGGUCCUAGGA
806

GUCCCGUCGGCGCUGAAUC
161
GUCCCGUCGGCGCUGAAUC
161
GAUUCAGCGCCGACGGGAC
807

AAGUGUUUGCUGACGCAAC
162
AAGUGUUUGCUGACGCAAC
162
GUUGCGUCAGCAAACACUU
808

CCUAGGACCCCUGCUCGUG
163
CCUAGGACCCCUGCUCGUG
163
CACGAGCAGGGGUCCUAGG
809

GUGUUUGCUGACGCAACCC
164
GUGUUUGCUGACGCAACCC
164
GGGUUGCGUCAGCAAACAC
810

AGUGUUUGCUGACGCAACC
165
AGUGUUUGCUGACGCAACC
165
GGUUGCGUCAGCAAACACU
811

CUAGGACCCCUGCUCGUGU
166
CUAGGACCCCUGCUCGUGU
166
ACACGAGCAGGGGUCCUAG
812

GUCCCCUAGAAGAAGAACU
167
GUCCCCUAGAAGAAGAACU
167
AGUUCUUCUUCUAGGGGAC
813

GUUUGCUGACGCAACCCCC
168
GUUUGCUGACGCAACCCCC
168
GGGGGUUGCGUCAGCAAAC
814

UGUUUGCUGACGCAACCCC
169
UGUUUGCUGACGCAACCCC
169
GGGGUUGCGUCAGCAAACA
815

UAGGACCCCUGCUCGUGUU
170
UAGGACCCCUGCUCGUGUU
170
AACACGAGCAGGGGUCCUA
816

UGCAACUUUUUCACCUCUG
171
UGCAACUUUUUCACCUCUG
171
CAGAGGUGAAAAAGUUGCA
817

CUGACGCAACCCCCACUGG
172
CUGACGCAACCCCCACUGG
172
CCAGUGGGGGUUGCGUCAG
818

AUGCAACUUUUUCACCUCU
173
AUGCAACUUUUUCACCUCU
173
AGAGGUGAAAAAGUUGCAU
819

UGCUGACGCAACCCCCACU
174
UGCUGACGCAACCCCCACU
174
AGUGGGGGUUGCGUCAGCA
820

GCUGACGCAACCCCCACUG
175
GCUGACGCAACCCCCACUG
175
CAGUGGGGGUUGCGUCAGC
821

GGGCGCACCUCUCUUUACG
176
GGGCGCACCUCUCUUUACG
176
CGUAAAGAGAGGUGCGCCC
822

GGCGCACCUCUCUUUACGC
177
GGCGCACCUCUCUUUACGC
177
GCGUAAAGAGAGGUGCGCC
823

GGCCAAAAUUCGCAGUCCC
178
GGCCAAAAUUCGCAGUCCC
178
GGGACUGCGAAUUUUGGCC
824

UGGCCAAAAUUCGCAGUCC
179
UGGCCAAAAUUCGCAGUCC
179
GGACUGCGAAUUUUGGCCA
825

UUACAGGCGGGGUUUUUCU
180
UUACAGGCGGGGUUUUUCU
180
AGAAAAACCCCGCCUGUAA
826

GGGGCGCACCUCUCUUUAC
181
GGGGCGCACCUCUCUUUAC
181
GUAAAGAGAGGUGCGCCCC
827

CGGGGCGCACCUCUCUUUA
182
CGGGGCGCACCUCUCUUUA
182
UAAAGAGAGGUGCGCCCCG
828

GUUACAGGCGGGGUUUUUC
183
GUUACAGGCGGGGUUUUUC
183
GAAAPACCCCGCCUGUAAC
829

CACCUCUGCCUAAUCAUCU
184
CACCUCUGCCUAAUCAUCU
184
AGAUGAUUAGGCAGAGGUG
830

UUUCACCUCUGCCUAAUCA
185
UUUCACCUCUGCCUAAUCA
185
UGAUUAGGCAGAGGUGAAA
831

UUCACCUCUGCCUAAUCAU
186
UUCACCUCUGCCUAAUCAU
186
AUGAUUAGGCAGAGGUGAA
832

GCGCACCUCUCUUUACGCG
187
GCGCACCUCUCUUUACGCG
187
CGCGUAAAGAGAGGUGCGC
833

CGUAGGGCUUUCCCCCACU
188
CGUAGGGCUUUCCGCCACU
188
AGUGGGGGAAAGCCCUACG
834

ACGGGGCGCACCUCUCUUU
189
ACGGGGCGCACCUCUCUUU
189
AAAGAGAGGUGCGCCCCGU
835

AUAAGAGGACUCUUGGACU
190
AUAAGAGGACUCUUGGACU
190
AGUCCAAGAGUCCUCUUAU
836

UCACCUCUGCCUAAUCAUC
191
UCACCUCUGCCUAAUCAUC
191
GAUGAUUAGGCAGAGGUGA
837

GUAGGGCUUUCCCCCACUG
192
GUAGGGCUUUCCCCCACUG
192
CAGUGGGGGAAAGCCCUAC
838

UAGGGCUUUCCCCCACUGU
193
UAGGGCUUUCCCCCACUGU
193
ACAGUGGGGGAAAGCCCUA
839

UUUUCACCUCUGCCUAAUC
194
UUUUCACCUCUGCCUAAUC
194
GAUUAGGCAGAGGUGAAAA
840

UUUUUCACCUCUGCCUAAU
195
UUUUUCACCUCUGCCUAAU
195
AUUAGGCAGAGGUGAAAAA
841

CCAUUUGUUCAGUGGUUCG
196
CCAUUUGUUCAGUGGUUCG
196
CGAACCACUGAACAAAUGG
842

GUGUUACAGGCGGGGUUUU
197
GUGUUACAGGCGGGGUUUU
197
AAAACCCCGCCUGUAACAC
843

CGUGUUACAGGCGGGGUUU
198
CGUGUUACAGGCGGGGUUU
198
AAACCCCGCCUGUAACACG
844

CCACGGGGCGCACCUCUCU
199
CCACGGGGCGCACCUCUCU
199
AGAGAGGUGCGCCCCGUGG
845

AGGCAGGUCCCCUAGAAGA
200
AGGCAGGUCCCCUAGAAGA
200
UCUUCUAGGGGACCUGCCU
846

CUCUCAAUUUUCUAGGGGG
201
CUCUCAAUUUUCUAGGGGG
201
CCCCCUAGAAAAUUGAGAG
847

GAGGCAGGUCCCCUAGAAG
202
GAGGCAGGUCCCCUAGAAG
202
CUUCUAGGGGACCUGCCUC
848

UGUAUUCCCAUCCCAUCAU
203
UGUAUUCCCAUCCCAUCAU
203
AUGAUGGGAUGGGAAUACA
849

GUAUUCCCAUCCCAUCAUC
204
GUAUUCCCAUCCCAUCAUC
204
GAUGAUGGGAUGGGAAUAC
850

CUCGUGUUACAGGCGGGGU
205
CUCGUGUUACAGGCGGGGU
205
ACCCCGCCUGUAACACGAG
851

UCGUGUUACAGGCGGGGUU
206
UCGUGUUACAGGCGGGGUU
206
AACCCCGCCUGUAACACGA
852

GUUGCCCGUUUGUCCUCUA
207
GUUGCCCGUUUGUCCUCUA
207
UAGAGGACAAACGGGCAAC
853

GCCAUUUGUUCAGUGGUUC
208
GCCAUUUGUUCAGUGGUUC
208
GAACCACUGAACAAAUGGC
854

CACGGGGCGCACCUCUCUU
209
CACGGGGCGCACCUCUCUU
209
AAGAGAGGUGCGCCCCGUG
855

UGUUACAGGCGGGGUUUUU
210
UGUUACAGGCGGGGUUUUU
210
AAAAACCCCGCCUGUAACA
856

GGCAGGUCCCCUAGAAGAA
211
GGCAGGUCCCCUAGAAGAA
211
UUCUUCUAGGGGACCUGCC
857

CUAUGCCUCAUCUUCUUGU
212
CUAUGCCUCAUCUUCUUGU
212
ACAAGAAGAUGAGGCAUAG
858

CUCAAUCGCCGCGUCGCAG
213
CUCAAUCGCCGCGUCGCAG
213
CUGCGACGCGGCGAUUGAG
859

UCAAUCGCCGCGUCGCAGA
214
UCAAUCGCCGCGUCGCAGA
214
UCUGCGACGCGGCGAUUGA
860

CACCAUAUUCUUGGGAACA
215
CACCAUAUUCUUGGGAACA
215
UGUUCCCAAGAAUAUGGUG
861

UCACCAUAUUCUUGGGAAC
216
UCACCAUAUUCUUGGGAAC
216
GUUCCCAAGAAUAUGGUGA
862

ACCAUAUUCUUGGGAACAA
217
ACCAUAUUCUUGGGAACAA
217
UUGUUCCCAAGAAUAUGGU
863

GCUAUGCCUCAUCUUCUUG
218
GCUAUGCCUCAUCUUCUUG
218
CAAGAAGAUGAGGCAUAGC
864

GCCGCGUCGCAGAAGAUCU
219
GCCGCGUCGCAGAAGAUCU
219
AGAUCUUCUGCGACGCGGC
865

AAUCGCCGCGUCGCAGAAG
220
AAUCGCCGCGUCGCAGAAG
220
CUUCUGCGACGCGGCGAUU
866

CGCCGCGUCGCAGAAGAUC
221
CGCCGCGUCGCAGAAGAUC
221
GAUCUUCUGCGACGCGGCG
867

GGCUCAGUUUACUAGUGCC
222
GGCUCAGUUUACUAGUGCC
222
GGCACUAGUAAACUGAGCC
868

AUCGCCGCGUCGCAGAAGA
223
AUCGCCGCGUCGCAGAAGA
223
UCUUCUGCGACGCGGCGAU
869

AGUGUGGAUUCGCACUCCU
224
AGUGUGGAUUCGCACUCCU
224
AGGAGUGCGAACCACACU
870

UCGCCGCGUCGCAGAAGAU
225
UCGCCGCGUCGCAGAAGAU
225
AUCUUCUGCGACGCGGCGA
871

CUCAUCUUCUUGUUGGUUC
226
CUCAUCUUCUUGUUGGUUC
226
GAACCAACAAGAAGAUGAG
872

CAUAUUCUUGGGAACAAGA
227
CAUAUUCUUGGGAACAAGA
227
UCUUGUUCCCAAGAAUAUG
873

AUGCCUCAUCUUCUUGUUG
228
AUGCCUCAUCUUCUUGUUG
228
CAACAAGAAGAUGAGGCAU
874

CUCCCCGUCUGUGCCUUCU
229
CUCCCCGUCUGUGCCUUCU
229
AGAAGGCACAGACGGGGAG
875

GCCUCAUCUUCUUGUUGGU
230
GCCUCAUCUUCUUGUUGGU
230
ACCAACAAGAAGAUGAGGC
876

UAUGCCUCAUCUUCUUGUU
231
UAUGCGUCAUCUUCUUGUU
231
AACAAGAAGAUGAGGCAUA
877

UCAUCUUCUUGUUGGUUCU
232
UCAUCUUCUUGUUGGUUCU
232
AGAACCAACAAGAAGAUGA
878

CCUCAUCUUCUUGUUGGUU
233
CCUCAUCUUCUUGUUGGUU
233
AACCAACAAGAAGAUGAGG
879

UCCCCGUCUGUGCCUUCUC
234
UCCCCGUCUGUGCCUUCUC
234
GAGAAGGCACAGACGGGGA
880

UGCCUCAUCUUCUUGUUGG
235
UGCCUCAUCUUCUUGUUGG
235
CCAACAAGAAGAUGAGGCA
881

UCUCAAUCGCCGCGUCGCA
236
UCUCAAUCGCCGCGUCGCA
236
UGCGACGCGGCGAUUGAGA
882

UCUUGUUGGUUCUUCUGGA
237
UCUUGUUGGUUCUUCUGGA
237
UCCAGAAGAACCAACAAGA
883

GGGUCACCAUAUUCUUGGG
238
GGGUCACCAUAUUCUUGGG
238
CCCAAGAAUAUGGUGACCC
884

UAUCGCUGGAUGUGUCUGC
239
UAUCGCUGGAUGUGUCUGC
239
GCAGACACAUCCAGCGAUA
885

CAUCUUCUUGUUGGUUCUU
240
CAUCUUCUUGUUGGUUCUU
240
AAGAACCAACAAGAAGAUG
886

GUCACCAUAUUCUUGGGAA
241
GUCACCAUAUUCUUGGGAA
241
UUCCCAAGAAUAUGGUGAC
887

CCAUAUUCUUGGGAACAAG
242
CCAUAUUCUUGGGAACAAG
242
CUUGUUCCCAAGAAUAUGG
888

GGUCACCAUAUUCUUGGGA
243
GGUCACCAUAUUCUUGGGA
243
UCCCAAGAAUAUGGUGACC
889

CUUCUUGUUGGUUCUUCUG
244
CUUCUUGUUGGUUCUUCUG
244
CAGAAGAACCAACAAGAAG
890

UUCUUGUUGGUUCUUCUGG
245
UUCUUGUUGGUUCUUCUGG
245
CCAGAAGAACCAACAAGAA
891

CAUGCAACUUUUUCACCUC
246
CAUGCAACUUUUUCACCUC
246
GAGGUGAAAAAGUUGCAUG
892

AUCGCUGGAUGUGUCUGCG
247
AUCGCUGGAUGUGUCUGCG
247
CGCAGACACAUCCAGCGAU
893

CAAUCGCCGGGUCGCAGAA
248
CAAUCGCCGCGUCGCAGAA
248
UUCUGCGACGCGGCGAUUG
894

UAGUGCCAUUUGUUCAGUG
249
UAGUGCCAUUUGUUCAGUG
249
CACUGAACAAAUGGCACUA
895

UCGCUGGAUGUGUCUGCGG
250
UCGCUGGAUGUGUCUGCGG
250
CGGGAGACACAUCCAGCGA
896

GUUUACUAGUGCCAUUUGU
251
GUUUACUAGUGCCAUUUGU
251
ACAAAUGGCACUAGUAAAC
897

CAGUUUACUAGUGCCAUUU
252
CAGUUUACUAGUGCCAUUU
252
AAAUGGCACUAGUAAACUG
898

CGCUGGAUGUGUCUGCGGC
253
CGCUGGAUGUGUCUGCGGC
253
GCCGCAGACACAUCCAGCG
899

UCUUCUUGUUGGUUCUUCU
254
UCUUCUUGUUGGUUCUUCU
254
AGAAGAACCAACAAGAAGA
900

CUAGUGCCAUUUGUUCAGU
255
CUAGUGCCAUUUGUUCAGU
255
ACUGAACAAAUGGCACUAG
901

AGUUUACUAGUGCCAUUUG
256
AGUUUACUAGUGCCAUUUG
256
CAAAUGGCACUAGUAAACU
902

GCUCGUGUUACAGGCGGGG
257
GCUCGUGUUACAGGCGGGG
257
CCCCGCCUGUAACACGAGC
903

CUUUUUCACCUCUGCCUAA
258
CUUUUUCACCUCUGCCUAA
258
UUAGGCAGAGGUGAAAAAG
904

ACUAGUGCCAUUUGUUCAG
259
ACUAGUGCCAUUUGUUCAG
259
CUGAACAAAUGGCACUAGU
905

AUCUUCUUGUUGGUUCUUC
260
AUCUUCUUGUUGGUUCUUC
