# Patent

## Title
Modulation of hepatitis B virus (HBV) expression

## Abstract

Disclosed herein are antisense compounds and methods for decreasing HBV mRNA, DNA and protein expression. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate HBV-related diseases, disorders or conditions.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 14/137,111, filed Dec. 20, 2013, which is a continuation of U.S. patent application Ser. No. 13/452,703, filed Apr. 20, 2012, now U.S. Pat. No. 8,642,752, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/478,040, filed Apr. 21, 2011; U.S. Provisional Patent Application No. 61/478,038, filed Apr. 21, 2011; U.S. Provisional Patent Application No. 61/596,690, filed Feb. 8, 2012; and U.S. Provisional Patent Application No. 61/596,692, filed Feb. 8, 2012, each of which is incorporated herein by reference in its entirety.


SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0175USC2.5T25.txt Aug. 20, 2014, which is approximately 276 KB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
FIELD
In certain embodiments provided are methods, compounds, and compositions for inhibiting expression of hepatitis B virus (HBV) mRNA and protein in an animal. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate HBV-related diseases and disorders.
BACKGROUND
Hepatitis B is a viral disease transmitted parenterally by contaminated material such as blood and blood products, contaminated needles, sexually and vertically from infected or carrier mothers to their offspring. It is estimated by the World Health Organization that more than 2 billion people have been infected worldwide, with about 4 million acute cases per year, 1 million deaths per year, and 350-400 million chronic carriers (World Health Organization: Geographic Prevalence of Hepatitis B Prevalence, 2004. http://www.who.int/vaccines-surveillance/graphics/htmls/hepbprev.htm).
The virus, HBV, is a double-stranded hepatotropic virus which infects only humans and non-human primates. Viral replication takes place predominantly in the liver and, to a lesser extent, in the kidneys, pancreas, bone marrow and spleen (Hepatitis B virus biology. Microbiol Mol Biol Rev. 64: 2000; 51-68.). Viral and immune markers are detectable in blood and characteristic antigen-antibody patterns evolve over time. The first detectable viral marker is HBsAg, followed by hepatitis B e antigen (HBeAg) and HBV DNA. Titers may be high during the incubation period, but HBV DNA and HBeAg levels begin to fall at the onset of illness and may be undetectable at the time of peak clinical illness (Hepatitis B virus infection—natural history and clinical consequences. N Engl J Med. 350: 2004; 1118-1129). HBeAg is a viral marker detectable in blood and correlates with active viral replication, and therefore high viral load and infectivity (Hepatitis B e antigen—the dangerous end game of hepatitis B N Engl J Med. 347: 2002; 208-210). The presence of anti-HBsAb and anti-HBcAb (IgG) indicates recovery and immunity in a previously infected individual.
Currently the recommended therapies for chronic HBV infection by the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) include interferon alpha (INFα), pegylated interferon alpha-2a (Peg-IFN2a), entecavir, and tenofovir. The nucleoside and nucleotide therapies, entecavir and tenofovir, are successful at reducing viral load, but the rates of HBeAg seroconversion and HBsAg loss are even lower than those obtained using IFNα therapy. Other similar therapies, including lamivudine (3TC), telbivudine (LdT), and adefovir are also used, but for nucleoside/nucleotide therapies in general, the emergence of resistance limits therapeutic efficacy.
Thus, there is a need in the art to discover and develop new anti-viral therapies. Additionally, there is a need for new anti-HBV therapies capable of increasing HBeAg and HBsAg seroconversion rates. Recent clinical research has found a correlation between seroconversion and reductions in HBeAg (Fried et al (2008) Hepatology 47:428) and reductions in HBsAg (Moucari et al (2009) Hepatology 49:1151). Reductions in antigen levels may have allowed immunological control of HBV infection because high levels of antigens are thought to induce immunological tolerance. Current nucleoside therapies for HBV are capable of dramatic reductions in serum levels of HBV but have little impact on HBeAg and HBsAg levels.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of HBV expression (See U.S. Patent Publication Nos. 2008/0039418 and 2007/0299027). Antisense therapy differs from nucleoside therapy in that it can directly target the transcripts for the HBV antigens and thereby reduce serum HBeAg and HBsAg levels. Because of the multiple, overlapping transcripts produced upon HBV infection, there is also an opportunity for a single antisense oligomer to reduce HBV DNA in addition to both HBeAg and HBsAg. Therefore, antisense technology is emerging as an effective means for reducing the expression of certain gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of HBV.
SUMMARY
Provided herein are methods, compounds, and compositions for modulating expression of HBV mRNA and protein. In certain embodiments, compounds useful for modulating expression of HBV mRNA and protein are antisense compounds. In certain embodiments, the antisense compounds are antisense oligonucleotides.
In certain embodiments, modulation can occur in a cell or tissue. In certain embodiments, the cell or tissue is in an animal. In certain embodiments, the animal is a human. In certain embodiments, HBV mRNA levels are reduced. In certain embodiments, HBV DNA levels are reduced. In certain embodiments, HBV protein levels are reduced. In certain embodiments, HBV antigen levels are reduced. In certain embodiments, HBV s-antigen (HBsAg) levels are reduced. In certain embodiments, HBV e-antigen (HBeAg) levels are reduced. Such reduction can occur in a time-dependent manner or in a dose-dependent manner.
Also provided are methods, compounds, and compositions useful for preventing, treating, and ameliorating diseases, disorders, and conditions. In certain embodiments, such HBV related diseases, disorders, and conditions are liver diseases. In certain embodiments, such liver diseases, disorders, and conditions includes jaundice, liver cancer, liver inflammation, liver fibrosis, inflammation, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, and liver disease-related transplantation. In certain embodiments, such HBV related diseases, disorders, and conditions are hyperproliferative diseases, disorders, and conditions. In certain embodiments such hyperproliferative diseases, disorders, and conditions include cancer as well as associated malignancies and metastases. In certain embodiments, such cancers include liver cancer and hepatocellular cancer (HCC).
Such diseases, disorders, and conditions can have one or more risk factors, causes, or outcomes in common. Certain risk factors and causes for development of liver disease or a hyperproliferative disease include growing older; tobacco use; exposure to sunlight and ionizing radiation; contact with certain chemicals; infection with certain viruses and bacteria; certain hormone therapies; family history of cancer; alcohol use; and certain lifestyle choices including poor diet, lack of physical activity, and/or being overweight. Certain symptoms and outcomes associated with development of a liver disease or a hyperproliferative disease include but are not limited to: flu-like illness, weakness, aches, headache, fever, loss of appetite, diarrhea, jaundice, nausea and vomiting, pain over the liver area of the body, clay- or grey-colored stool, itching all over, and dark-colored urine.
In certain embodiments, methods of treatment include administering a HBV antisense compound to an individual in need thereof. In certain embodiments, methods of treatment include administering a HBV antisense oligonucleotide to an individual in need thereof.


DETAILED DESCRIPTION
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.
DEFINITIONS
Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are incorporated by reference for the portions of the document discussed herein, as well as in their entirety.
Unless otherwise indicated, the following terms have the following meanings:
“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH2)2—OCH3) refers to an O-methoxy-ethyl modification at the 2′ position of a furanose ring. A 2′-O-methoxyethyl modified sugar is a modified sugar.
“2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means a nucleoside comprising a 2′-MOE modified sugar moiety.
“2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position of the furanosyl ring other than H or OH. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications.
“3′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 3′-most nucleotide of a particular antisense compound.
“5′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 5′-most nucleotide of a particular antisense compound.
“5-methylcytosine” means a cytosine modified with a methyl group attached to the 5 position. A 5-methylcytosine is a modified nucleobase.
“About” means within ±7% of a value. For example, if it is stated, “the compounds affected at least about 70% inhibition of HBV”, it is implied that the HBV levels are inhibited within a range of 63% and 77%.
“Acceptable safety profile” means a pattern of side effects that is within clinically acceptable limits.
“Active pharmaceutical agent” means the substance or substances in a pharmaceutical composition that provide a therapeutic benefit when administered to an individual. For example, in certain embodiments an antisense oligonucleotide targeted to HBV is an active pharmaceutical agent.
“Active target region” means a target region to which one or more active antisense compounds is targeted. “Active antisense compounds” means antisense compounds that reduce target nucleic acid levels or protein levels.
“Acute hepatitis B infection” results when a person exposed to the hepatitis B virus begins to develop the signs and symptoms of viral hepatitis. This period of time, called the incubation period, is an average of 90 days, but could be as short as 45 days or as long as 6 months. For most people this infection will cause mild to moderate discomfort but will go away by itself because of the body's immune response succeeds in fighting the virus. However, some people, particularly those with compromised immune systems, such as persons suffering from AIDS, undergoing chemotherapy, taking immunosuppressant drugs, or taking steroids, have very serious problems as a result of the acute HBV infection, and go on to more severe conditions such as fulminant liver failure.
“Administered concomitantly” refers to the co-administration of two agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive.
“Administering” means providing a pharmaceutical agent to an individual, and includes, but is not limited to administering by a medical professional and self-administering.
“Agent” means an active substance that can provide a therapeutic benefit when administered to an animal. “First Agent” means a therapeutic compound described herein. For example, a first agent can be an antisense oligonucleotide targeting HBV. “Second agent” means a second therapeutic compound described herein (e.g. a second antisense oligonucleotide targeting HBV) and/or a non-HBV therapeutic compound.
“Amelioration” refers to a lessening of at least one indicator of the severity of a condition or disease. The severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art.
“Animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
“Antibody” refers to a molecule characterized by reacting specifically with an antigen in some way, where the antibody and the antigen are each defined in terms of the other. Antibody may refer to a complete antibody molecule or any fragment or region thereof, such as the heavy chain, the light chain, Fab region, and Fc region.
“Antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.
“Antisense compound” means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, antisense oligonucleotides, siRNAs, shRNAs, snoRNAs, miRNAs, and satellite repeats.
“Antisense inhibition” means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.
“Antisense mechanisms” are all those mechanisms involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.
“Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.
“Base complementarity” refers to the capacity for the precise base pairing of nucleobases of an antisense oligonucleotide with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases.
“Bicyclic sugar” means a furanose ring modified by the bridging of two non-geminal carbon atoms. A bicyclic sugar is a modified sugar.
“Body weight” refers to an animal's whole body weight, inclusive of all tissues including adipose tissue.
“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.
“cEt” or “constrained ethyl” means a bicyclic sugar moiety comprising a bridge connecting the 4′-carbon and the 2′-carbon, wherein the bridge has the formula: 4′-CH(CH3)—O-2′.
“Constrained ethyl nucleoside” (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)—O-2′ bridge.
“Chemically distinct region” refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2′-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2′-O-methoxyethyl modifications.
“Chimeric antisense compounds” means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits.
“Chronic hepatitis B infection” occurs when a person initially suffers from an acute infection but is then unable to fight off the infection. Whether the disease becomes chronic or completely resolves depends mostly on the age of the infected person. About 90% of infants infected at birth will progress to chronic disease. However, as a person ages, the risk of chronic infection decreases such that between 20%-50% of children and less than 10% of older children or adults will progress from acute to chronic infection. Chronic HBV infections are the primary treatment goal for embodiments of the present invention, although ASO compositions of the present invention are also capable of treating HBV-related conditions, such as inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, and more.
“Co-administration” means administration of two or more pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses administration in parallel or sequentially.
“Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.
“Comply” means the adherence with a recommended therapy by an individual.
“Comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
“Contiguous nucleobases” means nucleobases immediately adjacent to each other.
“Cure” means a method or course that restores health or a prescribed treatment for an illness.
“Deoxyribonucleotide” means a nucleotide having a hydrogen at the 2′ position of the sugar portion of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of substituents.
“Designing” or “Designed to” refer to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.
“Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, in drugs that are injected, the diluent may be a liquid, e.g. saline solution.
“Dosage unit” means a form in which a pharmaceutical agent is provided, e.g. pill, tablet, or other dosage unit known in the art.