260
GAAGAACCAACAAGAAGAU
906

UGCUCGUGUUACAGGCGGG
261
UGCUCGUGUUACAGGCGGG
261
CCCGCCUGUAACACGAGCA
907

UGAAUCCCGCGGACGACCC
262
UGAAUCCCGCGGACGACCC
262
GGGUCGUCCGCGGGAUUCA
908

AGUGCCAUUUGUUCAGUGG
263
AGUGCCAUUUGUUCAGUGG
263
CCACUGAACAAAUGGCACU
909

GCUCAGUUUACUAGUGCCA
264
GCUCAGUUUACUAGUGCCA
264
UGGGACUAGUAAACUGAGC
910

UUUACUAGUGCCAUUUGUU
265
UUUACUAGUGCCAUUUGUU
265
AACAAAUGGCACUAGUAAA
911

UACUAGUGCCAUUUGUUCA
266
UACUAGUGCCAUUUGUUCA
266
UGAACAAAUGGCACUAGUA
912

UCAGUUUACUAGUGCCAUU
267
UCAGUUUACUAGUGCCAUU
267
AAUGGCACUAGUPAACUGA
913

UUACUAGUGCCAUUUGUUC
268
UUACUAGUGCCAUUUGUUC
268
GAACAAAUGGCACUAGUAA
914

CUCAGUUUACUAGUGCCAU
269
CUCAGUUUACUAGUGCCAU
269
AUGGCACUAGUAAACUGAG
915

UCUCAAUUUUCUAGGGGGA
270
UCUCAAUUUUCUAGGGGGA
270
UCCCCCUAGAAAAUUGAGA
916

CUGAAUCCCGCGGACGACC
271
CUGAAUCCCGCGGACGACC
271
GGUCGUCCGCGGGAUUCAG
917

GCUGGAUGUGUCUGCGGCG
272
GCUGGAUGUGUCUGCGGCG
272
CGCCGCAGACACAUCCAGC
918

GCUGAAUCCCGCGGACGAC
273
GCUGAAUCCCGCGGACGAC
273
GUCGUCCGCGGGAUUCAGC
919

CUGGAUGUGUCUGCGGCGU
274
CUGGAUGUGUCUGCGGCGU
274
ACGCCGCAGACACAUCCAG
920

UGUGCUGCCAACUGGAUCC
275
UGUGCUGCCAACUGGAUCC
275
GGAUCCAGUUGGCAGCACA
921

GUGCCAUUUGUUCAGUGGU
276
GUGCCAUUUGUUCAGUGGU
276
ACCACUGAACAAAUGGCAC
922

UGCCAUUUGUUCAGUGGUU
277
UGCCAUUUGUUCAGUGGUU
277
AACCACUGAACAAAUGGCA
923

CCAUGCAACUUUUUCACCU
278
CCAUGCAACUUUUUCACCU
278
AGGUGAAAAAGUUGCAUGG
924

GUGCUGCCAACUGGAUCCU
279
GUGCUGCCAACUGGAUCCU
279
AGGAUCCAGUUGGCAGCAC
925

CAUGGAGACCACCGUGAAC
280
CAUGGAGACCACCGUGAAC
280
GUUCACGGUGGUCUCCAUG
926

UACAGGCGGGGUUUUUCUU
281
UACAGGCGGGGUUUUUCUU
281
AAGAAAAACCCCGCCUGUA
927

GCGCUGAAUCCCGCGGACG
282
GCGCUGAAUCCCGCGGACG
282
CGUCCGCGGGAUUCAGCGC
928

CUUGUUGGUUCUUCUGGAC
283
CUUGUUGGUUCUUCUGGAC
283
GUCCAGAAGAACCAACAAG
929

UUGUUGGUUCUUCUGGACU
284
UUGUUGGUUCUUCUGGACU
284
AGUCCAGAAGAACCAACAA
930

CGCUGAAUCCCGCGGACGA
285
CGCUGAAUCCCGCGGACGA
285
UCGUCCGCGGGAUUCAGCG
931

GCAUGGAGACCACCGUGAA
286
GCAUGGAGACCACCGUGAA
286
UUCACGGUGGUCUCCAUGC
932

ACAGGCGGGGUUUUUCUUG
287
ACAGGCGGGGUUUUUCUUG
287
CAAGAAAAACCCCGCCUGU
933

ACCACGGGGCGCACCUCUC
288
ACCACGGGGCGCACCUCUC
288
GAGAGGUGCGCCCCGUGGU
934

UGUUGGUUCUUCUGGACUA
289
UGUUGGUUCUUCUGGACUA
289
UAGUCCAGAAGAACCAACA
935

CGGCGCUGAAUCCCGCGGA
290
CGGCGCUGAAUCCCGCGGA
290
UCCGCGGGAUUCAGCGCCG
936

GGGGUUUUUCUUGUUGACA
291
GGGGUUUUUCUUGUUGACA
291
UGUCAACAAGAAAAACCCC
937

AUGGAGACCACCGUGAACG
292
AUGGAGACCACCGUGAACG
292
CGUUCACGGUGGUCUCCAU
938

UCGCCAACUUACAAGGCCU
293
UCGCCAACUUACAAGGCCU
293
AGGCCUUGUAAGUUGGCGA
939

CCGUCGGCGCUGAAUCCCG
294
CCGUCGGCGCUGAAUCCCG
294
CGGGAUUCAGCGCCGACGG
940

CAGGCGGGGUUUUUCUUGU
295
CAGGCGGGGUUUUUCUUGU
295
ACAAGAAAAACCCCGCCUG
941

GGCGCUGAAUCCCGCGGAC
296
GGCGCUGAAUCCCGCGGAC
296
GUCCGCGGGAUUCAGCGCC
942

CAGCACCAUGCAACUUUUU
297
CAGCACCAUGCAACUUUUU
297
AAAAAGUUGCAUGGUGCUG
943

CCAGCACCAUGCAACUUUU
298
CCAGCACCAUGCAACUUUU
298
AAAAGUUGCAUGGUGCUGG
944

CGUCGGCGCUGAAUCCCGC
299
CGUCGGCGCUGAAUCCCGC
299
GCGGGAUUCAGCGCCGACG
945

GGGUUUUUCUUGUUGACAA
300
GGGUUUUUCUUGUUGACAA
300
UUGUCAACAAGAAAAACCC
946

CCCGUCGGCGCUGAAUCCC
301
CCCGUCGGCGCUGAAUCCC
301
GGGAUUCAGCGCCGACGGG
947

ACCAGCACCAUGCAACUUU
302
ACCAGCACCAUGCAACUUU
302
AAAGUUGCAUGGUGCUGGU
948

GCGGGGUUUUUCUUGUUGA
303
GCGGGGUUUUUCUUGUUGA
303
UCAACAAGAAAAACCCCGC
949

AGACCACCAAAUGCCCCUA
304
AGACCACCAAAUGCCCCUA
304
UAGGGGCAUUUGGUGGUCU
950

CGCCAACUUACAAGGCCUU
305
CGCCAACUUACAAGGCCUU
305
AAGGCCUUGUAAGUUGGCG
951

GACCACCAAAUGCCCCUAU
306
GACCACCAAAUGCCCCUAU
306
AUAGGGGCAUUUGGUGGUC
952

GGCGGGGUUUUUCUUGUUG
307
GGCGGGGUUUUUCUUGUUG
307
CAACAAGAAAAACCCCGCC
953

AGGCGGGGUUUUUCUUGUU
308
AGGCGGGGUUUUUCUUGUU
308
AACAAGAAAAACCCCGCCU
954

UCGGCGCUGAAUCCCGCGG
309
UCGGCGCUGAAUCCCGCGG
309
CCGCGGGAUUCAGCGCCGA
955

ACCACCAAAUGCCCCUAUC
310
ACCACCAAAUGCCCCUAUC
310
GAUAGGGGCAUUUGGUGGU
956

CGGGGUUUUUCUUGUUGAC
311
CGGGGUUUUUCUUGUUGAC
311
GUCAACAAGAAAAACCCCG
957

ACCAUGCAACUUUUUCACC
312
ACCAUGCAACUUUUUCACC
312
GGUGAAAAAGUUGCAUGGU
958

CUGUAGGCAUAAAUUGGUC
313
CUGUAGGCAUAAAUUGGUC
313
GACCAAUUUAUGCCUACAG
959

GUGUGGAUUCGCACUCCUC
314
GUGUGGAUUCGCACUCCUC
314
GAGGAGUGCGAAUCCACAC
960

UGUAGGCAUAAAUUGGUCU
315
UGUAGGCAUAAAUUGGUCU
315
AGACCAAUUUAUGCCUACA
961

CACCAUGCAACUUUUUCAC
316
CACCAUGCAACUUUUUCAC
316
GUGAAAAAGUUGCAUGGUG
962

GUCGGCGCUGAAUCCCGCG
317
GUCGGCGCUGAAUCCCGCG
317
CGCGGGAUUCAGCGCCGAC
963

AUACUGCGGAACUCCUAGC
318
AUACUGCGGAACUCCUAGC
318
GCUAGGAGUUCCGCAGUAU
964

UACCAAUUUUCUUUUGUCU
319
UACCAAUUUUCUUUUGUCU
319
AGACAAAAGAAAAUUGGUA
965

AUGCCCCUAUCUUAUGAAC
320
AUGCCCCUAUCUUAUCAAC
320
GUUGAUAAGAUAGGGGCAU
966

CCAUACUGCGGAAGUCCUA
321
CCAUACUGCGGAACUCCUA
321
UAGGAGUUCCGCAGUAUGG
967

GUAGGCAUAAAUUGGUCUG
322
GUAGGCAUAAAUUGGUCUG
322
CAGACCAAUUUAUGCCUAC
968

CAUACUGCGGAACUCCUAG
323
CAUACUGCGGAACUCCUAG
323
CUAGGAGUUCCGCAGUAUG
969

AAAUGCCCCUAUCUUAUCA
324
AAAUGCCCCUAUCUUAUCA
324
UGAUAAGAUAGGGGCAUUU
970

AAUGCCCCUAUCUUAUCAA
325
AAUGCCCCUAUCUUAUCAA
325
UUGAUAAGAUAGGGGCAUU
971

CACCAGCACCAUGCAACUU
326
CACCAGCACCAUGCAACUU
326
AAGUUGCAUGGUGCUGGUG
972

UGAACCUUUACCCCGUUGC
327
UGAACCUUUACCCCGUUGC
327
GCAACGGGGUAAAGGUUCA
973

UGGAGACCACCGUGAACGC
328
UGGAGACCACCGUGAACGC
328
GCGUUCACGGUGGUCUCCA
974

GCCAACUUACAAGGCCUUU
329
GCCAACUUACAAGGCCUUU
329
AAAGGCCUUGUAAGUUGGC
975

UUACCAAUUUUCUUUUGUC
330
UUACCAAUUUUCUUUUGUC
330
GACAAAAGAAAAUUGGUAA
976

UCCUGCUGGUGGCUCCAGU
331
UCCUGCUGGUGGCUCCAGU
331
ACUGGAGCCACCAGGAGGA
977

UGUGCCUUCUCAUCUGCCG
332
UGUGCCUUCUCAUCUGCCG
332
CGGCAGAUGAGAAGGCACA
978

CCCCGUCUGUGCCUUCUCA
333
CCCCGUCUGUGCCUUCUCA
333
UGAGAAGGCACAGACGGGG
979

UUCGUAGGGCUUUCCCCCA
334
UUCGUAGGGCUUUCCCCCA
334
UGGGGGAAAGCCCUACGAA
980

CAGAAGAUCUCAAUCUCGG
335
CAGAAGAUCUCAAUCUCGG
335
CCGAGAUUGAGAUCUUCUG
981

UGCCCCUAUCUUAUCAACA
336
UGCCCCUAUCUUAUCAACA
336
UGUUGAUAAGAUAGGGGCA
982

GCAGAAGAUCUCAAUCUCG
337
GCAGAAGAUCUCAAUCUCG
337
CGAGAUUGAGAUCUUCUGC
983

AGCACCAUGCAACUUUUUC
338
AGCACCAUGCAACUUUUUC
338
GAAAAAGUUGCAUGGUGCU
984

CGUCUGUGCCUUCUCAUCU
339
CGUCUGUGCCUUCUCAUCU
339
AGAUGAGAAGGCACAGACG
985

CAGUGGUUCGUAGGGCUUU
340
CAGUGGUUCGUAGGGCUUU
340
AAAGCCCUACGAACCACUG
986

UGGUUCGUAGGGCUUUCCC
341
UGGUUCGUAGGGCUUUCCC
341
GGGAAAGCCCUACGAACCA
987

GCCCCUAUCUUAUCAACAC
342
GCCCCUAUCUUAUCAACAC
342
GUGUUGAUAAGAUAGGGGC
988

CCCGUCUGUGCCUUCUCAU
343
CCCGUCUGUGCCUUCUCAU
343
AUGAGAAGGCACAGACGGG
989

GCACCAUGCAACUUUUUCA
344
GCACCAUGCAACUUUUUCA
344
UGAAAAAGUUGCAUGGUGC
990

AGUGGUUCGUAGGGCUUUC
345
AGUGGUUCGUAGGGCUUUC
345
GAAAGCCCUACGAACCACU
991

GGUUCGUAGGGCUUUCCCC
346
GGUUCGUAGGGCUUUCCCC
346
GGGGAAAGCCCUACGAACC
992

GUGGUUCGUAGGGCUUUCC
347
GUGGUUCGUAGGGCUUUCC
347
GGAAAGCCCUACGAACCAC
993

AGAAGAUCUCAAUCUCGGG
348
AGAAGAUCUCAAUCUCGGG
348
CCCGAGAUUGAGAUCUUCU
994

CCGUCUGUGCCUUCUCAUC
349
CCGUCUGUGCCUUCUCAUC
349
GAUGAGAAGGCACAGACGG
995

UGGGGUGGAGCCCUCAGGC
350
UGGGGUGGAGCCCUCAGGC
350
GCCUGAGGGCUCCACCCCA
996

GACCACGGGGCGCACCUCU
351
GACCACGGGGCGCACCUCU
351
AGAGGUGCGCCCGGUGGUC
997

UUGUUCAGUGGUUCGUAGG
352
UUGUUCAGUGGUUCGUAGG
352
CCUACGAACCACUGAACAA
998

UGUUCAGUGGUUCGUAGGG
353
UGUUCAGUGGUUCGUAGGG
353
CCCUACGAACCACUGAACA
999

GUUCGUAGGGCUUUCCCCC
354
GUUCGUAGGGCUUUCCCCC
354
GGGGGAAAGCCCUACGAAC
1000

GGAGACCACCGUGAACGCC
355
GGAGACCACCGUGAACGCC
355
GGCGUUCACGGUGGUCUCC
1001

UUUGUUCAGUGGUUCGUAG
356
UUUGUUCAGUGGUUCGUAG
356
CUACGAACCACUGAACAAA
1002

CCGACCACGGGGCGCACCU
357
CCGACCACGGGGCGCACCU
357
AGGUGCGCCCCGUGGUCGG
1003

UCCCUCGCCUCGCAGACGA
358
UCCCUCGCCUCGCAGACGA
358
UCGUCUGCGAGGCGAGGGA
1004

GUGCCUUCUCAUCUGCCGG
359
GUGCCUUCUCAUCUGCCGG
359
CCGGCAGAUGAGAAGGCAC
1005

UUCAGUGGUUCGUAGGGCU
360
UUCAGUGGUUCGUAGGGCU
360
AGCCCUACGAACCACUGAA
1006

CGACCACGGGGCGCACCUC
361
CGACCACGGGGCGCACCUC
361
GAGGUGCGCCCCGUGGUCG
1007

GUUCAGUGGUUCGUAGGGC
362
GUUCAGUGGUUCGUAGGGC
362
GCCCUACGAACCACUGAAC
1008

UCAGUGGUUCGUAGGGCUU
363
UCAGUGGUUCGUAGGGCUU
363
AAGCCCUACGAACCACUGA
1009

UUCCUGCUGGUGGCUCCAG
364
UUCCUGCUGGUGGCUCCAG
364
CUGGAGCCACCAGCAGGAA
1010

CCCUCGCCUCGCAGACGAA
365
CCCUCGCCUCGCAGACGAA
365
UUCGUCUGCGAGGCGAGGG
1011

CCUCGCCUCGCAGACGAAG
366
CCUCGCCUCGCAGACGAAG
366
CUUCGUCUGCGAGGCGAGG
1012

CUGUGCCUUCUCAUCUGCC
367
CUGUGCCUUCUCAUCUGCC
367
GGCAGAUGAGAAGGCACAG
1013

ACCUCUGCCUAAUCAUCUC
368
ACCUCUGCCUAAUCAUCUC
368
GAGAUGAUUAGGCAGAGGU
1014

UCGUAGGGCUUUCCGCCAC
369
UCGUAGGGCUUUCCCCCAC
369
GUGGGGGAAAGCCCUACGA
1015

ACUUCCGGAAACUACUGUU
370
ACUUCCGGAAACUACUGUU
370
AACAGUAGUUUCCGGAAGU
1016

UCUGUGCCUUCUCAUCUGC
371
UCUGUGCCUUCUCAUCUGC
371
GCAGAUGAGAAGGCACAGA
1017

GUCUGUGCCUUCUCAUCUG
372
GUCUGUGCCUUCUCAUCUG
372
CAGAUGAGAAGGCACAGAC
1018

ACCUCUGCACGUCGCAUGG
373
ACCUCUGCACGUCGCAUGG
373
CCAUGCGACGUGCAGAGGU
1019

CAACGACCGACCUUGAGGC
374
CAACGACCGACCUUGAGGC
374
GCCUCAAGGUCGGUCGUUG
1020

UCAACGACCGACCUUGAGG
375
UCAACGACCGACCUUGAGG
375
CCUCAAGGUCGGUCGUUGA
1021

UCACCUCUGCACGUCGCAU
376
UCACCUCUGCACGUCGCAU
376
AUGCGACGUGCAGAGGUGA
1022

GUCAACGACCGACCUUGAG
377
GUCAACGACCGACCUUGAG
377
CUCAAGGUCGGUCGUUGAC
1023

CAAAUGCCCCUAUCUUAUC
378
CAAAUGCCCCUAUCUUAUC
378
GAUAAGAUAGGGGCAUUUG
1024

CACCUCUGCACGUCGCAUG
379
CACCUCUGCACGUCGCAUG
379
CAUGCGACGUGCAGAGGUG
1025

CACUUCCGGAAACUACUGU
380
CACUUCCGGAAACUACUGU
380
ACAGUAGUUUCCGGAAGUG
1026

ACACUUCCGGAAACUACUG
381
ACACUUCCGGAAACUACUG
381
CAGUAGUUUCCGGAAGUGU
1027

UGUCAACGACCGACCUUGA
382
UGUCAACGACCGACCUUGA
382
UCAAGGUCGGUCGUUGACA
1028

AUGUCAACGACCGACCUUG
383
AUGUCAACGACCGACCUUG
383
CAAGGUCGGUCGUUGACAU
1029

GCGCAUGCGUGGAACCUUU
384
GCGCAUGCGUGGAACCUUU
384
AAAGGUUCCACGCAUGCGC
1030

UCUUAUCAACACUUCCGGA
385
UCUUAUCAACACUUCCGGA
385
UCCGGAAGUGUUGAUAAGA
1031

AUUUGUUCAGUGGUUCGUA
386
AUUUGUUCAGUGGUUCGUA
386
UACGAACCACUGAACAAAU
1032

CCCUAUCUUAUCAACACUU
387
CCCUAUCUUAUCAACACUU
387
AAGUGUUGAUAAGAUAGGG
1033

UAUCUUAUCAACACUUCCG
388
UAUCUUAUCAACACUUCCG
388
CGGAAGUGUUGAUAAGAUA
1034

CCUCUGCACGUCGCAUGGA
389
CCUCUGCACGUCGCAUGGA
389
UCCAUGCGACGUGCAGAGG
1035

UGUGGAUUCGCACUCCUCC
390
UGUGGAUUCGCACUCCUCC
390
GGAGGAGUGCGAAUCCACA
1036

CCCCUAUCUUAUCAACACU
391
CCCCUAUCUUAUCAACACU
391
AGUGUUGAUAAGAUAGGGG
1037

CCUAUCUUAUCAACACUUC
392
CCUAUCUUAUCAACACUUC
392
GAAGUGUUGAUAAGAUAGG
1038

UUCACCUCUGCACGUCGCA
393
UUCACCUCUGCACGUCGCA
393
UGCGACGUGCAGAGGUGAA
1039

CUAUCUUAUCAACACUUCC
394
CUAUCUUAUCAACACUUCC
394
GGAAGUGUUGAUAAGAUAG
1040

AUCUUAUCAACACUUCCGG
395
AUCUUAUCAACACUUCCGG
395
CCGGAAGUGUUGAUAAGAU
1041

CAUUUGUUCAGUGGUUCGU
396
CAUUUGUUCAGUGGUUCGU
396
ACGAACCACUGAACAAAUG
1042

GGAAACUACUGUUGUUAGA
397
GG~AACUACUGUUGUUAGA
397
UCUAACAACAGUAGUUUCC
1043

UCCGGAAACUACUGUUGUU
398
UCCGGAAACUACUGUUGUU
398
AACAACAGUAGUUUCCGGA
1044

CCAACUUACAAGGCCUUUC
399
CCAACUUACAAGGCCUUUC
399
GAAAGGCCUUGUAAGUUGG
1045

CGGAAACUACUGUUGUUAG
400
CGGAAACUACUGUUGUUAG
400
CUAACAACAGUAGUUUCCG
1046

GAGACCACCGUGAACGCCC
401
GAGACCACCGUGAACGCCC
401
GGGCGUUCACGGUGGUCUC
1047

CUUCACCUCUGCACGUCGC
402
CUUCACCUCUGCACGUCGC
402
GCGACGUGCAGAGGUGAAG
1048

CCGGAAACUACUGUUGUUA
403
CCGGAAACUACUGUUGUUA
403
UAACAACAGUAGUUUCCGG
1049

CAACUUACAAGGCCUUUCU
404
CAACUUACAAGGCCUUUCU
404
AGAAAGGCCUUGUAAGUUG
1050

CGCUUCACCUCUGCACGUC
405
CGCUUCACCUCUGCACGUC
405
GACGUGCAGAGGUGAAGCG
1051

CAUAAGAGGACUCUUGGAC
406
CAUAAGAGGACUCUUGGAC
406
GUCCAAGAGUCCUCUUAUG
1052

GCUUCACCUCUGCACGUCG
407
GCUUCACCUCUGCACGUCG
407
CGACGUGCAGAGGUGAAGC
1053

AAGAUCUCAAUCUCGGGAA
408
AAGAUCUCAAUCUCGGGAA
408
UUCCCGAGAUUGAGAUCUU
1054

UUGGAGUGUGGAUUCGCAC
409
UUGGAGUGUGGAUUCGCAC
409
GUGCGAAUCCACACUCCAA
1055

UUUGGAGUGUGGAUUCGCA
410
UUUGGAGUGUGGAUUCGCA
410
UGCGAAUCCACACUCCAAA
1056

UUCCGGAAACUACUGUUGU
411
UUCCGGAAACUACUGUUGU
411
ACAACAGUAGUUUCCGGAA
1057

GAAACUACUGUUGUUAGAC
412
GAAACUACUGUUGUUAGAC
412
GUCUAACAACAGUAGUUUC
1058

GAAGAUCUCAAUCUCGGGA
413
GAAGAUCUCAAUCUCGGGA
413
UCCCGAGAUUGAGAUCUUC
1059

UGGGGGCCAAGUCUGUACA
414
UGGGGGCCAAGUCUGUACA
414
UGUACAGACUUGGCCCCCA
1060

CUUCCGGAAACUACUGUUG
415
CUUCCGGAAACUACUGUUG
415
CAACAGUAGUUUCCGGAAG
1061

CCAAAUGCCCCUAUCUUAU
416
CCAAAUGCCCCUAUCUUAU
416
AUAAGAUAGGGGCAUUUGG
1062

AACUACUGUUGUUAGACGA
417
AACUACUGUUGUUAGACGA
417
UCGUCUAACAACAGUAGUU
1063

GUCCUACUGUUCAAGCCUC
418
GUCCUACUGUUCAAGCCUC
418
GAGGCUUGAACAGUAGGAC
1064

CCUACUGUUCAAGCCUCCA
419
CCUACUGUUCAAGCCUCCA
419
UGGAGGCUUGAACAGUAGG
1065

AAUGUCAACGACCGACCUU
420
AAUGUCAACGACCGACCUU
420
AAGGUCGGUCGUUGACAUU
1066

UCCUACUGUUCAAGCCUCC
421
UCCUACUGUUCAAGCCUCC
421
GGAGGCUUGAACAGUAGGA
1067

AAACUACUGUUGUUAGACG
422
AAACUACUGUUGUUAGACG
422
CGUCUAACAACAGUAGUUU
1068

CUACUGUUCAAGCCUCCAA
423
CUACUGUUCAAGCCUCCAA
423
UUGGAGGCUUGAACAGUAG
1069

UGUCCUACUGUUCAAGCCU
424
UGUCCUACUGUUCAAGCCU
424
AGGCUUGAACAGUAGGACA
1070

UACUGUUCAAGCCUCCAAG
425
UACUGUUCAAGCCUCCAAG
425
CUUGGAGGCUUGAACAGUA
1071

GUGGGCCUCAGUCCGUUUC
426
GUGGGCCUCAGUCCGUUUC
426
GAAACGGACUGAGGCCCAC
1072

CUCCUGCCUCCACCAAUCG
427
CUCCUGCCUCCACCAAUCG
427
CGAUUGGUGGAGGCAGGAG
1073

UGGGCCUCAGUCCGUUUCU
428
UGGGCCUCAGUCCGUUUCU
428
AGAAACGGACUGAGGCCCA
1074

UGGGAGUGGGCCUCAGUCC
429
UGGGAGUGGGCCUCAGUCC
429
GGACUGAGGCCCACUCCCA
1075

CCUCCUGCCUCCACCAAUC
430
CCUCCUGCCUCCACCAAUC
430
GAUUGGUGGAGGCAGGAGG
1076

UAUGGGAGUGGGCCUCAGU
431
UAUGGGAGUGGGCCUCAGU
431
ACUGAGGCCCACUCCCAUA
1077

GCAUGCGUGGAACCUUUGU
432
GCAUGCGUGGAACCUUUGU
432
ACAAAGGUUCCACGCAUGC
1078

AUAAGGUGGGAAACUUUAC
433
AUAAGGUGGGAAACUUUAC
433
GUAAAGUUUCCCACCUUAU
1079

CGCAUGCGUGGAACCUUUG
434
CGCAUGCGUGGAACCUUUG
434
CAAAGGUUCCACGCAUGCG
1080

AUGUCCUACUGUUCAAGCC
435
AUGUCCUACUGUUCAAGCC
435
GGCUUGAACAGUAGGACAU
1081

UUUUUGCCUUCUGACUUCU
436
UUUUUGCCUUCUGACUUCU
436
AGAAGUCAGAAGGCAAAAA
1082

GGGCCUCAGUCCGUUUCUC
437
GGGCCUCAGUCCGUUUCUC
437
GAGAAACGGACUGAGGCCC
1083

CAUAAGGUGGGAAACUUUA
438
CAUAAGGUGGGAAACUUUA
438
UAAAGUUUCCCACCUUAUG
1084

GGAGUGGGCCUCAGUCCGU
439
GGAGUGGGCCUCAGUCCGU
439
ACGGACUGAGGCCCACUCC
1085

UGGAGUGUGGAUUCGCACU
440
UGGAGUGUGGAUUCGCACU
440
AGUGCGAAUCCACACUCCA
1086

AUGGGAGUGGGCCUCAGUC
441
AUGGGAGUGGGCCUCAGUC
441
GACUGAGGCCCACUCCCAU
1087

GAGUGGGCCUCAGUCCGUU
442
GAGUGGGCCUCAGUCCGUU
442
AACGGACUGAGGCCCACUC
1088

CAUGUCCUACUGUUCAAGC
443
CAUGUCCUACUGUUCAAGC
443
GCUUGAACAGUAGGACAUG
1089

GGGAGUGGGCCUCAGUCCG
444
GGGAGUGGGCCUCAGUCCG
444
CGGACUGAGGCCCACUCGC
1090

AGUGGGCCUCAGUCCGUUU
445
AGUGGGCCUCAGUCCGUUU
445
AAACGGACUGAGGCCCACU
1091

CCACCAAAUGCCCCUAUCU
446
CCACCAAAUGCCCCUAUCU
446
AGAUAGGGGCAUUUGGUGG
1092

UGUUCAUGUCCUACUGUUC
447
UGUUCAUGUCCUACUGUUC
447
GAACAGUAGGACAUGAACA
1093

ACCAAAUGCCCCUAUCUUA
448
ACCAAAUGCCCCUAUCUUA
448
UAAGAUAGGGGCAUUUGGU
1094

CACCAAAUGCCCCUAUCUU
449
CACCAAAUGCCCCUAUCUU
449
AAGAUAGGGGCAUUUGGUG
1095

UUGGGGGCCAAGUCUGUAC
450
UUGGGGGCCAAGUCUGUAC
450
GUACAGACUUGGCCCCCAA
1096

GUUCAUGUCCUACUGUUCA
451
GUUCAUGUCCUACUGUUCA
451
UGAACAGUAGGACAUGAAC
1097

UCAUGUCCUACUGUUCAAG
452
UCAUGUCCUACUGUUCAAG
452
CUUGAACAGUAGGACAUGA
1098

UUCUCGCCAACUUACAAGG
453
UUCUCGCCAACUUACAAGG
453
CCUUGUAAGUUGGCGAGAA
1099

UUUUGCCUUCUGACUUCUU
454
UUUUGCCUUCUGACUUCUU
454
AAGAAGUCAGAAGGCAAAA
1100

UCCUCAGGCCAUGCAGUGG
455
UCCUCAGGCCAUGCAGUGG
455
CCACUGCAUGGCCUGAGGA
1101

CAUGCGUGGAACCUUUGUG
456
CAUGCGUGGAACCUUUGUG
456
CACAAAGGUUCCACGCAUG
1102

UUCAUGUCCUACUGUUCAA
457
UUCAUGUCCUACUGUUCAA
457
UUGAACAGUAGGACAUGAA
1103

UGGACUCAUAAGGUGGGAA
458