“Dose” means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual. In other embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week or month.
“Dosing regimen” is a combination of doses designed to achieve one or more desired effects.
“Duration” means the period of time during which an activity or event continues. In certain embodiments, the duration of treatment is the period of time during which doses of a pharmaceutical agent are administered.
“Effective amount” in the context of modulating an activity or of treating or preventing a condition means the administration of that amount of active ingredient to a subject in need of such modulation, treatment or prophylaxis, either in a single dose or as part of a series, that is effective for modulation of that effect, or for treatment or prophylaxis or improvement of that condition. The effective amount will vary depending upon the health and physical condition of the subject to be treated, the taxonomic group of subjects to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors.
“Efficacy” means the ability to produce a desired effect.
“Expression” includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.
“Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.
“Fully modified motif” refers to an antisense compound comprising a contiguous sequence of nucleosides wherein essentially each nucleoside is a sugar modified nucleoside having uniform modification.
“Gapmer” means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.”
“Gap-widened” means an antisense compound having a gap segment of 12 or more contiguous 2′-deoxyribonucleotides positioned between 5′ and 3′ wing segments having from one to six nucleotides having modified sugar moieties.
“HBV” means mammalian hepatitis B virus, including human hepatitis B virus. The term encompasses geographical genotypes of hepatitis B virus, particularly human hepatitis B virus, as well as variant strains of geographical genotypes of hepatitis B virus.
“HBV antigen” means any hepatitis B virus antigen or protein, including core proteins such as “hepatitis B core antigen” or “HBcAG” and “hepatitis B E antigen” or “HBeAG” and envelope proteins such as “HBV surface antigen”, or “HBsAg” or “HBsAG”.
“Hepatitis B E antigen” or “HBeAg” or “HBeAG” is a secreted, non-particulate form of HBV core protein. HBV antigens HBeAg and HBcAg share primary amino acid sequences, so show cross-reactivity at the T cell level. HBeAg is not required for viral assembly or replication, although studies suggest they may be required for establishment of chronic infection. Neonatal infection with HBeAg-negative mutant often results in fulminant acute rather than chronic HBV infection (Terezawa et al (1991) Pediatr. Res. 29:5), whereas infection of young woodchucks with WHeAg-negative mutant results in a much lower rate of chronic WHV infection (Cote et al (2000) Hepatology 31:190). HBeAg may possibly function as a toleragen by inactivating core specific T cells through deletion or clonal anergy (Milich et al (1998) J. Immunol. 160:8102). There is a positive correlation between reduction of HBV viral load and antigens, and a decrease of expression, by T cells, of the inhibitory receptor programmed death-1 (PD-1; also known as PDCD1), a negative regulator of activated T cells, upon antiviral therapy and HBeAg seroconversion (Evans et al (2008) Hepatology 48:759).
“HBV mRNA” means any messenger RNA expressed by hepatitis B virus.
“HBV nucleic acid” or ‘HBV DNA” means any nucleic acid encoding HBV. For example, in certain embodiments, a HBV nucleic acid includes, without limitation, any viral DNA sequence encoding a HBV genome or portion thereof, any RNA sequence transcribed from a viral DNA including any mRNA sequence encoding a HBV protein.
“HBV protein” means any protein secreted by hepatitis B virus The term encompasses various HBV antigens, including core proteins such as “Hepatitis E antigen”, “HBeAg” or “HBeAG” and envelope proteins such as “HBV surface antigen”, or “HBsAg”.
“HBV surface antigen”, or “HBsAg”, or “HBsAG” is the envelope protein of infectious HBV viral particles but is also secreted as a non-infectious particle with serum levels 1000-fold higher than HBV viral particles. The serum levels of HBsAg in an infected person or animal can be as high as 1000 μg/mL (Kann and Gehrlich (1998) Topley & Wilson's Microbiology and Microbial Infections, 9th ed. 745). In acute HBV infections, the half-life of HBsAg in the serum, or serum t1/2, is 8.3 days (Chulanov et al (2003) J. Med. Virol. 69: 313). Internalization of HBsAg by myeloid dendritic cells inhibits up-regulation of co-stimulatory molecules (i.e. B7) and inhibits T cell stimulatory capacity (den Brouw et al (2008) Immunology 126:280), and dendritic cells from chronically infected patients also show deficits in expression of co-stimulatory molecules, secretion of IL-12, and stimulation of T cells in the presence of HBsAg (Zheng et al (2004) J. Viral Hepatitis 11:217). HBsAg specific CD8 cells from CHB patients show altered tetramer binding. These CD8 cells are not anergic but may have TCR topology that confers partial tolerance or ignorance (Reignat et al (2002) J. Exp. Med. 195:1089). Moreover, reduction in serum HBsAg >1 log at week 24 has a high predictive value (92%) for sustained virological response (SVR—defined as nondetectable HBV DNA by PCR at 1 year after treatment) during Peg-IFNα2a therapy (Moucari et al (2009) Hepatology 49:1151).
“Hepatitis B-related condition” or “HBV-related condition” means any disease, biological condition, medical condition, or event which is exacerbated, caused by, related to, associated with, or traceable to a hepatitis B infection, exposure, or illness. The term hepatitis B-related condition includes chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, liver disease related to transplantation, and conditions having symptoms which may include any or all of the following: flu-like illness, weakness, aches, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain over the liver area of the body, clay- or grey-colored stool, itching all over, and dark-colored urine, when coupled with a positive test for presence of a hepatitis B virus, a hepatitis B viral antigen, or a positive test for the presence of an antibody specific for a hepatitis B viral antigen.
“Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.
“Identifying an animal having an HBV infection” means identifying an animal having been diagnosed with an HBV; or, identifying an animal having any symptom of an HBV infection including, but not limited to chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, liver disease related to transplantation, and conditions having symptoms which may include any or all of the following: flu-like illness, weakness, aches, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain over the liver area of the body, clay- or grey-colored stool, itching all over, and dark-colored urine, when coupled with a positive test for presence of a hepatitis B virus, a hepatitis B viral antigen, or a positive test for the presence of an antibody specific for a hepatitis B viral antigen, when coupled with a positive test for presence of a hepatitis B virus, a hepatitis B viral antigen, or a positive test for the presence of an antibody specific for a hepatitis B viral antigen.
“Immediately adjacent” means there are no intervening elements between the immediately adjacent elements.
“Individual” means a human or non-human animal selected for treatment or therapy.
“Individual compliance” means adherence to a recommended or prescribed therapy by an individual.
“Induce”, “inhibit”, “potentiate”, “elevate”, “increase”, “decrease” or the like, generally denote quantitative differences between two states. Such terms may refer to a statistically significant difference between the two states. For example, “an amount effective to inhibit the activity or expression of HBV” means that the level of activity or expression of HBV in a treated sample will quantitatively differ, and may be statistically significant, from the level of HBV activity or expression in untreated cells. Such terms are applied to, for example, levels of expression, and levels of activity.
“Inhibiting HBV” means reducing the level or expression of an HBV mRNA, DNA and/or protein. In certain embodiments, HBV is inhibited in the presence of an antisense compound targeting HBV, including an antisense oligonucleotide targeting HBV, as compared to expression of HBV mRNA, DNA and/or protein levels in the absence of a HBV antisense compound, such as an antisense oligonucleotide.
“Inhibiting the expression or activity” refers to a reduction, blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity.
“Injection site reaction” means inflammation or abnormal redness of skin at a site of injection in an individual.
“Internucleoside linkage” refers to the chemical bond between nucleosides.
“Intraperitoneal administration” means administration through infusion or injection into the peritoneum.
“Intravenous administration” means administration into a vein.
“Lengthened” antisense oligonucleotides are those that have one or more additional nucleosides relative to an antisense oligonucleotide disclosed herein.
“Linked deoxynucleoside” means a nucleic acid base (A, G, C, T, U) substituted by deoxyribose linked by a phosphate ester to form a nucleotide.
“Linked nucleosides” means adjacent nucleosides linked together by an internucleoside linkage.
“Locked nucleic acid” or “LNA” or “LNA nucleosides” means nucleic acid monomers having a bridge connecting two carbon atoms between the 4′ and 2′ position of the nucleoside sugar unit, thereby forming a bicyclic sugar. Examples of such bicyclic sugar include, but are not limited to A) α-L-Methyleneoxy (4′-CH2—O-2′) LNA, (B) β-D-Methyleneoxy (4′-CH2—O-2′) LNA, (C) Ethyleneoxy (4′-(CH2)2—O-2′) LNA, (D) Aminooxy (4′-CH2—O—N(R)-2′) LNA and (E) Oxyamino (4′-CH2—N(R)—O-2′) LNA, as depicted below.




As used herein, LNA compounds include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the sugar wherein each of the bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(R1)(R2)]n—, —C(R1)═C(R2)—, —C(R1)═N—, —C(═NR1)—, —C(═O)—, —C(═S)—, —O—, —Si(R1)2—, —S(═O)x— and —N(R1)—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R1 and R2 is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, a heterocycle radical, a substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)2-J1), or sulfoxyl (S(═O)-J1); and each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group.
Examples of 4′-2′ bridging groups encompassed within the definition of LNA include, but are not limited to one of formulae: —[C(R1)(R2)]n—, —[C(R1)(R2)]n—O—, —C(R1R2)—N(R1)—O— or —C(R1R2)—O—N(R1)—. Furthermore, other bridging groups encompassed with the definition of LNA are 4′-CH2-2′,4′-(CH2)2-2′,4′-(CH2)3-2′,4′-CH2—O-2′,4′-(CH2)2—O-2′,4′-CH2—O—N(R1)-2′ and 4′-CH2—N(R1)—O-2′-bridges, wherein each R1 and R2 is, independently, H, a protecting group or C1-C12 alkyl.
Also included within the definition of LNA according to the invention are LNAs in which the 2′-hydroxyl group of the ribosyl sugar ring is connected to the 4′ carbon atom of the sugar ring, thereby forming a methyleneoxy (4′-CH2—O-2′) bridge to form the bicyclic sugar moiety. The bridge can also be a methylene (—CH2—) group connecting the 2′ oxygen atom and the 4′ carbon atom, for which the term methyleneoxy (4′-CH2—O-2′) LNA is used. Furthermore; in the case of the bicyclic sugar moiety having an ethylene bridging group in this position, the term ethyleneoxy (4′-CH2CH2—O-2′) LNA is used. α-L-methyleneoxy (4′-CH2—O-2′), an isomer of methyleneoxy (4′-CH2—O-2′) LNA is also encompassed within the definition of LNA, as used herein.
“Mismatch” or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.
“Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).
“Modified nucleobase” means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
“Modified nucleoside” means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase.
“Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.
“Modified oligonucleotide” means an oligonucleotide comprising at least one modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.
“Modified sugar” means substitution and/or any change from a natural sugar moiety.
“Monomer” refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.
“Motif” means the pattern of unmodified and modified nucleosides in an antisense compound.
“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA (2′-OH).
“Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.
“Non-complementary nucleobase” refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.
“Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).
“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid.
“Nucleobase complementarity” refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.
“Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.
“Nucleoside” means a nucleobase linked to a sugar.
“Nucleoside mimetic” includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics, e.g., non furanose sugar units. Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system. “Mimetic” refers to groups that are substituted for a sugar, a nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.
“Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
“Off-target effect” refers to an unwanted or deleterious biological effect associated with modulation of RNA or protein expression of a gene other than the intended target nucleic acid.
“Oligomeric compound” means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.
“Oligonucleoside” means an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.
“Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.
“Parenteral administration” means administration through injection (e.g., bolus injection) or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g., intrathecal or intracerebroventricular administration.
“Peptide” means a molecule formed by linking at least two amino acids by amide bonds. Without limitation, as used herein, “peptide” refers to polypeptides and proteins.
“Pharmaceutically acceptable carrier” means a medium or diluent that does not interfere with the structure of the oligonucleotide. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject.