UGGACUCAUAAGGUGGGAA
458
UUCCCACCUUAUGAGUCCA
1104

UUUCUCGCCAACUUACAAG
459
UUUCUCGCCAACUUACAAG
459
CUUGUAAGUUGGCGAGAAA
1105

UGCGCGGGACGUCCUUUGU
460
UGCGCGGGACGUCCUUUGU
460
ACAAAGGACGUCCCGCGCA
1106

CCGGACCGUGUGCACUUCG
461
CCGGACCGUGUGCACUUCG
461
CGAAGUGCACACGGUCCGG
1107

CAUCCUCAGGCCAUGCAGU
462
CAUCCUCAGGCCAUGCAGU
462
ACUGCAUGGCCUGAGGAUG
1108

GGACUCAUAAGGUGGGAAA
463
GGACUCAUAAGGUGGGAAA
463
UUUCCCACCUUAUGAGUCC
1109

CUGCGCGGGACGUCCUUUG
464
CUGCGCGGGACGUCCUUUG
464
CAAAGGACGUCCCGCGCAG
1110

AUCCUCAGGCCAUGCAGUG
465
AUCCUCAGGCCAUGCAGUG
465
CACUGCAUGGCCUGAGGAU
1111

GACCGUGUGCACUUCGCUU
466
GACCGUGUGCACUUCGCUU
466
AAGCGAAGUGCACACGGUC
1112

ACUUUCUCGCCAACUUACA
467
ACUUUCUCGCCAACUUACA
467
UGUAAGUUGGCGAGAAAGU
1113

GGACCGUGUGCACUUCGCU
468
GGACCGUGUGCACUUCGCU
468
AGCGAAGUGCACACGGUCC
1114

CUUUCUCGCCAACUUACAA
469
CUUUCUCGCCAACUUACAA
469
UUGUAAGUUGGCGAGAAAG
1115

ACCGUGUGCACUUCGCUUC
470
ACCGUGUGCACUUCGCUUC
470
GAAGCGAAGUGCACACGGU
1116

UGCUGCCAACUGGAUCCUG
471
UGCUGCCAACUGGAUCCUG
471
CAGGAUCCAGUUGGCAGCA
1117

GCUGCCAACUGGAUCCUGC
472
GCUGCCAACUGGAUCCUGC
472
GCAGGAUCCAGUUGGCAGC
1118

CGGACCGUGUGCACUUCGC
473
CGGACCGUGUGCACUUCGC
473
GCGAAGUGCACACGGUCCG
1119

GCUGGUGGCUCCAGUUCAG
474
GCUGGUGGCUCCAGUUCAG
474
CUGAACUGGAGCCACCAGC
1120

UGCCUUCUGACUUCUUUCC
475
UGCCUUCUGACUUCUUUCC
475
GGAAAGAAGUCAGAAGGCA
1121

UCUCGCCAACUUACAAGGC
476
UCUCGCCAACUUACAAGGC
476
GCCUUGUAAGUUGGCGAGA
1122

CUCUUCAUCCUGCUGCUAU
477
CUCUUCAUCCUGCUGCUAU
477
AUAGCAGCAGGAUGAAGAG
1123

UGCCAACUGGAUCCUGCGC
478
UGCCAACUGGAUCCUGCGC
478
GCGCAGGAUCCAGUUGGCA
1124

CUUCAUCCUGCUGCUAUGC
479
CUUCAUCCUGCUGCUAUGC
479
GCAUAGCAGCAGGAUGAAG
1125

CCAACUGGAUCCUGCGCGG
480
CCAACUGGAUCCUGCGCGG
480
CCGCGCAGGAUCCAGUUGG
1126

GGUGGAGCCCUCAGGCUCA
481
GGUGGAGCCCUCAGGCUCA
481
UGAGCCUGAGGGCUCCACC
1127

UGCUGGUGGCUCCAGUUCA
482
UGCUGGUGGCUCCAGUUCA
482
UGAACUGGAGCCACCAGCA
1128

UCAUCCUGCUGCUAUGCCU
483
UCAUCCUGCUGCUAUGCCU
483
AGGCAUAGCAGCAGGAUGA
1129

GGGUGGAGCCCUCAGGCUC
484
GGGUGGAGCCCUCAGGCUC
484
GAGCCUGAGGGCUCCACCC
1130

GGCCAUCAGCGCAUGCGUG
485
GGCCAUCAGCGCAUGCGUG
485
CACGCAUGCGCUGAUGGCC
1131

UUCAUCCUGCUGCUAUGCC
486
UUCAUCCUGCUGCUAUGCC
486
GGCAUAGCAGCAGGAUGAA
1132

UCUUCAUCCUGCUGCUAUG
487
UCUUCAUCCUGCUGCUAUG
487
CAUAGCAGCAGGAUGAAGA
1133

GCCAACUGGAUCCUGCGCG
488
GCCAACUGGAUCCUGCGCG
488
CGCGCAGGAUCCAGUUGGC
1134

CUGCCAACUGGAUCCUGCG
489
CUGCCAACUGGAUCCUGCG
489
CGCAGGAUCCAGUUGGCAG
1135

CUCGCCAACUUACAAGGCC
490
CUCGCCAACUUACAAGGCC
490
GGCCUUGUAAGUUGGCGAG
1136

CCUCUUCAUCCUGCUGCUA
491
CCUCUUCAUCCUGCUGCUA
491
UAGCAGCAGGAUGAAGAGG
1137

ACUGGAUCCUGCGCGGGAC
492
ACUGGAUCCUGCGCGGGAC
492
GUCCCGCGCAGGAUCCAGU
1138

GGGGUGGAGCCCUCAGGCU
493
GGGGUGGAGCCCUCAGGCU
493
AGCCUGAGGGCUCCACCCC
1139

AACUGGAUCCUGCGCGGGA
494
AACUGGAUCCUGCGCGGGA
494
UCCCGCGCAGGAUCCAGUU
1140

CUUGGACUCAUAAGGUGGG
495
CUUGGACUCAUAAGGUGGG
495
CCCACCUUAUGAGUCCAAG
1141

CUGCCGGACCGUGUGCACU
496
CUGCCGGACCGUGUGCACU
496
AGUGCACACGGUCCGGCAG
1142

CCUGCGCGGGACGUCCUUU
497
CCUGCGCGGGACGUCCUUU
497
AAAGGACGUCCCGCGCAGG
1143

GAUCCUGCGCGGGACGUCC
498
GAUCCUGCGCGGGACGUCC
498
GGACGUCCCGCGCAGGAUC
1144

CCUUGGACUCAUAAGGUGG
499
CCUUGGACUCAUAAGGUGG
499
CCACCUUAUGAGUCCAAGG
1145

UGCCGGACCGUGUGCACUU
500
UGCCGGACCGUGUGCACUU
500
AAGUGCACACGGUCCGGCA
1146

AUCCUGCGCGGGACGUCCU
501
AUCCUGCGCGGGACGUCCU
501
AGGACGUCCCGCGCAGGAU
1147

GCCAUCAGCGCAUGCGUGG
502
GCCAUCAGCGCAUGCGUGG
502
CCACGCAUGCGCUGAUGGC
1148

UUGCCUUCUGACUUCUUUC
503
UUGCCUUCUGACUUCUUUC
503
GAAAGAAGUCAGAAGGCAA
1149

CAACUGGAUCCUGCGCGGG
504
CAACUGGAUCCUGCGCGGG
504
CCCGCGCAGGAUCCAGUUG
1150

UGGAUCCUGCGCGGGACGU
505
UGGAUCCUGCGCGGGACGU
505
ACGUCCCGCGCAGGAUCCA
1151

UGCUCAAGGAACCUCUAUG
506
UGCUCAAGGAACCUCUAUG
506
CAUAGAGGUUCCUUGAGCA
1152

UCCUGCGCGGGACGUCCUU
507
UCCUGCGCGGGACGUCCUU
507
AAGGACGUCCCGCGCAGGA
1153

UUUGCCUUCUGACUUCUUU
508
UUUGCCUUCUGACUUCUUU
508
AAAGAAGUCAGAAGGCAAA
1154

GCCGGACCGUGUGCACUUC
509
GCCGGACCGUGUGCACUUC
509
GAAGUGCACACGGUCCGGC
1155

GGAUCCUGCGCGGGACGUC
510
GGAUCCUGCGCGGGACGUC
510
GACGUCCCGCGCAGGAUCC
1156

UCCUCUUCAUCCUGCUGCU
511
UCCUCUUCAUCCUGCUGCU
511
AGCAGCAGGAUGAAGAGGA
1157

CUGGAUCCUGCGCGGGACG
512
CUGGAUCCUGCGCGGGACG
512
CGUCCGGCGGAGGAUCCAG
1158

GCUCAAGGAACCUCUAUGU
513
GCUCAAGGAACCUCUAUGU
513
ACAUAGAGGUUCCUUGAGC
1159

UCAUCCUCAGGCCAUGCAG
514
UCAUCCUCAGGCCAUGCAG
514
CUGCAUGGCCUGAGGAUGA
1160

UUCCUCUUCAUCCUGCUGC
515
UUCCUCUUCAUCCUGCUGC
515
GCAGCAGGAUGAAGAGGAA
1161

UCCUUUGUUUACGUCCCGU
516
UCCUUUGUUUACGUCCCGU
516
ACGGGACGUAAACAAAGGA
1162

GAGCCCUCAGGCUCAGGGC
517
GAGCCCUCAGGCUCAGGGC
517
GCCCUGAGCCUGAGGGCUC
1163

CCUUUGUUUACGUCCCGUC
518
CCUUUGUUUACGUCCCGUC
518
GACGGGACGUAAACAAAGG
1164

UUGGGGUGGAGCCCUCAGG
519
UUGGGGUGGAGCCCUCAGG
519
CCUGAGGGCUCCACCCCAA
1165

AUCAACACUUCCGGAAACU
520
AUCAACACUUCCGGAAACU
520
AGUUUCCGGAAGUGUUGAU
1166

ACGUCCUUUGUUUACGUCC
521
ACGUCCUUUGUUUACGUCC
521
GGACGUAAACAAAGGACGU
1167

GGACGUCCUUUGUUUACGU
522
GGACGUCCUUUGUUUACGU
522
ACGUAAACAAAGGACGUCC
1168

GGAGCCCUCAGGCUCAGGG
523
GGAGCCCUCAGGCUCAGGG
523
CCCUGAGCCUGAGGGCUCC
1169

GUCCUUUGUUUACGUCCCG
524
GUCCUUUGUUUACGUCCCG
524
CGGGACGUAAACAPAGGAC
1170

AUGAUGUGGUAUUGGGGGC
525
AUGAUGUGGUAUUGGGGGC
525
GCCCCCAAUACCACAUCAU
1171

UCUGCCGGACGGUGUGGAC
526
UCUGCCGGACCGUGUGCAC
526
GUGCACACGGUCCGGCAGA
1172

UAUCAACACUUCCGGAAAC
527
UAUCAACACUUCCGGAAAC
527
GUUUCCGGAAGUGUUGAUA
1173

CGUCCUUUGUUUACGUCCC
528
CGUCCUUUGUUUACGUCCC
528
GGGACGUPAACAAAGGACG
1174

GAUGAUGUGGUAUUGGGGG
529
GAUGAUGUGGUAUUGGGGG
529
CCCCCAAUACCACAUCAUC
1175

GACGUCCUUUGUUUACGUC
530
GACGUCCUUUGUUUACGUC
530
GACGUAAACAAAGGACGUC
1176

GGAUGAUGUGGUAUUGGGG
531
GGAUGAUGUGGUAUUGGGG
531
CCCCAAUACCACAUCAUCC
1177

UGGAUGAUGUGGUAUUGGG
532
UGGAUGAUGUGGUAUUGGG
532
CCCAAUACCACAUCAUCCA
1178

AUGGAUGAUGUGGUAUUGG
533
AUGGAUGAUGUGGUAUUGG
533
CCAAUACCACAUCAUCCAU
1179

GGGACGUCCUUUGUUUACG
534
GGGACGUCCUUUGUUUACG
534
CGUAAACAAAGGACGUCCC
1180

AUCAAGGUAUGUUGCCCGU
535
AUCAAGGUAUGUUGCCCGU
535
ACGGGCAACAUACCUUGAU
1181

ACCUGUAUUCCCAUCCCAU
536
ACCUGUAUUCCCAUCCCAU
536
AUGGGAUGGGAAUACAGGU
1182

UAUCAAGGUAUGUUGCCCG
537
UAUCAAGGUAUGUUGCCCG
537
CGGGCAACAUACCUUGAUA
1183

CACCUGUAUUCCCAUCCCA
538
CACCUGUAUUCCCAUCCCA
538
UGGGAUGGGAAUACAGGUG
1184

UGCACCUGUAUUCCCAUCC
539
UGCACCUGUAUUCCCAUCC
539
GGAUGGGAAUACAGGUGCA
1185

UAUAUGGAUGAUGUGGUAU
540
UAUAUGGAUGAUGUGGUAU
540
AUACCACAUCAUCCAUAUA
1186

UAUGGAUGAUGUGGUAUUG
541
UAUGGAUGAUGUGGUAUUG
541
CAAUACCACAUCAUCCAUA
1187

UUGGACUCAUAAGGUGGGA
542
UUGGACUCAUAAGGUGGGA
542
UCCCACCUUAUGAGUCCAA
1188

UGGAGCCCUCAGGCUCAGG
543
UGGAGCCCUCAGGCUCAGG
543
CCUGAGCCUGAGGGCUCCA
1189

CCUGUAUUCCCAUCCCAUC
544
CCUGUAUUCCCAUCCCAUC
544
GAUGGGAUGGGAAUACAGG
1190

CGGGACGUCCUUUGUUUAC
545
CGGGACGUCCUUUGUUUAC
545
GUAAACAAAGGACGUCCCG
1191

AUAUGGAUGAUGUGGUAUU
546
AUAUGGAUGAUGUGGUAUU
546
AAUACCACAUCAUCCAUAU
1192

GCACCUGUAUUCCCAUCCC
547
GCACCUGUAUUCCCAUCCC
547
GGGAUGGGAAUACAGGUGC
1193

GUGGAGCCCUCAGGCUCAG
548
GUGGAGCCCUCAGGCUCAG
548
CUGAGCCUGAGGGCUCCAC
1194

CGCGGGACGUCCUUUGUUU
549
CGCGGGACGUCCUUUGUUU
549
AAACAAAGGACGUCCCGCG
1195

GCUCCUCUGCCGAUCCAUA
550
GCUCCUCUGCCGAUCCAUA
550
UAUGGAUCGGCAGAGGAGC
1196

UGAUGUGGUAUUGGGGGCC
551
UGAUGUGGUAUUGGGGGCC
551
GGCCCCCAAUACCACAUCA
1197

CAGCGCAUGCGUGGAACCU
552
CAGCGCAUGCGUGGAACCU
552
AGGUUCCACGCAUGCGCUG
1198

AGCGCAUGCGUGGAACCUU
553
AGCGCAUGCGUGGAACCUU
553
AAGGUUCCACGCAUGCGCU
1199

AUGUGGUAUUGGGGGCCAA
554
AUGUGGUAUUGGGGGCCAA
554
UUGGCCCCCAAUACCACAU
1200

UUUCCUGCUGGUGGCUCCA
555
UUUCCUGCUGGUGGCUCCA
555
UGGAGCCACCAGCAGGAAA
1201

GAUCUCAAUCUCGGGAAUC
556
GAUCUCAAUCUCGGGAAUC
556
GAUUCCCGAGAUUGAGAUC
1202

GCGGGACGUCCUUUGUUUA
557
GCGGGACGUCCUUUGUUUA
557
UAAACAAAGGACGUCCCGC
1203

GAUGUGGUAUUGGGGGCCA
558
GAUGUGGUAUUGGGGGCCA
558
UGGCCCCCAAUACCACAUC
1204

CUUAUCAACACUUCCGGAA
559
CUUAUCAACACUUCCGGAA
559
UUCCGGAAGUGUUGAUAAG
1205

GGCUCCUCUGCCGAUCCAU
560
GGCUCCUCUGCCGAUCCAU
560
AUGGAUCGGCAGAGGAGCC
1206

CAAUGUCAACGACCGACCU
561
CAAUGUCAACGACCGACCU
561
AGGUCGGUCGUUGACAUUG
1207

CUGGUGGCUCCAGUUCAGG
562
CUGGUGGCUCCAGUUCAGG
562
CCUGAACUGGAGCCACCAG
1208

UCCCCAACCUCCAAUCACU
563
UCCCCAACCUCCAAUCACU
563
AGUGAUUGGAGGUUGGGGA
1209

CCAUCAGCGCAUGCGUGGA
564
CCAUCAGCGCAUGCGUGGA
564
UCCACGCAUGCGCUGAUGG
1210

UUAUCAACACUUCCGGAAA
565
UUAUCAACACUUCCGGAAA
565
UUUCCGGAAGUGUUGAUAA
1211

UCAACACUUCCGGAAACUA
566
UCAACACUUCCGGAAACUA
566
UAGUUUCCGGAAGUGUUGA
1212

CAACACUUCCGGAAACUAC
567
CAACACUUCCGGAAACUAC
567
GUAGUUUCCGGAAGUGUUG
1213

GCAGUCCCCAACCUCCAAU
568
GCAGUCCCCAACCUCCAAU
568
AUUGGAGGUUGGGGACUGC
1214

AACACUUCCGGAAACUACU
569
AACACUUCCGGAAACUACU
569
AGUAGUUUCCGGAAGUGUU
1215

CAGUCCCCAACCUCCAAUC
570
CAGUCCCCAACCUCCAAUC
570
GAUUGGAGGUUGGGGACUG
1216

GUCCCCAACCUCCAAUCAC
571
GUCCCCAACCUCCAAUCAC
571
GUGAUUGGAGGUUGGGGAC
1217

AUUUUCUUUUGUCUUUGGG
572
AUUUUCUUUUGUCUUUGGG
572
CCCAAAGACAAAAGAAAAU
1218

AGUCCCCAACCUCCAAUCA
573
AGUCCCCAACCUCCAAUCA
573
UGAUUGGAGGUUGGGGACU
1219

GUGCCUUGGGUGGCUUUGG
574
GUGCCUUGGGUGGCUUUGG
574
CCAAAGCCACCCAAGGCAC
1220

CCACCAAUCGGCAGUCAGG
575
CCACCAAUCGGCAGUCAGG
575
CCUGACUGCCGAUUGGUGG
1221

UGCCUUGGGUGGCUUUGGG
576
UGCCUUGGGUGGCUUUGGG
576
CCCAAAGCCACCCAAGGCA
1222

UGUGCCUUGGGUGGCUUUG
577
UGUGCCUUGGGUGGCUUUG
577
CAAAGCCACCCAAGGCACA
1223

CCUGCCUCCACCAAUCGGC
578
CCUGCCUCCACCAAUCGGC
578
GCCGAUUGGUGGAGGCAGG
1224

GUUAUAUGGAUGAUGUGGU
579
GUUAUAUGGAUGAUGUGGU
579
ACCACAUCAUCCAUAUAAC
1225

AUCAGCGCAUGCGUGGAAC
580
AUCAGCGCAUGCGUGGAAC
580
GUUCCACGCAUGCGCUGAU
1226

UGGCUUUCAGUUAUAUGGA
581
UGGCUUUCAGUUAUAUGGA
581
UCCAUAUAACUGAAAGCCA
1227

GGCUUUCAGUUAUAUGGAU
582
GGCUUUCAGUUAUAUGGAU
582
AUCCAUAUAACUGAAAGCC
1228

AGAUCUCAAUCUCGGGAAU
583
AGAUCUCAAUCUCGGGAAU
583
AUUCCCGAGAUUGAGAUCU
1229

UCCUGCCUCCACCAAUCGG
584
UCCUGCCUCCACCAAUCGG
584
CCGAUUGGUGGAGGCAGGA
1230

UCAGCGCAUGCGUGGAACC
585
UCAGCGCAUGCGUGGAACC
585
GGUUCCACGCAUGCGCUGA
1231

CAUCAGCGCAUGCGUGGAA
586
CAUCAGCGCAUGCGUGGAA
586
UUCCACGCAUGCGCUGAUG
1232

CUGCCUCCACCAAUCGGCA
587
CUGCCUCCACCAAUCGGCA
587
UGCCGAUUGGUGGAGGGAG
1233

UCCACCAAUCGGCAGUCAG
588
UCCACCAAUCGGCAGUCAG
588
CUGACUGCCGAUUGGUGGA
1234

UCAAGGUAUGUUGCCCGUU
589
UCAAGGUAUGUUGCCCGUU
589
AACGGGCAACAUACCUUGA
1235

GCUUUCAGUUAUAUGGAUG
590
GCUUUCAGUUAUAUGGAUG
590
CAUCCAUAUAACUGAAAGC
1236

UGCCUCCACCAAUCGGCAG
591
UGCCUCCACCAAUCGGCAG
591
CUGCCGAUUGGUGGAGGCA
1237

GCCUCCACCAAUCGGCAGU
592
GCCUCCACCAAUCGGCAGU
592
ACUGCCGAUUGGUGGAGGC
1238

UUUCAGUUAUAUGGAUGAU
593
UUUCAGUUAUAUGGAUGAU
593
AUCAUCCAUAUAACUGAAA
1239

AGUUAUAUGGAUGAUGUGG
594
AGUUAUAUGGAUGAUGUGG
594
CCACAUCAUCCAUAUAACU
1240

CUUUCAGUUAUAUGGAUGA
595
CUUUCAGUUAUAUGGAUGA
595
UCAUCCAUAUAACUGAAAG
1241

UCUGCACGUCGCAUGGAGA
596
UCUGCACGUCGCAUGGAGA
596
UCUCCAUGCGACGUGCAGA
1242

UUCUUUUGUCUUUGGGUAU
597
UUCUUUUGUCUUUGGGUAU
597
AUACCCAAAGACAAAAGAA
1243

UUUCUUUUGUCUUUGGGUA
598
UUUCUUUUGUCUUUGGGUA
598
UACCCAAAGACAAAAGAAA
1244

CACGUCGCAUGGAGACCAC
599
CACGUCGCAUGGAGACCAC
599
GUGGUCUCCAUGCGACGUG
1245

CUUUGUUUACGUCCCGUCG
600
CUUUGUUUACGUCCCGUCG
600
CGACGGGACGUAAACAAAG
1246

UUUGUUUACGUCCCGUCGG
601
UUUGUUUACGUCCCGUCGG
601
CCGACGGGACGUAAACAAA
1247

UGCACGUCGCAUGGAGACC
602
UGCACGUCGCAUGGAGACC
602
GGUCUCCAUGCGACGUGCA
1248

GCACGUCGCAUGGAGACCA
603
GCACGUCGCAUGGAGACCA
603
UGGUCUCCAUGCGACGUGC
1249

CGCAUGGAGACCACCGUGA
604
CGCAUGGAGACCACGGUGA
604
UCACGGUGGUCUCCAUGCG
1250

UCGCAUGGAGACCACCGUG
605
UCGCAUGGAGACCACCGUG
605
CACGGUGGUCUCCAUGCGA
1251

UUUUCUUUUGUCUUUGGGU
606
UUUUCUUUUGUCUUUGGGU
606
ACCCAAAGACAAAAGAAAA
1252

GUCGCAUGGAGACCACCGU
607
GUCGCAUGGAGACCACCGU
607
ACGGUGGUCUCCAUGCGAC
1253

CUCUGCACGUCGCAUGGAG
608
CUCUGCACGUCGCAUGGAG
608
CUCCAUGCGACGUGCAGAG
1254

GCAAUGUCAACGACCGACC
609
GCAAUGUCAACGACCGACC
609
GGUCGGUCGUUGACAUUGC
1255

CUGCACGUCGCAUGGAGAC
610
CUGCACGUCGCAUGGAGAC
610
GUCUCCAUGCGACGUGCAG
1256

CGUCGCAUGGAGACCACCG
611
CGUCGCAUGGAGACCACCG
611
CGGUGGUCUCCAUGCGACG
1257

ACGUCGCAUGGAGACCACC
612
ACGUCGCAUGGAGACCACC
612
GGUGGUCUCCAUGCGACGU
1258

UUUGUCUUUGGGUAUACAU
613
UUUGUCUUUGGGUAUACAU
613
AUGUAUACCCAAAGACAAA
1259

UGUGGUUUCACAUUUCCUG
614
UGUGGUUUCACAUUUCCUG
614
CAGGAAAUGUGAAACCACA
1260

UCUUUUGUCUUUGGGUAUA
615
UCUUUUGUCUUUGGGUAUA
615
UAUACCCAAAGACAAAAGA
1261

CUUUUGUCUUUGGGUAUAC
616
CUUUUGUCUUUGGGUAUAC
616
GUAUACCCAAAGACAAAAG
1262

UUUUGUCUUUGGGUAUACA
617
UUUUGUCUUUGGGUAUACA
617
UGUAUACCCAAAGACAAAA
1263

CCUUCUCAUCUGCCGGACC
618
CCUUCUCAUCUGCCGGACC
618
GGUCCGGCAGAUGAGAAGG
1264

AUCUGCCGGACCGUGUGCA
619
AUCUGCCGGACCGUGUGCA
619
UGCACACGGUCCGGCAGAU
1265

CUCGCCUCGCAGACGAAGG
620
CUCGCCUCGCAGACGAAGG
620
CCUUCGUCUGCGAGGCGAG
1266

UCAUCUGCCGGACCGUGUG
621
UCAUCUGCCGGACGGUGUG
621
CACACGGUCCGGCAGAUGA
1267

UUGUUUACGUCCCGUCGGC
622
UUGUUUACGUCCCGUCGGC
622
GCCGACGGGACGUAAACAA
1268

UGUUUACGUCCCGUCGGCG
623
UGUUUACGUCCCGUCGGCG
623
CGCCGACGGGACGUAAACA
1269

CACCAAUCGGCAGUCAGGA
624
CACCAAUCGGCAGUCAGGA
624
UCCUGACUGCCGAUUGGUG
1270

UUUACGUCCCGUCGGCGCU
625
UUUACGUCCCGUCGGCGCU
625
AGCGCCGACGGGACGUAAA
1271

GUUUACGUCCCGUCGGCGC
626
GUUUACGUCCCGUCGGCGC
626
GCGCCGACGGGACGUAAAC
1272

CAUCUGCCGGACCGUGUGC
627
CAUCUGCCGGACGGUGUGC
627
GCACACGGUCCGGCAGAUG
1273

UCUUUUGGAGUGUGGAUUC
628
UCUUUUGGAGUGUGGAUUC
628
GAAUCCACACUCCAAAAGA
1274

UCGCCUCGCAGACGAAGGU
629
UCGCCUCGCAGACGAAGGU
629
ACCUUCGUCUGCGAGGCGA
1275

GCCUCGCAGACGAAGGUCU
630
GCCUCGCAGACGAAGGUCU
630
AGACCUUCGUCUGCGAGGC
1276

CUCAUCUGCCGGACCGUGU
631
CUCAUCUGCCGGACCGUGU
631
ACACGGUCCGGCAGAUGAG
1277

UGAGGCAUACUUCAAAGAC
632
UGAGGCAUACUUCAAAGAC
632
GUCUUUGAAGUAUGCCUCA
1278

UGGCUUUGGGGCAUGGACA
633
UGGCUUUGGGGCAUGGACA
633
UGUCCAUGCCCCAAAGCCA
1279

GGCUUUGGGGCAUGGACAU
634
GGCUUUGGGGCAUGGACAU
634
AUGUCCAUGCCCCAAAGCC
1280

CUUUUGGAGUGUGGAUUCG
635
CUUUUGGAGUGUGGAUUCG
635
CGAAUCCACACUCCAAAAG
1281

UCUCUUUUUUGCCUUCUGA
636
UCUCUUUUUUGCCUUCUGA
636
UCAGAAGGCAAAAAAGAGA
1282

ACCAAUUUUCUUUUGUCUU
637
ACCAAUUUUCUUUUGUCUU
637
AAGACAAAAGAAAAUUGGU
1283

CUUCUCAUCUGCCGGACCG
638
CUUCUCAUCUGCCGGACCG
638
CGGUCCGGCAGAUGAGAAG
1284

CCUCCUCCUGCCUCCACCA
639
CCUCCUCCUGCCUCCACCA
639
UGGUGGAGGCAGGAGGAGG
1285

UUUUGGAGUGUGGAUUCGC
640
UUUUGGAGUGUGGAUUCGC
640
GCGAAUCCACACUCCAAAA
1286

UUCUCAUCUGCCGGACCGU
641
UUCUCAUCUGCCGGACCGU
641
ACGGUCCGGCAGAUGAGAA
1287

AAUUUUCUUUUGUCUUUGG
642
AAUUUUCUUUUGUCUUUGG
642
CCAAAGACAAAAGAAAAUU
1288

CGCCUCGCAGACGAAGGUC
643
CGCCUCGCAGACGAAGGUC
643
GACCUUCGUCUGCGAGGCG
1289

CCAAUUUUCUUUUGUCUUU
644
CCAAUUUUCUUUUGUCUUU
644
AAAGACAAAAGAAAAUUGG
1290

CAAUUUUCUUUUGUCUUUG
645
CAAUUUUCUUUUGUCUUUG
645
CAAAGACAAAAGAAAAUUG
1291

UCUCAUCUGCCGGACCGUG
646
UCUCAUCUGCCGGACCGUG
646
CACGGUCCGGCAGAUGAGA
1292

[0325] HBV Composite

[0326] The 3′-ends of the Upper sequence and the Lower sequence of the siRNA construct can include an overhang sequence, for example about 1, 2, 3, or 4 nucleotides in length, preferably 2 nucleotides in length, wherein the overhanging sequence of the lower sequence is optionally complementary to a portion of the target sequence. The upper sequence is also referred to as the sense strand, whereas the lower sequence is also referred to as the antisense strand. The upper and lower sequences in the Table can further comprise a chemical modification having Formula I-VII.