“Pharmaceutically acceptable derivative” encompasses pharmaceutically acceptable salts, conjugates, prodrugs or isomers of the compounds described herein.
“Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.
“Pharmaceutical agent” means a substance that provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, an antisense oligonucleotide targeted to HBV is a pharmaceutical agent.
“Pharmaceutical composition” means a mixture of substances suitable for administering to a subject. For example, a pharmaceutical composition may comprise an antisense oligonucleotide and a sterile aqueous solution. In certain embodiments, a pharmaceutical composition shows activity in free uptake assay in certain cell lines.
“Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.
“Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.
“Prevention” or “preventing” refers to delaying or forestalling the onset or development of a condition or disease for a period of time from hours to days, preferably weeks to months.
“Prodrug” means a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
“Prophylactically effective amount” refers to an amount of a pharmaceutical agent that provides a prophylactic or preventative benefit to an animal.
“Recommended therapy” means a therapeutic regimen recommended by a medical professional for the treatment, amelioration, or prevention of a disease.
“Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.
“Ribonucleotide” means a nucleotide having a hydroxy at the 2′ position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.
“Salts” mean a physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.
“Segments” are defined as smaller or sub-portions of regions within a target nucleic acid.
“Seroconversion” is defined as serum HBeAg absence plus serum HBeAb presence, if monitoring HBeAg as the determinant for seroconversion, or defined as serum HBsAg absence, if monitoring HBsAg as the determinant for seroconversion, as determined by currently available detection limits of commercial ELISA systems.
“Shortened” or “truncated” versions of antisense oligonucleotides taught herein have one, two or more nucleosides deleted.
“Side effects” means physiological responses attributable to a treatment other than desired effects. In certain embodiments, side effects include, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.
“Significant,” as used herein means measurable or observable, e.g, a significant result, such as, a significant improvement or significant reduction generally refers to a measurable or observable result, such as a measurable or observable improvement or reduction.
“Sites,” as used herein, are defined as unique nucleobase positions within a target nucleic acid.
“Slows progression” means decrease in the development of the said disease.
“Specifically hybridizable” refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments. “Stringent hybridization conditions” or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.
“Statistically Significant,” as used herein means a measurable or observable parameter that is unlikely to occur by chance.
“Subcutaneous administration” means administration just below the skin.
“Subject” means a human or non-human animal selected for treatment or therapy.
“Target” refers to a protein, the modulation of which is desired.
“Target gene” refers to a gene encoding a target.
“Targeting” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.
“Target nucleic acid,” “target RNA,” “target RNA transcript” and “nucleic acid target” all mean a nucleic acid capable of being targeted by antisense compounds.
“Target region” means a portion of a target nucleic acid to which one or more antisense compounds is targeted.
“Target segment” means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. “5′ target site” refers to the 5′-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment.
“Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.
“Treatment” refers to administering a composition to effect an alteration or improvement of the disease or condition.
“Unmodified” nucleobases mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
“Unmodified nucleotide” means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).
“Validated target segment” is defined as at least an 8-nucleobase portion (i.e. 8 consecutive nucleobases) of a target region to which an active oligomeric compound is targeted.
“Wing segment” means a plurality of nucleosides modified to impart to an oligonucleotide properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.
Certain Embodiments
Certain embodiments provide methods, compounds, and compositions for inhibiting HBV mRNA expression.
Certain embodiments provide antisense compounds targeted to a HBV nucleic acid. In certain embodiments, the HBV nucleic acid is the sequences set forth in GENBANK Accession No. U95551.1 (incorporated herein as SEQ ID NO: 1).
In certain embodiments, the compounds provided herein are or comprise a modified oligonucleotide. In certain embodiments the compounds comprise a modified oligonucleotide and a conjugate as described herein. In certain embodiments, the modified oligonucleotide is a pharmaceutically acceptable derivative.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. The HBV target can have a sequence recited in SEQ ID NO: 1 or a portion thereof or a variant thereof.
In certain embodiments, the compounds or modified oligonucleotides provided herein are 10 to 30 linked nucleosides in length and are targeted to HBV. In certain embodiments, the HBV target has the sequence recited in SEQ ID NO: 1. In certain embodiments, such compounds or oligonucleotides target one of the following nucleotide regions of HBV: CCTGCTGGTGGCTCCAGTTC (SEQ ID NO: 1273); AGAGTCTAGACTCGTGGTGGACTTCTCTCA (SEQ ID NO: 1354); CATCCTGCTGCTATGCCTCATCTTCTT (SEQ ID NO: 1276); CAAGGTATGTTGCCCGT (SEQ ID NO: 1277); CCTATGGGAGTGGGCCTCAG (SEQ ID NO: 1279; TGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCG (SEQ ID NO: 1287); TATATGGATGATGTGGT (SEQ ID NO:1359); TGCCAAGTGTTTGCTGA (SEQ ID NO:1360); TGCCGATCCATACTGCGGAACTCCT (SEQ ID NO: 1361); CCGTGTGCACTTCGCTTCACCTCTGCACGT (SEQ ID NO:1352); GGAGGCTGTAGGCATAAATTGGT (SEQ ID NO:1353); CTTTTTCACCTCTGCCTA (SEQ ID NO:1362); TTCAAGCCTCCAAGCTGTGCCTTGG (SEQ ID NO:1363); AGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGG (SEQ ID NO: 1274); TGGATGTGTCTGCGGCGTTTTATCAT (SEQ ID NO: 1275); TGTATTCCCATCCCATC (SEQ ID NO: 1278); TGGCTCAGTTTACTAGTGC (SEQ ID NO: 1280); GGGCTTTCCCCCACTGT (SEQ ID NO: 1281); TCCTCTGCCGATCCATACTGCGGAACTCCT (SEQ ID NO: 1282); CGCACCTCTCTTTACGCGG (SEQ ID NO: 1283); GGAGTGTGGATTCGCAC (SEQ ID NO: 1284); or GAAGAAGAACTCCCTCGCCT (SEQ ID NO: 1285). In certain embodiments, such compounds or oligonucleotides have a gap segment of 9, 10, or more linked deoxynucleosides. In certain embodiments, such compounds or oligonucleotides have a gap segment of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 linked nucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 1, 2, 3, 4, 5, 6, 7, or 8 linked modified nucleosides. In certain embodiments, one or more modified nucleosides in the wing segment have a modified sugar. In certain embodiments, the modified sugar is a bicyclic sugar. In certain embodiments, the modified nucleoside is an LNA nucleoside. In certain embodiments, the modified nucleoside is a 2′-substituted nucleoside. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications. In certain embodiments, the modified nucleoside is a 2′-MOE nucleoside. In certain embodiments, the modified nucleoside is a constrained ethyl (cEt) nucleoside.
In certain embodiments, the compounds or compositions comprise modified oligonucleotides consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising a portion at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleobases complementary to an equal length portion of any of the nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1. In certain embodiments, such oligonucleotides have a gap segment of 9, 10, or more linked deoxynucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 1, 2, 3, 4, 5, 6, 7, or 8 linked modified nucleosides. In certain embodiments, one or more modified nucleosides in the wing segment have a modified sugar. In certain embodiments, the modified sugar is a bicyclic sugar. In certain embodiments, the modified nucleoside is an LNA nucleoside. In certain embodiments, the modified nucleoside is a 2′-substituted nucleoside. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications. In certain embodiments, the modified nucleoside is a 2′-MOE nucleoside. In certain embodiments, the modified nucleoside is a constrained ethyl (cEt) nucleoside. In certain embodiments, each modified nucleoside in each wing segment is independently a 2′-MOE nucleoside or a nucleoside with a bicyclic sugar modification such as a constrained ethyl (cEt) nucleoside or LNA nucleoside.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases of any of SEQ ID NOs: 5-310, 321-802, 804-1272, 1288-1350, 1364-1372, 1375, 1376, or 1379. In certain embodiments, such oligonucleotides have a gap segment of 9, 10, or more linked deoxynucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 1-5, 1-4, 1-3, 2-5, 2-4 or 2-3 linked modified nucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 1, 2, 3, 4, 5, 6, 7, or 8 linked modified nucleosides. In certain embodiments, one or more modified nucleosides in the wing segment have a modified sugar. In certain embodiments, the modified sugar is a bicyclic sugar. In certain embodiments, the modified nucleoside is an LNA nucleoside. In certain embodiments, the modified nucleoside is a 2′-substituted nucleoside. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications. In certain embodiments, the modified nucleoside is a 2′-MOE nucleoside. In certain embodiments, the modified nucleoside is a constrained ethyl (cEt) nucleoside.
In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, or 4 sugar modified nucleosides. In certain embodiments, each sugar modified nucleoside is independently a 2′-MOE nucleoside or a nucleoside with a bicyclic sugar moiety such as a constrained ethyl (cEt) nucleoside or LNA nucleoside. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 9 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, or 5 sugar modified nucleosides. In certain embodiments, each sugar modified nucleoside is independently a 2′-MOE nucleoside or a bicyclic nucleoside such as a constrained ethyl (cEt) nucleoside or LNA nucleoside.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases of any of SEQ ID NOs: 5-310, 321-802, 804-1272, 1288-1350, 1364-1372, 1375, 1376, or 1379. In certain embodiments, such oligonucleotides have a gap segment of 10 or more linked deoxynucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 1-5, 1-4, 1-3, 2-5, 2-4 or 2-3 linked modified nucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 2, 3, 4, 5, 6, 7, or 8 linked modified nucleosides. In certain embodiments, one or more modified nucleosides in the wing segment have a modified sugar. In certain embodiments, the modified sugar is a bicyclic sugar. In certain embodiments, the modified nucleoside is an LNA nucleoside. In certain embodiments, the modified nucleoside is a 2′-substituted nucleoside. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications. In certain embodiments, the modified nucleoside is a 2′-MOE nucleoside.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases of any of SEQ ID NOs: 5, 15, 16, 33, 39-95, 123-135, 163-175, 180-310, 321-406, 413-455, 461-802, or 804-1272. In certain embodiments, such oligonucleotides have a gap segment of 10 or more linked deoxynucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 1-5, 1-4, 1-3, 2-5, 2-4 or 2-3 linked modified nucleosides. In certain embodiments, one or more modified nucleosides in the wing segment have a modified sugar. In certain embodiments, the modified sugar is a bicyclic sugar. In certain embodiments, the modified nucleoside is an LNA nucleoside. In certain embodiments, the modified nucleoside is a 2′-substituted nucleoside. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications. In certain embodiments, the modified nucleoside is a 2′-MOE nucleoside.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases of any of SEQ ID NOs: 6-14, 17-32, 34-38, 96-122, 136-162, 176-179, 407-412, 456-462, 523-538. In certain embodiments, such oligonucleotides have a gap segment of 10 or more linked deoxynucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 1-5, 1-4, 1-3, 2-5, 2-4 or 2-3 linked modified nucleosides. In certain embodiments, one or more modified nucleosides in the wing segment have a modified sugar. In certain embodiments, the modified nucleoside is a 2′-substituted nucleoside. In certain embodiments, the modified nucleoside is a 2′-MOE nucleoside. In certain embodiments, the modified oligonucleotide is 14 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-3 or 2 sugar modified nucleosides.
In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 9 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, or 5 sugar modified nucleosides, such as 2′-MOE nucleosides.
In certain embodiments, the modified oligonucleotide is 17 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3-4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, or 6 sugar modified nucleosides, such as 2′-MOE nucleosides.
In certain embodiments, the modified oligonucleotide is 18 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 3-5, or 4 sugar modified nucleosides.
In certain embodiments, the modified oligonucleotide is 20 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, or 5 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, 6, 7, or 8 sugar modified nucleosides, such as 2′-MOE nucleosides.
In certain embodiments, the compounds or compositions comprise a salt of the modified oligonucleotide.
In certain embodiments, the compounds or compositions further comprise a pharmaceutically acceptable carrier or diluent.