3TABLE IIIHBV Synthetic siRNA constructsTarget SequenceSeq IDAliasesSequenceSeq IDHBV (HepBzyme site) as siRNA str1 (sense)B UGGACUUCUCUCAAUUUUCUA B1319HBV (HepBzyme site) as siRNA str2B UAGAAAAUUGAGAGAAGUCCA B1320(antisense)HBV18371 site as siRNA str1 (sense)B UUUUUCACCUCUGCCUAAUCA B1321HBV18371 site as siRNA str2 (antisense)B UGAUUAGGCAGAGGUGAAAAA B1322HBV16372-18373 site as siRNA str1 (sense)B CAAGCCUCCAAGCUGUGCCUU B1323HBV16372-18373 site as siRNA str2B AAGGCACAGCUUGGAGGCUUG B1324(antisense)HBV (HepBzyme site) as siRNA str1 (sense)B UAGAAAAUUGAGAGAAGUCCA B1320Inverted ControlHBV (HepBzyme site) as siRNA str1 (sense)B UGGACUUCUCUCAAUUUUCUA B1319Inverted Control ComplimentHBV (HepBzyme site) as siRNAUGGACUUCUCUCAAUUUUCUAUU1325str1 (sense) + 2U overhangHBV (HepBzyme site) as siRNA str2UAGAAAAUUGAGAGAAGUCCAUU1326(antisense) + 2U overhangHBV18371 site as siRNA strl (sense) +UUUUUCACCUCUGCCUAAUCAUU13272U overhangHBV18371 site as siRNA str2 (antisense) +UGAUUAGGCAGAGGUGAAAAAUU13282U overhangHBV16372-18373 site as siRNACAAGCCUCCAAGCUGUGCCUUUU1329str1 (sense)-s-2U overhangHBV16372-18373 site as siRNA str 2AAGGCACAGCUUGGAGGCUUGUU1330(antisense) + 2U overhangHBV (HepBzyme site) as siRNABUGGACUUCUCUCAAUUUUCUAUUB1331str1 (sense) + 2U overhangHBV (HepBzyme site) as siRNA str2BUAGAAAAUUGAGAGAAGUCCAUUB1332(antisense) + 2U overhangHBV18371 site as siRNA str1 (sense) +BUUUUUCACCUCUGCCUAAUCAUUB13332U overhangHBV1 8371 site as siRNA str2 (antisense) +BUGAUUAGGCAGAGGUGAAAAAUUB13342U overhangHBV16372-18373 site as siRNABCAAGCCUCCAAGCUGUGCCUUUUB1335str1 (sense) + 2U overhangHBV1 6372-18373 site as siRNA str2BAAGGCACAGCUUGGAGGCUUGUUB1336(antisense) + 2U overhangGAGUCUAGACUCGUGGUGGACUU1293HBV:248U21 siRNA posGUCUAGACUCGUGGUGGACTT1337AUCCUGCUGCUAUGCCUCAUCUU1294HBV:414U21 siRNA posCCUGCUGCUAUGCCUCAUCTT1338UUCAAGCCUCCAAGCUGUGCCUU1295HBV:1867U21 siRNA posCAAGCCUCCAAGCUGUGCCTT1339CAAGCUGUGCCUUGGGUGGCUUU1296HBV:1877U21 siRNA posAGCUGUGCCUUGGGUGGCUTT1340GAGUCUAGACUCGUGGUGGACUU1293HBV:228L21 sIRNA neg (248C)GUCCACCACGAGUCUAGACTT1341AUCCUGCUGCUAUGCCUCAUCUU1294HBV:394L21 siRNA neg (414C)GAUGAGGCAUAGCAGCAGGTT1342UUCAAGCCUCCAAGCUGUGCCUU1295HBV:1847L21 siRNA neg (1867C)GGCACAGCUUGGAGGCUUGTT1343CAAGCUGUGCCUUGGGUGGCUUU1296HBV:1857L21 siRNA neg (18770)AGCCACCCAAGGCACAGCUTT1344GAGUCUAGACUCGUGGUGGACUU1293HBV:248U21 siRNA pos invCAGGUGGUGCUCAGAUCUGTT1345AUCCUGCUGCUAUGCCUCAUCUU1294HBV:414U21 siRNA pos invCUAOUCCGUAUCGUCGUCGTT1346UUCAAGCCUCCAAGCUGUGCCUU1295HBV:1867U21 siRNA pos invCCGUGUCGAACCUCCGAACTT1347CAAGCUGUGCCUUGGGUGGCUUU1296HBV:1877U21 siRNApos invUCGGUGGGUUCCGUGUCGATT1348GAGUCUAGACUCGUGGUGGACUU1293HBV:228L21 siRNA neg (2480) invCAGAUCUGAGCACCACCUGTT1349AUCCUGCUGCUAUGCCUCAUCUU1294HBV:394L21 siRNA neg (4140) invGGACGACGAUACGGAGUAGTT1350UUCAAGCCUCCAAGCUGUGCCUU1295HBV:1847L21 siRNA neg (18670) invGUUCGGAGGUUCGACACGGTT1351CAAGCUGUGCCUUGGGUGGCUUU1296HBV:1857L21 siRNA neg (18770) invUCGACACGGAACCCACCGATT1352AUCCUGCUGCUAUGCCUCAUCUU1294HBV:414U21 siRNA pos stab3cScSuSGScuGcuAuGccucASuScSTST1353AUCCUGCUGCUAUGCCUCAUCUU1294HBV:414U21 siRNA pos stab4B ccuGcuGcuAuGccucAucTTB1354AUCCUGCUGCUAUGCCUCAUCUU1294HBV:414U21 siRNApos stab6BccuGcuGcuAuGccucAucTTB1355AUCCUGCUGCUAUGCCUCAUCUU1294HBV:394L21 siRNA neg (414C) stab2GSASUSGSASGSGSCSASUSASGSCSASGSCSASGSGSTST1356AUCCUGCUGCUAUGCCUCAUCUU1294HBV:394L21 siRNA neg (414C) stab5GAUGAGGCAUAGCAGCAGGTST1357AUCCUGCUGCUAUGCCUCAUCUU1294HBV:414U21 siRNApos inv stab3cSuSAScSuccGuAucGucGSuScScSTST1358AUCCUGCUGCUAUGCCUCAUCUU1294HBV:414U21 siRNApos inv stab4BcUAcuccGuAucGucGuccTTB1359AUCCUGCUGCUAUGCCUCAUCUU1294HBV:414U21 siRNApos inv stab6BcUAcuccGuAucGucGuccTTB1360AUCCUGCUGCUAUGCCUGAUCUU1294HBV:394L21 siRNA neg (414C) inv stab2GSGSASCSGSASCSGSASUSASCSGSGSASGSUSASGSTST1361AUCCUGCUGCUAUGCCUCAUCUU1294HBV:394L21 siRNA neg (414C) inv stabSGGAcGAcGAuAcGGAGuAGTST1362AUCCUGCUGCUAUGCCUCAUCUU1294HBV:414U21 siRNApos stab3cScSuSGScuGcuAuGccucASuScSTSTS1353AUCCUGCUGCUAUGCCUGAUCUU1294HBV:414U21 siRNApos stab4BccuGcuGcuAuGccucAucTTB1354AU0OUGCUGCUAUGCCUCAUCUU1294HBV:414U21 siRNApos stab6BccuGcuGcuAuGccucAucTTB1355AUCCUGCUGCUAUGCCUCAUCUU1294HBV:394L21 siRNA neg (4140) stab2GSASUSGSASGSGSCSASUSASGSCSASGSCSASGSGSTST1356AUCCUGCUGCUAUGCCUCAUCUU1294HBV:394L21 siRNA neg (4140) stab5GAuGAGGcAuAGcAGcAGGTST1357AUCCUGCUGCUAUGCCUCAUCUU1294HBV:414U21 siRNApos inv stab3CSUSAScSuccGuAucGucGSuScScSTST1358AUOOUGOUGCUAUGCCUCAUCUU1294HBV:414U21 siRNApos inv stab4B cuAcuccGuAucGucGuccTT B1359AUCGUGCUGCUAUGCCUCAUCUU1294HBV:414U21 siRNA P05 inv stab6B cuAcuccGuAucGucGuccTT B1360AUCCUGCUGOUAUGCCUCAUCUU1294HBV:394L21 siRNA neg (4140) inv stab2GSGSASCSGSASCSGSASUSASCSGSGSASGSUSASGSTST1361AUCCUGCUGCUAUGCCUCAUCUU1294HBV:394L21 siRNA neg (4140) inv stab5GGAcGAcGAuAcGGAGuAGTST1362GGUGGACUUCUCUGAAUUUUCUA1297HBV:262U21 siRNAUGGACUUCUCUCAAUUUUCUA1363GGACUUCUCUGAAUUUUCUAGGG1298HBV:265U21 siRNAACUUCUCUGAAUUUUCUAGGG1364GAUGUGUCUGCGGCGUUUUAUCA1299HBV:380U21 siRNAUGUGUCUGCGGCGUUUUAUCA1365CAUCCUGCUGCUAUGCCUCAUCU1300HBV:413U21 siRNAUCCUGCUGCUAUGCCUCAUCU1366GGUAUGUUGCCCGUUUGUCGUCU1301HBV:462U21 siRNAUAUGUUGCCCGUUUGUCCUCU1367CGUGUGCACUUCGCUUCACCUCU1302HBV:1580U21 siRNAUGUGCACUUCGCUUCACCUCU1368CACUUCGCUUCACCUCUGCACGU1303HBV:1586U21 siRNACUUCGCUUCACCUCUGCACGU1369GGAGGCUGUAGGCAUAAAUUGGU1304HBV:1780U21 siRNAAGGCUGUAGGGAUAAAUUGGU1370CUCCAAGCUGUGCCUUGGGUGGC1305HBV:1874U21 siRNACGAAGCUGUGOCUUGGGUGGC1371CCCUAGAAGAAGAACUCGCUCGC1306HBV:2369U21 siRNACUAGAAGAAGAACUOOCUCGO1372GAAGAAGAACUCCCUCGCCUCGC1307HBV:2374U21 siRNAAGAAGAACUCCCUCG0CUCGC1373GGUGGACUUCUCUCAAUUUUCUA1297HBV:280L21 siRNA (262C)GAAAAUUGAGAGAAGUCCACC1374GGACUUCUCUCAAUUUUCUAGGG1298HBV:283L21 siRNA (265C)CUAGAAAAUUGAGAGAAGUCC1375GAUGUGUCUGCGGCGUUUUAUCA1299HBV:398L21 siRNA (380C)AUAAAACGCGGGAGACACAUC1376GAUCCUGCUGCUAUGCCUCAUCU1300HBV:431L21 siRNA (413C)AUGAGGCAUAGCAGCAGGAUG1377GGUAUGUUGCCGGUUUGUCCUCU1301HBV:480L21 siRNA (462C)AGGACAAACGGGCAACAUACC1378CGUGUGCACUUCGCUUCACCUCU1302HBV:1598L21 siRNA (1580C)AGGUGAAGCGAAGUGCACACG1379CACUUCGCUUCACCUCUGCACGU1303HBV:1604L21 siRNA (1586C)GUGCAGAGGUGAAGCGAAGUG1380GGAGGCUGUAGGCAUAAAUUGGU1304HBV:1798L21 siRNA (1780C)CAAUUUAUGCCUACAGCCUC01381CUCCAAGCUGUGCCUUGGGUGGC1305HBV:1892L21 siRNA (1874C)ACCCAAGGGACAGCUUGGAG1382CCCUAGAAGAAGAACUCCGUCGC1306HBV:2387L21 siRNA (2369C)GAGGGAGUUCUUCUUCUAGGG1383GAAGAAGAACUCCCUCGCCUCGC1307HBV:2392L21 siRNA (2374C)GAGGCGAGGGAGUUCUUOUUC1384AUOUUUUAACUCUCUUGAGGUGG1308HBV:260U21 siRNA invCUUUUAACUCUOUUGAGGUGG1385GGGAUCUUUUAACUCUCUUCAGG1309HBV:263U21 siRNA invGAUCUUUUAACUOUOUUGAGG1386AOUAUUUUGCGGCGUOUGUGUAG1310HBV:378U21 siRNA invUAUUUUGCGGCGUCUGUGUAG1387UCUACUCCGUAUCGUCGUCGUAC1311HBV:411U21 siRNA invUACUCCGUAUCGUCGUCCUAC1388UCUCCUGUUUGCCCGUUGUAUGG1312HBV:460U21 siRNA invUCGUGUUUGCCCGUUGUAUGG1389UCUCGACUUCGCUUCACGUGUGC1313HBV:1578U21 siRNA invUCGACUUCGCUUCACGUGUGC1390UGCACGUCUCCACUUCGCUUCAC1314HBV:1584U21 siRNA invGACGUCUCGACUUCGCUUCAC1391UGGUUAAAUACGGAUGUCGGAGG1315HBV:1778U21 siRNA invGUUAAAUACGGAUGUCGGAGG1392CGGUGGGUUCCGUGUCGAACCUC1316HBV:1872U21 siRNA invGUGGGUUCCGUGUCGAACCUC1393CGCUCCCUGAAGAAGAAGAUCCC1317HBV:2367U21 siRNA invCUCCCUGAAGAAGAAGAUCCC1394CGCUCCGCUCCCUGAAGAAGAAG1318HBV:2372U21 siRNA invCUCCGCUCCCUGAAGAAGAAG1395AUCUUUUAACUCUCUUGAGGUGG1308bogus HBV:282L21 siRNA (260C) invACCUGAAGAGAGUUAAAAGAU1396GGGAUCUUUUAACUCUCUUCAGG1309HBV:285L21 siRNA (263C) invUGAAGAGAGUUAAAAGAUCCG1397ACUAUUUUGCGGCGUCUGUGUAG1310HBV:400L21 siRNA (378C) invAGAGAGACGCCGGAAAAUAGU1398UCUACUCCGUAUCGUCGUCCUAC1311HBV:433L21 siRNA (411C) invAGGACGACGAUACGGAGUAGA1399UCUCCUGUUUGCCCGUUGUAUGG1312HBV:482L21 siRNA (460C) invAUAGAACGGGGAAAGAGGAGA1400UCUCCACUUCGCUUGACGUGUGC1313HBV:1600L21 siRNA (1578C) invAGACGUGAAGCGAAGUGGAGA1401UGCACGUCUCCACUUCGCUUCAC1314HBV:1606L21 siRNA (1584C) invGAAGCGAAGUGGAGACGUGCA1402UGGUUAAAUACGGAUGUCGGAGG1315HBV:1800L21 siRNA (1778C) invUCCGACAUCCGUAUUUAACCA1403CGGUGGGUUCCGUGUCGAACCUC1316HBV:1894L21 siRNA (1872C) invGGUUCGACACGGAACCCACCG1404CGCUCCCUCAAGAAGAAGAUCCC1317HBV:2389L21 siRNA (2367C) invGAUCUUCUUCUUGAGGGAGCG1405CGCUCCGCUCCCUCAAGAAGAAG1318HBV:2394L21 siRNA (2372C) invUCUUCUUGAGGGAGCGGAGCG1406GGUGGACUUCUCUCAAUUUUCUA1297HBV:262U21 siRNA stab4BuGGAcuucucucAAuuuucuAB1407GGACUUCUCUCAAUUUUCUAGGG1298HBV:265U21 