In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary to SEQ ID NO: 1, as measured over the entirety of the modified oligonucleotide.
In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary to any one of SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, as measured over the entirety of the modified oligonucleotide.
In certain embodiments, the compound or modified oligonucleotide is single-stranded.
In certain embodiments, the modified oligonucleotide consists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 18 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 14 linked nucleosides.
In certain embodiments, at least one internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.
In certain embodiments, at least one nucleoside of the modified oligonucleotide comprises a modified sugar. In certain embodiments, at least one modified sugar comprises a 2′-O-methoxyethyl group (2′—O(CH2)2—OCH3). In certain embodiments, the modified sugar comprises a 2′-O—CH3 group.
In certain embodiments, at least one modified sugar is a bicyclic sugar. In certain embodiments, at least one modified sugar the bicyclic sugar comprises a 4′-(CH2)n—O-2′ bridge, wherein n is 1 or 2. In certain embodiments, the bicyclic sugar comprises a 4′-CH2—O-2′ bridge. In certain embodiments, the bicyclic sugar comprises a 4′-CH(CH3)—O-2′ bridge.
In certain embodiments, at least one nucleoside of said modified oligonucleotide comprises a modified nucleobase. In certain embodiments, the modified nucleobase is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of a single-stranded modified oligonucleotide.
In certain embodiments, the modified oligonucleotide comprises: a) a gap segment consisting of linked deoxynucleosides; b) a 5′ wing segment consisting of linked nucleosides; and c) a 3′ wing segment consisting of linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment and each nucleoside of each wing segment comprises a modified sugar.
In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of 10 linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of three linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar and/or a constrained ethyl (cEt) sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine. In some aspects, each of the three linked nucleosides of the 5′ wing segment is a 2′-O-methoxyethyl sugar and each of the three linked nucleosides of the 3′ wing segment is a constrained ethyl (cEt) sugar. In other aspects, the three linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and the three linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a constrained ethyl (cEt) sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction. In other aspects, the three linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and the three linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction.
In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of 10 linked deoxynucleosides, the 5′ wing segment consisting of two linked nucleosides, the 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar and a constrained ethyl (cEt) sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine. In some aspects, the two linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and the four linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction.
In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of 9 linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of two linked nucleosides; the five linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the two linked nucleosides of the 3′ wing segment are a 2′-O-methoxyethyl sugar and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of 8 linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of five linked nucleosides; the three linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the five linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of 8 linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of five linked nucleosides; each of the three linked nucleosides of the 5′ wing segment is a constrained ethyl (cEt) sugar; each of the five linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 9 linked deoxynucleosides, the 5′ wing segment consisting of four linked nucleosides, the 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar and a constrained ethyl (cEt) sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine. In some aspects, the four linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and the four linked nucleosides of the 3′ wing segment are a 2′-O-methoxyethyl sugar, constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction.
In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 9 linked deoxynucleosides, the 5′ wing segment consisting of four linked nucleosides, the 3′ wing segment consisting of four linked nucleosides; the four linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the four linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, and a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 9 linked deoxynucleosides, the 5′ wing segment consisting of four linked nucleosides, the 3′ wing segment consisting of four linked nucleosides; the four linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 8 linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of four linked nucleosides; the five linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 8 linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of four linked nucleosides; the five linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 7 linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of five linked nucleosides; the five linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the five linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, and a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 7 linked deoxynucleosides, the 5′ wing segment consisting of six linked nucleosides, the 3′ wing segment consisting of four linked nucleosides; the six linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 7 linked deoxynucleosides, the 5′ wing segment consisting of six linked nucleosides, the 3′ wing segment consisting of four linked nucleosides; the six linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of 10 linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of five linked nucleosides, each nucleoside of each wing segment comprises a constrained ethyl (cEt) sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of 10 linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of five linked nucleosides, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine, wherein the five linked nucleosides of the 5′ wing are a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and each of the five linked nucleosides of the 3′ wing are a 2′-O-methoxyethyl sugar.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of three linked nucleosides. In some aspects, the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; each of the three linked nucleosides of the 5′ wing segment is a 2′-O-methoxyethyl sugar and each of the three linked nucleosides of the 3′ wing segment is a constrained ethyl (cEt) sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine residue is a 5-methylcytosine. In other aspects, the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; the three linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the three linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a constrained ethyl (cEt) sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; each internucleoside linkage is a phosphorothioate linkage; and each cytosine residue is a 5-methylcytosine. In other aspects, the three linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and the three linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of two linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides. In some aspects, the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; the two linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the four linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, 2′-O-methoxyethyl sugar, constrained ethyl (cEt) sugar, and 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; each internucleoside linkage is a phosphorothioate linkage; and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) gap segment consisting of 9 linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of two linked nucleosides, wherein the five linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the two linked nucleosides of the 3′ wing segment are a 2′-O-methoxyethyl sugar and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 8 linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides, wherein the three linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the five linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 8 linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides, wherein each of the three linked nucleosides of the 5′ wing segment is a constrained ethyl (cEt) sugar; each of the five linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 9 linked deoxynucleosides; b) a 5′ wing segment consisting of four linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar and a constrained ethyl (cEt) sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine. In some aspects, the four linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and the four linked nucleosides of the 3′ wing segment are a 2′-O-methoxyethyl sugar, constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 9 linked deoxynucleosides; b) a 5′ wing segment consisting of four linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides, wherein the four linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the four linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, and a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 9 linked deoxynucleosides; b) a 5′ wing segment consisting of four linked nucleosides; and c) the 3′ wing segment consisting of four linked nucleosides, wherein the four linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 8 linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides, wherein the five linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 8 linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides, wherein the five linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 7 linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides, wherein the five linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the five linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, and a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 7 linked deoxynucleosides; b) a 5′ wing segment consisting of six linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides, wherein the six linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 7 linked deoxynucleosides; b) a 5′ wing segment consisting of six linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides, wherein the six linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 10 linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides, each nucleoside of each wing segment comprises a constrained ethyl (cEt) sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 10 linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine, wherein the five linked nucleosides of the 5′ wing are a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and each of the five linked nucleosides of the 3′ wing are a 2′-O-methoxyethyl sugar.
In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of five linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of two linked nucleosides, the 3′ wing segment consisting of eight linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of eight linked nucleosides, the 3′ wing segment consisting of two linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of seven linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of seven linked nucleosides, the 3′ wing segment consisting of three linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of four linked nucleosides, the 3′ wing segment consisting of six linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of six linked nucleosides, the 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 18 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of four linked nucleosides, the 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of two linked nucleosides, the 3′ wing segment consisting of six linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of six linked nucleosides, the 3′ wing segment consisting of two linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of four linked nucleosides, the 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of three linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of five linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of three linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of two linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of two linked nucleosides, the 3′ wing segment consisting of five linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of four linked nucleosides, the 3′ wing segment consisting of three linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 14 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of two linked nucleosides, the 3′ wing segment consisting of two linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of two linked nucleosides; and c) a 3′ wing segment consisting of eight linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of eight linked nucleosides; and c) a 3′ wing segment consisting of two linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of seven linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of seven linked nucleosides; and c) a 3′ wing segment consisting of three linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of four linked nucleosides; and c) a 3′ wing segment consisting of six linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of six linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 18 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of four linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of two linked nucleosides; and c) a 3′ wing segment consisting of six linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of six linked nucleosides; and c) a 3′ wing segment consisting of two linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of four linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of three linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of three linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of two linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of two linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of four linked nucleosides; and c) a 3′ wing segment consisting of three linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 14 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of two linked nucleosides; and c) a 3′ wing segment consisting of two linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.
In certain embodiments, the provided methods, compounds, and compositions inhibit HBV mRNA expression and/or DNA levels and or protein levels and/or antigen levels.
Another embodiment provides a method for treating a HBV-related diseases, disorders, and conditions in a mammal, the method comprising administering a therapeutically effective amount of any pharmaceutical composition as described above to a mammal in need thereof, so as to treat the HBV-related diseases, disorders, and condition. In related embodiments, the mammal is a human and the HBV-related disease, disorder, and condition is a hepatitis B virus infection from a human hepatitis B virus. More particularly, the human hepatitis B virus may be any of the human geographical genotypes: A (Northwest Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, Korea, China, Japan, Polynesia, Vietnam); D (Mediterranean area, Middle East, India); E (Africa); F (Native Americans, Polynesia); G (United States, France); or H (Central America).
In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid is complementary within the following nucleotide regions of SEQ ID NO: 1:1-20, 10-29, 10-56, 13-38, 13-35, 19-38, 25-47, 25-50, 25-56, 43-68, 43-63, 55-74, 58-73, 58-74, 58-77, 58-79, 58-80, 58-84, 59-74, 59-75, 59-80, 60-75, 60-76, 60-79, 61-76, 61-77, 61-80, 62-77, 63-84, 68-114, 101-123, 98-123, 113-138, 116-138, 131-150, 137-162, 152-186, 158-177, 167-186, 191-215, 196-224, 196-215, 196-218, 199-228, 199-218, 199-224, 200-224, 205-224, 206-228, 218-237, 224-243, 233-264, 242-263, 243-262, 244-263, 245-274; 245-260, 245-264, 246-266, 247-266, 247-269, 247-270, 245-267, 251-267, 245-266, 250-269, 251-266, 251-268, 251-269, 245-269, 245-266, 245-261, 250-265, 250-266, 250-267, 250-268, 250-269, 251-270, 252-267, 253-268, 253-269, 253-272, 253-274, 254-269, 254-270, 254-274, 255-270, 255-271, 255-274, 255-401, 255-400, 255-274, 255-273, 255-272, 255-271, 256-271, 256-275, 255-276, 256-272, 256-276, 253-275, 256-279, 257-276, 258-273, 259-274, 260-279, 262-281, 262-321, 262-315, 262-312, 265-312, 266-288, 266-291, 266-285, 281-321, 281-303, 290-321, 290-312, 292-311, 290-312, 293-312, 293-315, 293-321, 296-321, 302-321, 324-343, 339-361, 339-367, 348-367, 342-367, 358-392, 358-378, 360-392, 360-383, 360-388, 360-385, 362-381, 366-388, 369-388, 366-385, 366-392, 370-389, 370-392, 380-399, 382-401, 384-433, 384-400, 384-401, 385-401, 405-424, 409-428, 405-428, 411-426, 411-427, 411-430, 411-431, 411-437, 412-431, 411-426, 411-427, 412-428, 412-431, 412-427, 413-433, 413-432, 413-428, 413-429, 413-432, 413-433, 414-427, 415-427, 414-429, 414-430, 414-433, 415-428, 415-429, 415-430, 415-431, 415-434, 416-431, 416-432, 416-429, 416-435, 417-432, 417-433, 417-436, 418-433, 418-435, 418-434, 418-437, 419-435, 419-434, 420-435, 419-432, 419-434, 421-436, 422-437, 422-441, 423-436, 425-465, 454-473, 454-472, 457-476, 457-472, 457-473, 454-476, 455-472, 457-485, 458-485, 458-483, 458-477, 458-473, 459-485, 460-485, 463-498, 463-485, 466-485, 463-482, 457-491, 458-491, 459-491, 460-491, 463-491, 466-491, 472-491, 472-493, 473-492, 475-491, 459-494, 460-494, 463-494, 466-494, 467-498, 472-494, 475-494, 457-473, 457-472, 458-494, 454-494, 457-494, 457-473, 485-513, 470-493, 476-519, 485-519, 500-519, 512-534, 512-550, 524-546, 536-559, 548-567, 548-570, 550-570, 548-594, 554-573, 548-576, 560-594, 584-606, 611-645, 617-363, 623-642, 617-645, 639-754, 639-658, 639-654, 641-656, 642-657, 643-658, 642-754, 653-672, 662-685, 665-685, 665-689, 668-687, 670-754, 670-706, 670-685, 670-686, 670-689, 671-690, 671-691, 671-686, 671-687, 672-693, 672-697, 672-707, 672-687, 672-688, 673-688, 674-693, 678-693, 679-694, 679-707, 679-698, 679-701, 679-702, 679-707, 680-695, 680-699, 679-699, 681-706, 681-696, 682-697, 682-706, 682-707, 682-702, 682-701, 683-698, 684-699, 685-700, 686-701, 687-754, 688-704, 689-709, 689-710, 690-705, 679-705, 679-710, 679-706, 690-710, 691-710, 690-754, 690-706, 684-703, 687-705, 687-702, 687-703, 687-706, 688-703, 688-704, 688-705, 689-704, 689-705, 689-708, 690-705, 690-706, 690-709, 691-706, 692-711, 693-716, 693-712, 695-715, 697-716, 697-716, 690-716, 724-746, 724-752, 724-754, 724-758, 733-752, 738-754, 738-753, 739-758, 739-754, 739-775, 739-754, 740-754, 742-785, 742-773, 757-776, 757-785, 790-815, 793-812, 811-833, 811-844, 814-833, 811-906, 820-839, 822-844, 822-867, 823-842, 845-864, 845-867, 854-906, 845-909, 845-906, 854-876, 863-882, 863-885, 878-900, 887-906, 899-918, 899-933, 899-958, 905-927, 905-933, 914-933, 936-958, 936-955, 945-964, 951-970, 951-985, 951-1044, 951-1024, 951-1056, 951-997, 960-985, 963-1044, 963-1024, 963-997, 972-1015, 1025-1044, 1031-1056, 1037-1056, 1046-1083, 1049-1068, 1070-1089, 1070-1095, 1082-1101, 1081-1134, 1081-1143, 1082-1101, 1088-1107, 1088-1134, 1094-1119, 1097-1119, 1112-1134, 1118-1143, 1118-1146, 1088-1146, 1121-1140, 1127-1146, 1127-1193, 1150-1193, 1156-1187, 1165-1187, 1170-1192, 1171-1191, 1172-1191, 1176-1192, 1176-1285, 1177-1192, 1176-1191, 1203-1297, 1206-1228, 1206-1255, 1209-1228, 1215-1255, 1245-1265, 1251-1280, 1262-1285, 1251-1285, 1259-1296, 1259-1290, 1259-1287, 1261-1296, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1296, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1264-1297, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1296, 1269-1284, 1269-1285, 1269-1288, 1270-1285, 1271-1290, 1271-1296, 1277-1296, 1261-1290, 1262-1290, 1268-1290, 1263-1305, 1259-1305, 1259-1305, 1266-1305, 1259-1302, 1275-1294, 1281-1306, 1281-1324, 1281-1336, 1282-1301, 1286-1306, 1290-1324, 1293-1318, 1290-1324, 1293-1315, 1296-1315, 1311-1336, 1311-1333, 1326-1345, 1353-1381, 1359-1378, 1395-1414, 1498-1532, 1498-1523, 1498-1535, 1510-1529, 1515-1535, 1515-1563, 1515-1596, 1515-1605, 1515-1602, 1515-1540, 1515-1535, 1518-1605, 1518-1602, 1518-1537, 1521-1563, 1521-1540, 1550-1655, 1550-1563, 1550-1569, 1553-1578, 1553-1599, 1553-1590, 1565-1584, 1571-1595, 1577-1605, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1578-1598, 1571-1598, 1579-1594, 1579-1594, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1580-1605, 1580-1602, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1582-1602, 1553-1655, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1586-1652, 1642-1664, 1651-1720, 1651-1673, 1655-1679, 1695-1720, 1716-1738, 1743-1763, 1743-1768, 1764-1783, 1773-1792, 1777-1796, 1777-1800, 1778-1800, 1778-1793, 1778-1794, 1778-1797, 1779-1798, 1779-1797, 1779-1796, 1779-1795, 1779-1794, 1780-1799, 1780-1796, 1780-1795, 1781-1797, 1781-1796, 1781-1796, 1781-1800, 1781-1797, 1782-1799, 1782-1797, 1782-1798, 1783-1798, 1783-1799, 1784-1800, 1784-1799, 1779-1799, 1778-1889, 1778-1794, 1779-1795, 1780-1799, 1785-1800, 1794-1813, 1806-1837, 1806-1828, 1806-1825, 1809-1828, 1812-1843, 1812-1837, 1812-1831, 1815-1843, 1815-1844, 1815-1840, 1815-1834, 1818-1837, 1821-1840, 1821-1844, 1821-1837, 1822-1843, 1822-1839, 1822-1837, 1823-1843, 1823-1838, 1824-1839, 1827-1846, 1861-1884, 1861-1880, 1865-1885, 1866-1881, 1867-1882, 1867-1886, 1868-1883, 1869-1885, 1869-1884, 1870-1885, 1871-1886, 1872-1887, 1874-1889, 1876-1895, 1888-1914, 1888-1908, 1891-1910, 1891-1914, 1895-1938, 1895-1935, 1913-1935, 1898-1920, 1907-1929, 1913-1935, 1918-1934, 1919-1938, 1919-1934, 1921-1934, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2368-2393, 2381-2397, 2368-2394, 2379-2394, 2381-2396, 2368-2397, 2368-2396, 2420-2439, 2458-2476, 2459-2478, 2819-2838, 2818-2838, 2873-2892, and 3161-3182.
In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid target the following nucleotide regions of SEQ ID NO: 1:1-20, 10-29, 10-56, 13-38, 13-35, 19-38, 25-47, 25-50, 25-56, 43-68, 43-63, 55-74, 58-73, 58-74, 58-77, 58-79, 58-80, 58-84, 59-74, 59-75, 59-80, 60-75, 60-76, 60-79, 61-76, 61-77, 61-80, 62-77, 63-84, 68-114, 101-123, 98-123, 113-138, 116-138, 131-150, 137-162, 152-186, 158-177, 167-186, 191-215, 196-224, 196-215, 196-218, 199-228, 199-218, 199-224, 200-224, 205-224, 206-228, 218-237, 224-243, 233-264, 242-263, 243-262, 244-263, 245-274; 245-260, 245-264, 246-266, 247-266, 247-269, 247-270, 245-267, 251-267, 245-266, 250-269, 251-266, 251-268, 251-269, 245-269, 245-266, 245-261, 250-265, 250-266, 250-267, 250-268, 250-269, 251-270, 252-267, 253-268, 253-269, 253-272, 253-274, 254-269, 254-270, 254-274, 255-270, 255-271, 255-274, 255-401, 255-400, 255-274, 255-273, 255-272, 255-271, 256-271, 256-275, 255-276, 256-272, 256-276, 253-275, 256-279, 257-276, 258-273, 259-274, 260-279, 262-281, 262-321, 262-315, 262-312, 265-312, 266-288, 266-291, 266-285, 281-321, 281-303, 290-321, 290-312, 292-311, 290-312, 293-312, 293-315, 293-321, 296-321, 302-321, 324-343, 339-361, 339-367, 348-367, 342-367, 358-392, 358-378, 360-392, 360-383, 360-388, 360-385, 362-381, 366-388, 369-388, 366-385, 366-392, 370-389, 370-392, 380-399, 382-401, 384-433, 384-400, 384-401, 385-401, 405-424, 409-428, 405-428, 411-426, 411-427, 411-430, 411-431, 411-437, 412-431, 411-426, 411-427, 412-428, 412-431, 412-427, 413-433, 413-432, 413-428, 413-429, 413-432, 413-433, 414-427, 415-427, 414-429, 414-430, 414-433, 415-428, 415-429, 415-430, 415-431, 415-434, 416-431, 416-432, 416-429, 416-435, 417-432, 417-433, 417-436, 418-433, 418-435, 418-434, 418-437, 419-435, 419-434, 420-435, 419-432, 419-434, 421-436, 422-437, 422-441, 423-436, 425-465, 454-473, 454-472, 457-476, 457-472, 457-473, 454-476, 455-472, 457-485, 458-485, 458-483, 458-477, 458-473, 459-485, 460-485, 463-498, 463-485, 466-485, 463-482, 457-491, 458-491, 459-491, 460-491, 463-491, 466-491, 472-491, 472-493, 473-492, 475-491, 459-494, 460-494, 463-494, 466-494, 467-498, 472-494, 475-494, 457-473, 457-472, 458-494, 454-494, 457-494, 457-473, 485-513, 470-493, 476-519, 485-519, 500-519, 512-534, 512-550, 524-546, 536-559, 548-567, 548-570, 550-570, 548-594, 554-573, 548-576, 560-594, 584-606, 611-645, 617-363, 623-642, 617-645, 639-754, 639-658, 639-654, 641-656, 642-657, 643-658, 642-754, 653-672, 662-685, 665-685, 665-689, 668-687, 670-754, 670-706, 670-685, 670-686, 670-689, 671-690, 671-691, 671-686, 671-687, 672-693, 672-697, 672-707, 672-687, 672-688, 673-688, 674-693, 678-693, 679-694, 679-707, 679-698, 679-701, 679-702, 679-707, 680-695, 680-699, 679-699, 681-706, 681-696, 682-697, 682-706, 682-707, 682-702, 682-701, 683-698, 684-699, 685-700, 686-701, 687-754, 688-704, 689-709, 689-710, 690-705, 679-705, 679-710, 679-706, 690-710, 691-710, 690-754, 690-706, 684-703, 687-705, 687-702, 687-703, 687-706, 688-703, 688-704, 688-705, 689-704, 689-705, 689-708, 690-705, 690-706, 690-709, 691-706, 692-711, 693-716, 693-712, 695-715, 697-716, 697-716, 690-716, 724-746, 724-752, 724-754, 724-758, 733-752, 738-754, 738-753, 739-758, 739-754, 739-775, 739-754, 740-754, 742-785, 742-773, 757-776, 757-785, 790-815, 793-812, 811-833, 811-844, 814-833, 811-906, 820-839, 822-844, 822-867, 823-842, 845-864, 845-867, 854-906, 845-909, 845-906, 854-876, 863-882, 863-885, 878-900, 887-906, 899-918, 899-933, 899-958, 905-927, 905-933, 914-933, 936-958, 936-955, 945-964, 951-970, 951-985, 951-1044, 951-1024, 951-1056, 951-997, 960-985, 963-1044, 963-1024, 963-997, 972-1015, 1025-1044, 1031-1056, 1037-1056, 1046-1083, 1049-1068, 1070-1089, 1070-1095, 1082-1101, 1081-1134, 1081-1143, 1082-1101, 1088-1107, 1088-1134, 1094-1119, 1097-1119, 1112-1134, 1118-1143, 1118-1146, 1088-1146, 1121-1140, 1127-1146, 1127-1193, 1150-1193, 1156-1187, 1165-1187, 1170-1192, 1171-1191, 1172-1191, 1176-1192, 1176-1285, 1177-1192, 1176-1191, 1203-1297, 1206-1228, 1206-1255, 1209-1228, 1215-1255, 1245-1265, 1251-1280, 1262-1285, 1251-1285, 1259-1296, 1259-1290, 1259-1287, 1261-1296, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1296, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1264-1297, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1296, 1269-1284, 1269-1285, 1269-1288, 1270-1285, 1271-1290, 1271-1296, 1277-1296, 1261-1290, 1262-1290, 1268-1290, 1263-1305, 1259-1305, 1259-1305, 1266-1305, 1259-1302, 1275-1294, 1281-1306, 1281-1324, 1281-1336, 1282-1301, 1286-1306, 1290-1324, 1293-1318, 1290-1324, 1293-1315, 1296-1315, 1311-1336, 1311-1333, 1326-1345, 1353-1381, 1359-1378, 1395-1414, 1498-1532, 1498-1523, 1498-1535, 1510-1529, 1515-1535, 1515-1563, 1515-1596, 1515-1605, 1515-1602, 1515-1540, 1515-1535, 1518-1605, 1518-1602, 1518-1537, 1521-1563, 1521-1540, 1550-1655, 1550-1563, 1550-1569, 1553-1578, 1553-1599, 1553-1590, 1565-1584, 1571-1595, 1577-1605, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1578-1598, 1571-1598, 1579-1594, 1579-1594, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1580-1605, 1580-1602, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1582-1602, 1553-1655, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1586-1652, 1642-1664, 1651-1720, 1651-1673, 1655-1679, 1695-1720, 1716-1738, 1743-1763, 1743-1768, 1764-1783, 1773-1792, 1777-1796, 1777-1800, 1778-1800, 1778-1793, 1778-1794, 1778-1797, 1779-1798, 1779-1797, 1779-1796, 1779-1795, 1779-1794, 1780-1799, 1780-1796, 1780-1795, 1781-1797, 1781-1796, 1781-1796, 1781-1800, 1781-1797, 1782-1799, 1782-1797, 1782-1798, 1783-1798, 1783-1799, 1784-1800, 1784-1799, 1779-1799, 1778-1889, 1778-1794, 1779-1795, 1780-1799, 1785-1800, 1794-1813, 1806-1837, 1806-1828, 1806-1825, 1809-1828, 1812-1843, 1812-1837, 1812-1831, 1815-1843, 1815-1844, 1815-1840, 1815-1834, 1818-1837, 1821-1840, 1821-1844, 1821-1837, 1822-1843, 1822-1839, 1822-1837, 1823-1843, 1823-1838, 1824-1839, 1827-1846, 1861-1884, 1861-1880, 1865-1885, 1866-1881, 1867-1882, 1867-1886, 1868-1883, 1869-1885, 1869-1884, 1870-1885, 1871-1886, 1872-1887, 1874-1889, 1876-1895, 1888-1914, 1888-1908, 1891-1910, 1891-1914, 1895-1938, 1895-1935, 1913-1935, 1898-1920, 1907-1929, 1913-1935, 1918-1934, 1919-1938, 1919-1934, 1921-1934, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2368-2393, 2381-2397, 2368-2394, 2379-2394, 2381-2396, 2368-2397, 2368-2396, 2420-2439, 2458-2476, 2459-2478, 2819-2838, 2818-2838, 2873-2892, and 3161-3182.