siRNA stab4BAcuucucucAAuuuucuAGGGB1408GAUGUGUCUGCGGCGUUUUAUCA1299HBV:380U21 siRNA stab4BuGuGucuGcGGcGuuuuAucAB1409CAUCCUGCUGCUAUGCCUCAUCU1300HBV:413U21 siRNA stab4BuccuGcuGcuAuGccucAucuB1410GGUAUGUUGCCCGUUUGUCCUCU1301HBV:462U21 siRNAstab4BuAuGuuGcccGuuuGuccucuB1411CGUGUGCACUUCGCUUCACCUCU1302HBV:1586U21 siRNA stab4BuGuGcAcuucGcuucAccucuB1412CACUUCGCUUCACCUCUGCACGU1303HBV:1586U21 siRNA stab4BcuucGcuucAccucuGcAcGuB1413GGAGGCUGUAGGCAUAAAUUGGU1304HBV:1780U21 siRNA stab4BAGGcuGuAGGcAuAAAuuGGuB1414CUCCAAGCUGUGCCUUGGGUGGC1305HBV:1874U21 siRNAstab4BccAAGcuGuGccuuGGGuGGcB1415CCCUAGAAGAAGAACUCCCUCGC1306HBV:2369U21 siRNA stab4BcuAGAAGAAGAAcucccucGcB1416GAAGAAGAACUCCCUCGCCUCGC1307HBV:2374U21 siRNAstab4BAGAAGAAcucccucGccucGcB1417GGUGGACUUCUCUCAAUUUUCUA1297HBV:280L21 siRNA (262C) stab5GAAAAuuGAGAGAAGuccATST1418GGACUUCUCUCAAUUUUCUAGGG1298HBV:283L21 siRNA (265C) stab5cuAGAAAAGuuGAGAGAAGuTST1419GAUGUGUCUGCGGCGUUUUAUGA1299HBV:398L21 siRNA (380C) stab5AuAAAAcGccGcAGAcAcATST1420GAUCCUGCUGCUAUGCCUCAUCU1300HBV:431L21 siRNA (413C) stab5AuGAGGcAUAGcAGcAGGATST1421GGUAUGUUGCCCGUUUGUCCUCU1301HBV:480L21 siRNA (462C) stab5AGGAcAAAcGGGcAACAUATST1422CGUGUGCACUUCGCUUCACCUCU1302HBV:1598L21 siRNA (158CC) stab5AGGuGAAGcGAAGuGcAcATST1423CACUUCGCUUGACCUCUGCACGU1303HBV:1604L21 siRNA (1586C) stab5GuGcAGAGGuGAAGcGAAGTST1424GGAGGCUGUAGGGAUAAAUUGGU1304HBV:1798L21 siRNA (178CC) stab5cAAuuuAuGccuAcAGccuTST1425CUCCAAGCUGUGCGUUGGGUGGC1305HBV:1892L21 siRNA (1874C) stab5cAcccAAGGcAcAGCuuGGTST1426CCCUAGAAGAAGAACUCCCUCGC1306HBV:2387L21 siRNA (2369C) stab5GAGGGAGuucuucuucuAGTST1427GAAGAAGAACUCCCUCGCCUCGC1307HBV:2392L21 siRNA (2374C) stab5GAGGcGAGGGAGuucuucuTST1428GGUGGACUUCUCUGAAUUUUCUA1297HBV:262U21 siRNA inv stab4BAucuuuuAAcucucuucAGGuB1429GGACUUCUCUGAAUUUUCUAGGG1298HBV:265U21 siRNA inv stab4BGGGAucuuuuAAcucucuucAB1430GAUGUGUCUGCGGCGUUUUAUGA1299HBV:380U21 siRNA inv stab4BAcuAuuuuGcGGcGucucGuGuB1431CAUCCUGCUGCUAUGCCUGAUCU1300HBV:413U21 siRNA inv stab4BucuAcuccGuAucGucGuccuB1432GGUAUGUUGCCGGUUUGUCCUCU1301HBV:462U21 siRNA inv stab4BucuccuGuuuGcccGuuGuAuB1433CGUGUGGACUUCGCUUCACCUCU1302HBV:1580U21 siRNA inv stab4BucuccAcuucGcuucAcGuGuB1434GACUUCGCUUGACCUCUGGACGU1303HBV:1586U21 siRNA inv stab4BuGcAcGucuccAcuucGcuucB1435GGAGGCUGUAGGGAUAAAUUGGU1304HBV:1780U21 siRNA inv stab4BuGGuuAAAuAcGGAuGucGGAB1436CUCGAAGCUGUGCCUUGGGUGGC1305HBV:1874U21 siRNA inv stab4BcGGuGGGuuccGuGucGAAccB1437CCCUAGAAGAAGAACUCCCUCGC1306HBV:2369U21 siRNA inv stab4BcGcucccucAAGAAGAAGAucB1438GAAGAAGAACUCCCUCGCCUCGC1307HBV:2374U21 siRNA inv stab4BcGcuccGcuccucAAGAAGAB1439GGUGGACUUCUCUCAAUUUUCUA1297HBV:280L21 siRNA (262C) inv stab5AccuGAAGAGAGuuAAAAGTST1440GGACUUCUCUCAAUUUUCUAGGG1298HBV:283L21 siRNA (265C) inv stab5uGAAGAGAGuuAAAAGAuCTST1441GAUGUGUCUGCGGCGUUUUAUCA1299HBV:398L21 siRNA (380C) inv stab5AcAcAGAcGCccGcAAAAuATST1442CAUCCUGCUGCUAUGCGUCAUCU1300HBV:431L21 siRNA (413C) inv stab5AGGAcGAcGAuAcGGAGuATST1443GGUAUGUUGCCCGUUUGUCCUCU1301HBV:480L21 siRNA (462C) inv stab5AuAcGAAcGGGcAAAcAGGATST1444CGUGUGCACUUCGCUUCACCUCU1302HBV:1598L21 siRNA (158CC) inv stab5AcAcGuGAAGcGAAGuGGATST1445CACUUCGCUUCACCUCUGGACGU1303HBV:1604L21 siRNA (1586C) inv stab5GAAGcGAAGuGGAGAcGuGTST1446GGAGGCUGUAGGCAUAAAUUGGU1304HBV:1798L21 siRNA (1780C) inv stab5uccGAcAuccGuAuuuAAcTST1447CUCCAAGCUGUGCCUUGGGUGGC1305HBV:1892L21 siRNA (1874C) inv stab5GGuucGAcAcGGAAcccAcTST1448CCCUAGAAGAAGAACUCCCUCGC1306HBV:2387L21 siRNA (2369C) inv stab5GAucuucuucuuGAGGGAGTST1449GAAGAAGAACUCCCUCGCCUCGC1307HBV:2392L21 siRNA (2374C) inv stab5ucuucuuGAGGGAGcGGAGTST1450GGUGGACUUCUCUCAAUUUUCUA1297HBV:262U21 siRNA invAUCUUUUAACUCUCUUCAGGU1451GGACUUCUCUCAAUUUUCUAGGG1298HBV:265U21 siRNA invGGGAUCUUUUAACUCUCUUCA1452GAUGUGUCUGCGGCGUUUUAUCA1299HBV:380U21 siRNA invACUAUUUUGCGGCGUCUGUGU1453CAUCCUGCUGCUAUGCGUGAUCU1300HBV:413U21 siRNA invUCUACUCCGUAUCGUCGUCCU1454GGUAUGUUGCGCGUUUGUCCUCU1301HBV:462U21 siRNA invUCUCCUGUUUGCCCGUUGUAU1455CGUGUGCACUUCGCUUGACCUCU1302HBV:1580U21 siRNA invUCUCCACUUCGCUUGACGUGU1456GACUUCGCUUCACCUCUGCACGU1303HBV:1586U21 siRNA invUGGACGUCUCGACUUCGCUUC1457GGAGGCUGUAGGGAUAAAUUGGU1304HBV:1780U21 siRNA invUGGUUAAAUACGGAUGUCGGA1458CUCCAAGCUGUGCCUUGGGUGGC1305HBV:1874U21 siRNA invCGGUGGGUUCCGUGUCGAACG1459CGCUAGAAGAAGAACUCCCUCGC1306HBV:2369U21 siRNA invCGCUCCCUGAAGAAGAAGAUC1460GAAGAAGAACUCGCUCGCGUCGC1307HBV:2374U21 siRNA invCGCUCCGCUCCCUGAAGAAGA1461GGUGGACUUCUCUCAAUUUUCUA1297HBV:280L21 siRNA (262C) invCGACCUGAAGAGAGUUAAAAG1462GGACUUCUCUGAAUUUUCUAGGG1298HBV:283L21 siRNA (265C) invCCUGAAGAGAGUUAAAAGAUC1463GAUGUGUCUGCGGCGUUUUAUGA1299HBV:398L21 siRNA (380C) invCUACAGAGACGCCGGAAAAUA1464GAUCCUGCUGCUAUGCCUGAUCU1300HBV:431L21 siRNA (413C) invGUAGGACGACGAUACGGAGUA1465GGUAUGUUGCCCGUUUGUCGUCU1301HBV:480L21 siRNA (462C) invCGAUAGAACGGGGAAAGAGGA1466CGUGUGGACUUCGCUUGACGUCU1302HBV:1598L21 siRNA (1580C) invGGAGACGUGAAGCGAAGUGGA1467GACUUCGCUUGACCUCUGCACGU1303HBV:1604L21 siRNA (1586C) invGUGAAGCGAAGUGGAGACGUG1468GGAGGCUGUAGGGAUAAAUUGGU1304HBV:1798L21 siRNA (1780C) invCCUCCGAGAUCCGUAUUUAAC1469CUCGAAGCUGUGCGUUGGGUGGC1305HBV:1892L21 siRNA (1874C) invGAGGUUCGAGACGGAACCCAC1470CCCUAGAAGAAGAACUCCCUCGC1306HBV:2387L21 siRNA (2369C) invGGGAUCUUCUUCUUGAGGGAG1471GAAGAAGAACUCCCUCGCCUCGC1307HBV:2392L21 siRNA (2374C) invCUUCUUCUUGAGGGAGCGGAG1472GGUGGACUUCUCUGAAUUUUCUA1297HBV:262U21 siRNA stab7BuGGAcuucucucAAuuuucTTB1473GAUGUGUCUGCGGCGUUUUAUCA1299HBV:380U21 siRNA stab7BuGuGucuGcGGcGuuuuAuTTB1474CAUCCUGCUGCUAUGCCUCAUCU1300HBV:413U21 siRNA stab7BuccuGcuGcuAuGccucAuTTB1475AUCCUGCUGCUAUGCCUCAUCUU1294HBV:414U21 siRNA stab7BccuGcuGcuAuGccucAucTTB1476GGUAUGUUGCCCGUUUGUCCUCU1301HBV:462U21 siRNAstab7BuAuGuuGcccGuuuGuccuTTB1477CGUGUGCACUUCGCUUCACCUCU1302HBV:1580U21 siRNA stab7BuGuGcAcuucGcuucAccuTTB1478CACUUCGCUUCACCUCUGCACGU1303HBV:1580U21 siRNA stab7BcuccuGcuucAccucuGcAcTTB1479GGAGGCUGUAGGCAUAAAUUGGU1304HBV:1780U21 siRNA stab7BAGGcuGuAGGcAuAAAuuGTTB1480GGUGGACUUCUCUCAAUUUUCUA1297HBV:280L21 siRNA (262C)stab8gaaaauugagagaaguccaTST1481GAUGUGUCUGCGGCGUUUUAUCA1299HBV:398L21 siRNA (380C)stab8auaaaacgccgcagacacaTST1482GAUCCUGCUGCUAUGCCUCAUCU1300HBV:431L21 siRNA (413C)stab8augaggcauagcagcaggaTST1483AUCCUGCUGCUAUGCCUCAUCUU1294HBV:394L21 siRNA (414C)stab8gaugaggcauagcagcaggTST1484GGUAUGUUGCCCGUUUGUCCUCU1301HBV:480L21 siRNA (462C)stab8aggacaaacgggcaacauaTST1485CGUGUGCACUUCGCUUCACCUCU1302HBV:1598L21 siRNA (1580C)stab8aggugaagcgaagugcacaTST1486CACUUCGCUUCACCUCUGCACGU1303HBV:1604L21 siRNA (1586C)stab8gugcagaggugaagcgaagTST1487GGAGGCUGUAGGCAUAAAUUGGU1304HBV:1798L21 siRNA (1780C)stab8caauuuaugccuacagccuTST1488GGUGGACUUCUCUCAAUUUUCUA1297HBV:262U21 siRNA inv stab7BAucuuuuAAcucucuucAGTTB1489GAUGUGUCUGCGGCGUUUUAUCA1299HBV:380U21 siRNA invstab7BAcuAuuuuGcGGcGucuGuTTB1490CAUCCUGCUGCUAUGCCUCAUCU1300HBV:413U21 siRNA inv stab7BucuAcuccGuAucGucGucTTB1491AUCCUGCUGCUAUGCCUCAUCUU1294HBV:414U21 siRNA inv stab7BcuAcuccGuAucGucGuccTTB1492GGUAUGUUGCCCGUUUGUCCUCU1301HBV:462U21 siRNA inv stab7BucuccuGuuuGcccGuuGuTTB1493CGUGUGCACUUCGCUUCACCUCU1302HBV:1580U21 siRNA inv stab7BucuccAcuucGcuucAcGuTTB1494CACUUCGCUUCACCUCUGCACGU1303HBV:1586U21 siRNA inv stab7BuGcAcGucuccAcuucGcuTTB1495GGAGGCUGUAGGCAUAAAUUGGU1304HBV:1780U21 siRNA inv stab7BuGGuuAAAuAcGGAuGucGTTB1496GGUGGACUUCUCUGAAUUUUCUA1297HBV:280L21 siRNA (262C) inv stab8ccaccugaagagaguuaaaTST1497GAUGUGUCUGCGGCGUUUUAUCA1299HBV:398L21 siRNA (380C) inv stab8cuacacagacgccgcaaaaTST1498CAUCCUGCUGCUAUGCCUCAUCU1300HBV:431L21 siRNA (413C) inv stab8guaggacgacgauacggagTST1499AUCGUGCUGCUAUGCCUCAUCUU1294HBV:394L21 siRNA (414C) inv stab8ggacgacgauacggaguagTST1500GGUAUGUUGCCCGUUUGUCCUCU1301HBV:480L21 siRNA (462C) inv stab8ccauacaacgggcaaacagTST1501CGUGUGCACUUCGCUUGACGUCU1302HBV:1598L21 siRNA (1580C) inv stab8gcacacgugaagcgaagugTST1502GACUUCGCUUGACCUCUGCACGU1303HBV:1604L21 siRNA (1586C) inv stab8gugaagcgaaguggagacgTST1503GGAGGCUGUAGGCAUAAAUUGGU1304HBV:1798L21 siRNA (1780C) inv stab8ccuccgacauccguauuuaTST1504A = Adenosine G = Guanosine C = Cytidine U = Uridine T = Thymidine A = 2′-deoxy Adenosine G = 2′-deoxy Guanosine U = 2′-deoxy-2′-fluoro uridine C = 2′-deoxy-2′-fluoro cytidine a = 2′-O-methyl uridine C = 2′-O-methyl cytidine B = inverted deoxy abasic ribose L = glyceryl moiety S = phosphorothioate internucleotide linkage