In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid is complementary within the HBV pre-S1 second portion gene region corresponding to nucleotide region 1-1932 of SEQ ID NO: 1. In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid is complementary within the HBV pre-S1 first portion gene region corresponding to nucleotide region 2831-3182 of SEQ ID NO: 1.
In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid target the HBV pre-S1 second portion gene region corresponding to nucleotide region 1-1932 of SEQ ID NO: 1. In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid target the HBV pre-S1 first portion gene region corresponding to nucleotide region 2831-3182 of SEQ ID NO: 1.
In certain embodiments, antisense compounds or oligonucleotides target a region of a HBV nucleic acid. In certain embodiments, such compounds or oligonucleotides targeted to a region of a HBV nucleic acid have a contiguous nucleobase portion that is complementary to an equal length nucleobase portion of the region. For example, the portion can be at least an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases portion complementary to an equal length portion of a region recited herein. In certain embodiments, such compounds or oligonucleotide target the following nucleotide regions of SEQ ID NO: 1: 1-20, 10-29, 10-56, 13-38, 13-35, 19-38, 25-47, 25-50, 25-56, 43-68, 43-63, 55-74, 58-73, 58-74, 58-77, 58-79, 58-80, 58-84, 59-74, 59-75, 59-80, 60-75, 60-76, 60-79, 61-76, 61-77, 61-80, 62-77, 63-84, 68-114, 101-123, 98-123, 113-138, 116-138, 131-150, 137-162, 152-186, 158-177, 167-186, 191-215, 196-224, 196-215, 196-218, 199-228, 199-218, 199-224, 200-224, 205-224, 206-228, 218-237, 224-243, 233-264, 242-263, 243-262, 244-263, 245-274; 245-260, 245-264, 246-266, 247-266, 247-269, 247-270, 245-267, 251-267, 245-266, 250-269, 251-266, 251-268, 251-269, 245-269, 245-266, 245-261, 250-265, 250-266, 250-267, 250-268, 250-269, 251-270, 252-267, 253-268, 253-269, 253-272, 253-274, 254-269, 254-270, 254-274, 255-270, 255-271, 255-274, 255-401, 255-400, 255-274, 255-273, 255-272, 255-271, 256-271, 256-275, 255-276, 256-272, 256-276, 253-275, 256-279, 257-276, 258-273, 259-274, 260-279, 262-281, 262-321, 262-315, 262-312, 265-312, 266-288, 266-291, 266-285, 281-321, 281-303, 290-321, 290-312, 292-311, 290-312, 293-312, 293-315, 293-321, 296-321, 302-321, 324-343, 339-361, 339-367, 348-367, 342-367, 358-392, 358-378, 360-392, 360-383, 360-388, 360-385, 362-381, 366-388, 369-388, 366-385, 366-392, 370-389, 370-392, 380-399, 382-401, 384-433, 384-400, 384-401, 385-401, 405-424, 409-428, 405-428, 411-426, 411-427, 411-430, 411-431, 411-437, 412-431, 411-426, 411-427, 412-428, 412-431, 412-427, 413-433, 413-432, 413-428, 413-429, 413-432, 413-433, 414-427, 415-427, 414-429, 414-430, 414-433, 415-428, 415-429, 415-430, 415-431, 415-434, 416-431, 416-432, 416-429, 416-435, 417-432, 417-433, 417-436, 418-433, 418-435, 418-434, 418-437, 419-435, 419-434, 420-435, 419-432, 419-434, 421-436, 422-437, 422-441, 423-436, 425-465, 454-473, 454-472, 457-476, 457-472, 457-473, 454-476, 455-472, 457-485, 458-485, 458-483, 458-477, 458-473, 459-485, 460-485, 463-498, 463-485, 466-485, 463-482, 457-491, 458-491, 459-491, 460-491, 463-491, 466-491, 472-491, 472-493, 473-492, 475-491, 459-494, 460-494, 463-494, 466-494, 467-498, 472-494, 475-494, 457-473, 457-472, 458-494, 454-494, 457-494, 457-473, 485-513, 470-493, 476-519, 485-519, 500-519, 512-534, 512-550, 524-546, 536-559, 548-567, 548-570, 550-570, 548-594, 554-573, 548-576, 560-594, 584-606, 611-645, 617-363, 623-642, 617-645, 639-754, 639-658, 639-654, 641-656, 642-657, 643-658, 642-754, 653-672, 662-685, 665-685, 665-689, 668-687, 670-754, 670-706, 670-685, 670-686, 670-689, 671-690, 671-691, 671-686, 671-687, 672-693, 672-697, 672-707, 672-687, 672-688, 673-688, 674-693, 678-693, 679-694, 679-707, 679-698, 679-701, 679-702, 679-707, 680-695, 680-699, 679-699, 681-706, 681-696, 682-697, 682-706, 682-707, 682-702, 682-701, 683-698, 684-699, 685-700, 686-701, 687-754, 688-704, 689-709, 689-710, 690-705, 679-705, 679-710, 679-706, 690-710, 691-710, 690-754, 690-706, 684-703, 687-705, 687-702, 687-703, 687-706, 688-703, 688-704, 688-705, 689-704, 689-705, 689-708, 690-705, 690-706, 690-709, 691-706, 692-711, 693-716, 693-712, 695-715, 697-716, 697-716, 690-716, 724-746, 724-752, 724-754, 724-758, 733-752, 738-754, 738-753, 739-758, 739-754, 739-775, 739-754, 740-754, 742-785, 742-773, 757-776, 757-785, 790-815, 793-812, 811-833, 811-844, 814-833, 811-906, 820-839, 822-844, 822-867, 823-842, 845-864, 845-867, 854-906, 845-909, 845-906, 854-876, 863-882, 863-885, 878-900, 887-906, 899-918, 899-933, 899-958, 905-927, 905-933, 914-933, 936-958, 936-955, 945-964, 951-970, 951-985, 951-1044, 951-1024, 951-1056, 951-997, 960-985, 963-1044, 963-1024, 963-997, 972-1015, 1025-1044, 1031-1056, 1037-1056, 1046-1083, 1049-1068, 1070-1089, 1070-1095, 1082-1101, 1081-1134, 1081-1143, 1082-1101, 1088-1107, 1088-1134, 1094-1119, 1097-1119, 1112-1134, 1118-1143, 1118-1146, 1088-1146, 1121-1140, 1127-1146, 1127-1193, 1150-1193, 1156-1187, 1165-1187, 1170-1192, 1171-1191, 1172-1191, 1176-1192, 1176-1285, 1177-1192, 1176-1191, 1203-1297, 1206-1228, 1206-1255, 1209-1228, 1215-1255, 1245-1265, 1251-1280, 1262-1285, 1251-1285, 1259-1296, 1259-1290, 1259-1287, 1261-1296, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1296, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1264-1297, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1296, 1269-1284, 1269-1285, 1269-1288, 1270-1285, 1271-1290, 1271-1296, 1277-1296, 1261-1290, 1262-1290, 1268-1290, 1263-1305, 1259-1305, 1259-1305, 1266-1305, 1259-1302, 1275-1294, 1281-1306, 1281-1324, 1281-1336, 1282-1301, 1286-1306, 1290-1324, 1293-1318, 1290-1324, 1293-1315, 1296-1315, 1311-1336, 1311-1333, 1326-1345, 1353-1381, 1359-1378, 1395-1414, 1498-1532, 1498-1523, 1498-1535, 1510-1529, 1515-1535, 1515-1563, 1515-1596, 1515-1605, 1515-1602, 1515-1540, 1515-1535, 1518-1605, 1518-1602, 1518-1537, 1521-1563, 1521-1540, 1550-1655, 1550-1563, 1550-1569, 1553-1578, 1553-1599, 1553-1590, 1565-1584, 1571-1595, 1577-1605, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1578-1598, 1571-1598, 1579-1594, 1579-1594, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1580-1605, 1580-1602, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1582-1602, 1553-1655, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1586-1652, 1642-1664, 1651-1720, 1651-1673, 1655-1679, 1695-1720, 1716-1738, 1743-1763, 1743-1768, 1764-1783, 1773-1792, 1777-1796, 1777-1800, 1778-1800, 1778-1793, 1778-1794, 1778-1797, 1779-1798, 1779-1797, 1779-1796, 1779-1795, 1779-1794, 1780-1799, 1780-1796, 1780-1795, 1781-1797, 1781-1796, 1781-1796, 1781-1800, 1781-1797, 1782-1799, 1782-1797, 1782-1798, 1783-1798, 1783-1799, 1784-1800, 1784-1799, 1779-1799, 1778-1889, 1778-1794, 1779-1795, 1780-1799, 1785-1800, 1794-1813, 1806-1837, 1806-1828, 1806-1825, 1809-1828, 1812-1843, 1812-1837, 1812-1831, 1815-1843, 1815-1844, 1815-1840, 1815-1834, 1818-1837, 1821-1840, 1821-1844, 1821-1837, 1822-1843, 1822-1839, 1822-1837, 1823-1843, 1823-1838, 1824-1839, 1827-1846, 1861-1884, 1861-1880, 1865-1885, 1866-1881, 1867-1882, 1867-1886, 1868-1883, 1869-1885, 1869-1884, 1870-1885, 1871-1886, 1872-1887, 1874-1889, 1876-1895, 1888-1914, 1888-1908, 1891-1910, 1891-1914, 1895-1938, 1895-1935, 1913-1935, 1898-1920, 1907-1929, 1913-1935, 1918-1934, 1919-1938, 1919-1934, 1921-1934, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2368-2393, 2381-2397, 2368-2394, 2379-2394, 2381-2396, 2368-2397, 2368-2396, 2420-2439, 2458-2476, 2459-2478, 2819-2838, 2818-2838, 2873-2892, and 3161-3182.
In certain embodiments, antisense compounds or oligonucleotides target a region of a HBV nucleic acid. In certain embodiments, such compounds or oligonucleotides targeted to a region of a HBV nucleic acid have a contiguous nucleobase portion that is complementary to an equal length nucleobase portion of the region. For example, the portion can be at least an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases portion complementary to an equal length portion of a region recited herein. In certain embodiments, such compounds or oligonucleotide target the following nucleotide regions of SEQ ID NO: 1: 58-73, 58-74, 58-77, 59-74, 60-75, 61-76, 62-77, 245-274; 245-260, 250-265, 251-266, 252-267, 253-268, 254-269, 255-270, 256-271, 256-272, 258-273, 259-274, 380-399, 382-401, 411-437, 411-427, 411-426, 412-427, 413-428, 413-432, 414-429, 415-430, 416-431, 417-432, 418-433, 419-434, 420-435, 421-436, 422-437, 457-472, 458-473, 639-754, 639-658, 639-654, 641-656, 642-657, 643-658, 670-754, 670-706, 670-685, 671-686, 672-687, 673-688, 678-693, 679-694, 680-695, 681-706, 681-696, 682-697, 683-698, 684-699, 685-700, 686-701, 687-702, 688-703, 689-704, 690-705, 691-706, 738-754, 738-753, 739-754, 1176-1285, 1176-1191, 1177-1192, 1261-1285, 1261-1276, 1262-1277, 1263-1278, 1264-1279, 1265-1280, 1266-1281, 1267-1282, 1268-1283, 1269-1284, 1270-1285, 1577-1606, 1577-1592, 1578-1593, 1579-1594, 1580-1595, 1581-1596, 1582-1597, 1583-1598, 1584-1599, 1585-1600, 1586-1601, 1587-1602, 1588-1603, 1589-1604, 1590-1605, 1591-1606, 1778-1889, 1778-1800, 1778-1793, 1779-1794, 1780-1799, 1780-1796, 1780-1795, 1781-1796, 1782-1797, 1783-1798, 1784-1799, 1785-1800, 1822-1839, 1822-1837, 1823-1838, 1824-1839, 1866-1881, 1867-1882, 1868-1883, 1869-1884, 1870-1885, 1871-1886, 1872-1887, or 1874-1889, and wherein at least one nucleoside of the compound or modified oligonucleotide comprises at least one 2′-O-methoxyethyl or constrained ethyl (cEt) sugar.
In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid is complementary within the following nucleotide regions of SEQ ID NO: 1: 58-73, 58-74, 58-77, 59-74, 59-75, 60-75, 60-76, 61-76, 61-77, 62-77, 253-272, 253-269, 254-270, 255-271, 256-272, 411-437, 411-426, 411-427, 411-430, 412-427, 412-428, 412-431, 413-428, 413-429, 413-432, 414-429, 414-430, 414-433, 415-430, 415-431, 415-434, 416-431, 416-432, 416-435, 417-432, 417-433, 417-436, 418-433, 418-434, 418-437, 457-472, 457-473, 458-473, 670-706, 670-685, 670-686, 671-686, 671-687, 672-687, 672-688, 673-688, 687-702, 687-703, 687-706, 688-703, 688-704, 689-704, 689-705, 690-705, 690-706, 691-706, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1269-1284, 1269-1285, 1270-1285, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1579-1594, 1579-1594, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1778-1800, 1778-1793, 1778-1794, 1778-1797, 1779-1794, 1779-1795, 1779-1798, 1780-1795, 1780-1796, 1780-1799, 1781-1796, 1781-1797, 1781-1800, 1782-1797, 1782-1798, 1783-1798, 1783-1799, 1784-1799, and 1784-1800.
In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid target the following nucleotide regions of SEQ ID NO: 1: 58-73, 58-74, 58-77, 59-74, 59-75, 60-75, 60-76, 61-76, 61-77, 62-77, 253-272, 253-269, 254-270, 255-271, 256-272, 411-437, 411-426, 411-427, 411-430, 412-427, 412-428, 412-431, 413-428, 413-429, 413-432, 414-429, 414-430, 414-433, 415-430, 415-431, 415-434, 416-431, 416-432, 416-435, 417-432, 417-433, 417-436, 418-433, 418-434, 418-437, 457-472, 457-473, 458-473, 670-706, 670-685, 670-686, 671-686, 671-687, 672-687, 672-688, 673-688, 687-702, 687-703, 687-706, 688-703, 688-704, 689-704, 689-705, 690-705, 690-706, 691-706, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1269-1284, 1269-1285, 1270-1285, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1579-1594, 1579-1594, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1778-1800, 1778-1793, 1778-1794, 1778-1797, 1779-1794, 1779-1795, 1779-1798, 1780-1795, 1780-1796, 1780-1799, 1781-1796, 1781-1797, 1781-1800, 1782-1797, 1782-1798, 1783-1798, 1783-1799, 1784-1799, and 1784-1800.
In certain embodiments, antisense compounds or oligonucleotides target a region of a HBV nucleic acid. In certain embodiments, such compounds or oligonucleotides targeted to a region of a HBV nucleic acid have a contiguous nucleobase portion that is complementary to an equal length nucleobase portion of the region. For example, the portion can be at least an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases portion complementary to an equal length portion of a region recited herein. In certain embodiments, such compounds or oligonucleotide target the following nucleotide regions of SEQ ID NO: 1: 1-20, 10-29, 10-56, 13-38, 13-35, 19-38, 25-47, 25-50, 25-56, 43-68, 43-63, 55-74, 58-73, 58-74, 58-77, 58-79, 58-80, 58-84, 59-74, 59-75, 59-80, 60-75, 60-76, 60-79, 61-76, 61-77, 61-80, 62-77, 63-84, 68-114, 101-123, 98-123, 113-138, 116-138, 131-150, 137-162, 152-186, 158-177, 167-186, 191-215, 196-224, 196-215, 196-218, 199-228, 199-218, 199-224, 200-224, 205-224, 206-228, 218-237, 224-243, 233-264, 242-263, 243-262, 244-263, 245-264, 246-266, 247-266, 247-269, 247-270, 245-267, 251-267, 245-266, 250-269, 251-268, 251-269, 245-269, 245-266, 245-261, 250-266, 250-267, 250-268, 250-269, 251-270, 253-269, 253-272, 253-274, 254-270, 254-274, 255-271, 255-274, 255-401, 255-400, 255-274, 255-273, 255-272, 255-271, 256-275, 255-276, 256-272, 256-276, 253-275, 256-279, 257-276, 260-279, 262-281, 262-321, 262-315, 262-312, 265-312, 266-288, 266-291, 266-285, 281-321, 281-303, 290-321, 290-312, 292-311, 290-312, 293-312, 293-315, 293-321, 296-321, 302-321, 324-343, 339-361, 339-367, 348-367, 342-367, 358-392, 358-378, 360-392, 360-383, 360-388, 360-385, 362-381, 366-388, 369-388, 366-385, 366-392, 370-389, 370-392, 384-433, 384-400, 384-401, 385-401, 405-424, 409-428, 405-428, 411-426, 411-427, 411-430, 411-431, 411-437, 412-431, 411-426, 411-427, 412-428, 412-431, 412-427, 413-433, 413-432, 413-428, 413-429, 413-432, 413-433, 414-427, 415-427, 414-429, 414-430, 414-433, 415-428, 415-429, 415-430, 415-431, 415-434, 416-431, 416-432, 416-429, 416-435, 417-432, 417-433, 417-436, 418-433, 418-435, 418-434, 418-437, 419-435, 419-434, 420-435, 419-432, 419-434, 422-441, 423-436, 425-465, 454-473, 454-472, 457-476, 457-472, 457-473, 454-476, 455-472, 457-485, 458-485, 458-483, 458-477, 458-473, 459-485, 460-485, 463-498, 463-485, 466-485, 463-482, 457-491, 458-491, 459-491, 460-491, 463-491, 466-491, 472-491, 472-493, 473-492, 475-491, 459-494, 460-494, 463-494, 466-494, 467-498, 472-494, 475-494, 457-473, 457-472, 458-494, 454-494, 457-494, 457-473, 485-513, 470-493, 476-519, 485-519, 500-519, 512-534, 512-550, 524-546, 536-559, 548-567, 548-570, 550-570, 548-594, 554-573, 548-576, 560-594, 584-606, 611-645, 617-363, 623-642, 617-645, 642-754, 653-672, 662-685, 665-685, 665-689, 668-687, 670-706, 670-685, 670-686, 670-689, 671-690, 671-691, 671-686, 671-687, 672-693, 672-697, 672-707, 672-687, 672-688, 673-688, 674-693, 679-707, 679-698, 679-701, 679-702, 679-707, 680-699, 679-699, 682-706, 682-707, 682-702, 682-701, 687-754, 688-704, 689-709, 689-710, 690-705, 679-705, 679-710, 679-706, 690-710, 691-710, 690-754, 690-706, 684-703, 687-705, 687-702, 687-703, 687-706, 688-703, 688-704, 688-705, 689-704, 689-705, 689-708, 690-705, 690-706, 690-709, 691-706, 692-711, 693-716, 693-712, 695-715, 697-716, 697-716, 690-716, 724-746, 724-752, 724-754, 724-758, 733-752, 738-754, 739-758, 739-754, 739-775, 739-754, 740-754, 742-785, 742-773, 757-776, 757-785, 790-815, 793-812, 811-833, 811-844, 814-833, 811-906, 820-839, 822-844, 822-867, 823-842, 845-864, 845-867, 854-906, 845-909, 845-906, 854-876, 863-882, 863-885, 878-900, 887-906, 899-918, 899-933, 899-958, 905-927, 905-933, 914-933, 936-958, 936-955, 945-964, 951-970, 951-985, 951-1044, 951-1024, 951-1056, 951-997, 960-985, 963-1044, 963-1024, 963-997, 972-1015, 1025-1044, 1031-1056, 1037-1056, 1046-1083, 1049-1068, 1070-1089, 1070-1095, 1082-1101, 1081-1134, 1081-1143, 1082-1101, 1088-1107, 1088-1134, 1094-1119, 1097-1119, 1112-1134, 1118-1143, 1118-1146, 1088-1146, 1121-1140, 1127-1146, 1127-1193, 1150-1193, 1156-1187, 1165-1187, 1170-1192, 1171-1191, 1172-1191, 1176-1192, 1177-1192, 1176-1191, 1203-1297, 1206-1228, 1206-1255, 1209-1228, 1215-1255, 1245-1265, 1251-1280, 1262-1285, 1251-1285, 1259-1296, 1259-1290, 1259-1287, 1261-1296, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1296, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1264-1297, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1296, 1269-1284, 1269-1285, 1269-1288, 1270-1285, 1271-1290, 1271-1296, 1277-1296, 1261-1290, 1262-1290, 1268-1290, 1263-1305, 1259-1305, 1259-1305, 1266-1305, 1259-1302, 1275-1294, 1281-1306, 1281-1324, 1281-1336, 1282-1301, 1286-1306, 1290-1324, 1293-1318, 1290-1324, 1293-1315, 1296-1315, 1311-1336, 1311-1333, 1326-1345, 1353-1381, 1359-1378, 1395-1414, 1498-1532, 1498-1523, 1498-1535, 1510-1529, 1515-1535, 1515-1563, 1515-1596, 1515-1605, 1515-1602, 1515-1540, 1515-1535, 1518-1605, 1518-1602, 1518-1537, 1521-1563, 1521-1540, 1550-1655, 1550-1563, 1550-1569, 1553-1578, 1553-1599, 1553-1590, 1565-1584, 1571-1595, 1577-1605, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1578-1598, 1571-1598, 1579-1594, 1579-1594, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1580-1605, 1580-1602, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1582-1602, 1553-1655, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1586-1652, 1642-1664, 1651-1720, 1651-1673, 1655-1679, 1695-1720, 1716-1738, 1743-1763, 1743-1768, 1764-1783, 1773-1792, 1777-1796, 1777-1800, 1778-1800, 1778-1793, 1778-1794, 1778-1797, 1779-1798, 1779-1797, 1779-1796, 1779-1795, 1779-1794, 1780-1799, 1780-1796, 1780-1795, 1781-1797, 1781-1796, 1781-1796, 1781-1800, 1781-1797, 1782-1799, 1782-1797, 1782-1798, 1783-1798, 1783-1799, 1784-1800, 1784-1799, 1779-1799, 1778-1794, 1779-1795, 1780-1799, 1794-1813, 1806-1837, 1806-1828, 1806-1825, 1809-1828, 1812-1843, 1812-1837, 1812-1831, 1815-1843, 1815-1844, 1815-1840, 1815-1834, 1818-1837, 1821-1840, 1821-1844, 1821-1837, 1822-1843, 1822-1839, 1823-1843, 1827-1846, 1861-1884, 1861-1880, 1865-1885, 1867-1886, 1869-1885, 1876-1895, 1888-1914, 1888-1908, 1891-1910, 1891-1914, 1895-1938, 1895-1935, 1913-1935, 1898-1920, 1907-1929, 1913-1935, 1918-1934, 1919-1938, 1919-1934, 1921-1934, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2368-2393, 2381-2397, 2368-2394, 2379-2394, 2381-2396, 2368-2397, 2368-2396, 2420-2439, 2458-2476, 2459-2478, 2819-2838, 2818-2838, 2873-2892, and 3161-3182.