[0327]

4

TABLE IV

A. 2.5 μmol Synthesis Cycle ABI 394 Instrument

Reagent
Equivalents
Amount
Wait Time* DNA
Wait Time* 2′-0-methyl
Wait Time*RNA

Phosphoramidites
6.5
163 μL
45 sec
2.5 min
7.5 min

S-Ethyl Tetrazole
23.8
238 μL
45 sec
2.5 min
7.5 min

Acetic Anhydride
100
233 μL
5 sec
5 sec
5 sec

N-Methyl
186
233 μL
5 sec
5 sec
5 sec

Imidazole

TCA
176
2.3 mL
21 sec
21 sec
21 sec

Iodine
11.2
1.7 mL
45 sec
45 sec
45 sec

Beaucage
12.9
645 μL
100 sec
300 sec
300 sec

Acetonitrile
NA
6.67 mL
NA
NA
NA

B. 0.2 μmol Synthesis Cycle ABI 394 Instrument

Reagent
Equivalents
Amount
Wait Time* DNA
Wait Time* 2′-O-methyl
Wait Time*RNA

Phosphoramidites
15
31 μL
45 sec
233 sec
465 sec

S-Ethyl Tetrazole
38.7
31 μL
45 sec
233 min
465 sec

Acetic Anhydride
655
124 μL
5 sec
5 sec
5 sec

N-Methyl
1245
124 μL
5 sec
5 sec
5 sec

Imidazole

TCA
700
732 μL
10 sec
10 sec
10 sec

Iodine
20.6
244 μL
15 sec
15 sec
15 sec

Beaucage
7.7
232 μL
100 sec
300 sec
300 sec

Acetonitrile
NA
2.64 mL
NA
NA
NA

C. 0.2 μmol Synthesis Cycle 96 well Instrument

Equivalents:DNA/
Amount: DNA/2′-O-

Wait Time* 2′-O-

Reagent
2′-O-methyl/Ribo
methyl/Ribo
Wait Time* DNA
methyl
Wait Time* Ribo

Phosphoramidites
22/33/66
40/60/120 μL
60 sec
180 sec
360 sec

S-Ethyl Tetrazole
70/105/210
40/60/120 μL
60 sec
180 min
360 sec

Acetic Anhydride
265/265/265
50/50/50 μL
10 sec
10 sec
10 sec

N-Methyl
502/502/502
50/50/50 μL
10 sec
10 sec
10 sec

Imidazole

TCA
238/475/475
250/500/500 μL
15 sec
15 sec
15 sec

Iodine
6.8/6.8/6.8
80/80/80 μL
30 sec
30 sec
30 sec

Beaucage
34/51/51
80/120/120
100 sec
200 sec
200 sec

Acetonitrile
NA
1150/1150/1150 μL
NA
NA
NA

Wait time does not include contact time during delivery.

Tandem synthesis utilizes double coupling of linker molecule

Number	Date	Country
60296876	Jun 2001	US
60335059	Oct 2001	US
60337055	Dec 2001	US
60358580	Feb 2002	US
60363124	Mar 2002	US
60386782	Jun 2002	US
60406784	Aug 2002	US
60408378	Sep 2002	US
60409293	Sep 2002	US

	Number	Date	Country
Parent	08193627	Feb 1994	US
Child	09436430	Nov 1999	US
Parent	07882712	May 1992	US
Child	08193627	Feb 1994	US

	Number	Date	Country
Parent	PCT/US02/09187	Mar 2002	US
Child	10244647	Sep 2002	US
Parent	09877478	Jun 2001	US
Child	10244647	Sep 2002	US
Parent	09696347	Oct 2000	US
Child	09877478	Jun 2001	US
Parent	09636385	Aug 2000	US
Child	09696347	Oct 2000	US
Parent	09531025	Mar 2000	US
Child	09636385	Aug 2000	US
Parent	09436430	Nov 1999	US
Child	09531025	Mar 2000	US

RNA interference mediated inhibition of hepatitis B virus (HBV) using short interfering nucleic acid (siNA)

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

PRIORITY

Provisional Applications (9)

Continuations (2)

Continuation in Parts (6)