In certain embodiments, antisense compounds or oligonucleotides target a region of a HBV nucleic acid. In certain embodiments, such compounds or oligonucleotides targeted to a region of a HBV nucleic acid have a contiguous nucleobase portion that is complementary to an equal length nucleobase portion of the region. For example, the portion can be at least an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases portion complementary to an equal length portion of a region recited herein. In certain embodiments, such compounds or oligonucleotide target the following nucleotide regions of SEQ ID NO: 1: 233-264, 242-263, 243-262, 244-263, 245-264, 246-266, 247-266, 247-269, 247-270, 245-267, 251-267, 245-266, 250-269, 251-268, 251-269, 245-269, 245-266, 245-261, 250-266, 250-267, 250-268, 250-269, 251-270, 253-272, 253-274, 254-274, 255-274, 255-401, 255-400, 255-274, 255-273, 255-272, 255-271, 256-275, 255-276, 256-276, 253-275, 256-279, 257-276, 260-279, 262-281, 262-321, 262-315, 262-312, 265-312, 266-288, 266-291, 266-285, 281-321, 281-303, 405-424, 409-428, 405-428, 411-430, 411-431, 411-431, 412-431, 411-426, 411-427, 412-428, 412-431, 412-427, 413-433, 413-432, 413-428, 413-433, 411-427, 414-427, 415-427, 415-428, 415-429, 416-432, 416-429, 418-435, 418-434, 419-435, 419-434, 420-435, 419-432, 419-434, 422-441, 423-436, 425-465, 584-606, 611-645, 617-363, 623-642, 617-645, 642-754, 653-672, and wherein at least one nucleoside of the compound or modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar.
In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 50% inhibition: 1-20, 10-29, 10-56, 13-38, 13-35, 19-38, 25-47, 25-50, 25-56, 43-68, 43-63, 55-74, 58-77, 58-74, 58-73, 58-79, 58-80, 58-84, 59-74, 59-75, 59-80, 60-79, 60-75, 60-76, 61-80, 61-76, 61-77, 62-77, 63-84, 68-114, 101-123, 98-123, 113-138, 116-138, 131-150, 137-162, 152-186, 158-177, 167-186, 191-215, 196-224, 196-215, 196-218, 199-228, 199-218, 199-224, 200-224, 205-224, 206-228, 218-237, 224-243, 233-264, 242-263, 243-262, 244-263, 245-264, 246-266, 247-266, 247-269, 247-270, 245-267, 251-267, 245-266, 250-269, 251-268, 251-269, 245-269, 245-266, 245-261, 250-265, 250-266, 250-267, 250-268, 250-269, 251-266, 251-270, 252-267, 253-268, 253-269, 253-272, 253-274, 254-269, 254-270, 254-274, 255-274, 255-401, 255-400, 255-274, 255-273, 255-272, 255-271, 255-270, 256-271, 256-272, 256-275, 255-276, 256-276, 253-275, 256-279, 257-276, 258-273, 259-274, 260-279, 262-281, 262-321, 262-315, 262-312, 265-312, 266-288, 266-291, 266-285, 281-321, 281-303, 290-321, 290-312, 292-311, 290-312, 293-312, 293-315, 293-321, 296-321, 302-321, 324-343, 339-361, 339-367, 348-367, 342-367, 358-392, 358-378, 360-392, 360-383, 360-388, 360-385, 362-381, 366-388, 369-388, 366-385, 366-392, 370-389, 370-392, 380-399, 382-401, 384-433, 384-400, 384-401, 385-401, 405-424, 409-428, 405-428, 411-430, 411-431, 411-431, 412-431, 411-426, 411-427, 411-430, 411-437, 412-428, 412-431, 412-427, 413-432, 413-428, 413-429, 413-433, 411-427, 414-427, 414-429, 414-430, 414-433, 415-427, 415-428, 415-429, 415-430, 415-431, 415-434, 416-435, 416-432, 416-431, 416-429, 417-432, 417-433, 417-436, 418-437, 418-435, 418-434, 418-433, 419-435, 419-434, 420-435, 419-432, 419-434, 421-436, 422-441, 422-437, 423-436, 425-465, 454-473, 454-472, 457-476, 454-476, 455-472, 457-485, 457-473, 457-472, 458-485, 458-483, 458-477, 458-473, 459-485, 460-485, 463-498, 463-485, 466-485, 463-482, 457-491, 458-491, 459-491, 460-491, 463-491, 466-491, 472-491, 472-493, 473-492, 475-491, 459-494, 460-494, 463-494, 466-494, 467-498, 472-494, 475-494, 457-473, 457-472, 458-494, 454-494, 457-494, 457-473, 485-513, 470-493, 476-519, 485-519, 500-519, 512-534, 512-550, 524-546, 536-559, 548-567, 548-570, 550-570, 548-594, 554-573, 548-576, 560-594, 584-606, 611-645, 617-363, 623-642, 617-645, 639-654, 641-656, 642-657, 642-754, 643-658, 653-672, 662-685, 665-685, 665-689, 668-687, 670-689, 670-706, 670-685, 670-686, 671-686, 671-687, 671-690, 671-691, 672-687, 672-688, 672-693, 672-697, 672-707, 673-688, 674-693, 678-693, 679-707, 679-694, 679-698, 679-701, 679-702, 679-707, 680-699, 679-699, 680-695, 681-696, 682-706, 682-707, 682-702, 682-701, 682-697, 683-698, 684-699, 685-700, 686-701, 687-702, 687-703, 687-706, 687-754, 688-703, 688-704, 689-704, 689-705, 689-709, 689-710, 690-705, 690-706, 691-706, 679-705, 679-710, 679-706, 690-710, 691-710, 690-754, 690-706, 684-703, 687-705, 687-703, 687-706, 688-705, 689-708, 690-709, 692-711, 693-716, 693-712, 695-715, 697-716, 697-716, 690-716, 724-746, 724-752, 724-754, 724-758, 733-752, 738-753, 738-754, 739-758, 739-754, 739-775, 739-754, 740-754, 742-785, 742-773, 757-776, 757-785, 790-815, 793-812, 811-833, 811-844, 814-833, 811-906, 820-839, 822-844, 822-867, 823-842, 845-864, 845-867, 854-906, 845-909, 845-906, 854-876, 854-873, 863-882, 863-885, 878-900, 878-897, 887-906, 899-918, 899-933, 899-958, 905-927, 905-933, 914-933, 936-958, 936-955, 945-964, 951-970, 951-985, 951-1044, 951-1024, 951-1056, 951-997, 960-985, 963-1044, 963-1024, 963-997, 966-985, 972-1015, 978-997, 1025-1044, 1031-1056, 1037-1056, 1046-1083, 1049-1068, 1070-1089, 1070-1095, 1082-1101, 1081-1134, 1081-1143, 1082-1101, 1088-1107, 1088-1134, 1094-1119, 1097-1119, 1112-1134, 1118-1143, 1118-1146, 1088-1146, 1121-1140, 1127-1146, 1127-1193, 1150-1193, 1156-1187, 1165-1187, 1170-1192, 1171-1191, 1172-1191, 1176-1192, 1177-1192, 1176-1191, 1203-1297, 1206-1228, 1206-1255, 1209-1228, 1215-1255, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1245-1265, 1251-1280, 1251-1285, 1251-1270, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1296, 1259-1290, 1259-1287, 1260-1279, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1261-1296, 1262-1277, 1262-1278, 1262-1281, 1262-1285, 1262-1296, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1264-1297, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1296, 1269-1284, 1269-1285, 1269-1288, 1270-1285, 1271-1290, 1271-1296, 1277-1296, 1261-1290, 1262-1290, 1268-1290, 1263-1305, 1259-1305, 1259-1305, 1266-1305, 1259-1302, 1275-1294, 1281-1306, 1281-1324, 1281-1336, 1782-1797, 1282-1301, 1286-1306, 1290-1324, 1293-1318, 1290-1324, 1293-1315, 1296-1315, 1311-1336, 1311-1333, 1326-1345, 1353-1381, 1359-1378, 1395-1414, 1498-1532, 1498-1523, 1498-1535, 1510-1529, 1515-1535, 1515-1563, 1515-1596, 1515-1605, 1515-1602, 1515-1540, 1515-1535, 1518-1605, 1518-1602, 1518-1537, 1521-1563, 1521-1540, 1550-1655, 1550-1563, 1550-1569, 1553-1578, 1553-1599, 1553-1590, 1565-1584, 1571-1595, 1577-1606, 1577-1605, 1577-1596, 1577-1592, 1577-1593, 1578-1593, 1578-1594, 1578-1597, 1578-1598, 1579-1594, 1579-1595, 1579-1598, 1571-1598, 1580-1605, 1580-1602, 1580-1595, 1580-1596, 1580-1599, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1582-1602, 1553-1655, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1586-1652, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1642-1664, 1651-1720, 1651-1673, 1655-1679, 1695-1720, 1716-1738, 1743-1763, 1743-1768, 1764-1783, 1773-1792, 1777-1796, 1777-1800, 1778-1800, 1778-1793, 1778-1794, 1778-1797, 1779-1798, 1779-1797, 1779-1796, 1779-1795, 1779-1794, 1780-1796, 1781-1797, 1781-1796, 1781-1800, 1781-1797, 1782-1799, 1784-1800, 1779-1799, 1778-1794, 1779-1795, 1780-1799, 1794-1813, 1780-1795, 1780-1796, 1780-1799, 1781-1796, 1781-1797, 1781-1800, 1782-1798, 1783-1799, 1784-1799, 1784-1800, 1785-1800, 1806-1837, 1806-1828, 1806-1825, 1809-1828, 1812-1843, 1812-1837, 1812-1831, 1815-1843, 1815-1844, 1815-1840, 1815-1834, 1818-1837, 1821-1840, 1821-1844, 1821-1837, 1822-1843, 1822-1839, 1823-1843, 1827-1846, 1861-1884, 1861-1880, 1865-1885, 1867-1886, 1869-1885, 1876-1895, 1888-1914, 1888-1908, 1891-1910, 1891-1914, 1895-1938, 1895-1935, 1913-1935, 1898-1920, 1907-1929, 1913-1935, 1918-1934, 1919-1938, 1919-1934, 1921-1934, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2278-2297, 2281-2300, 2284-2303, 2368-2393, 2381-2397, 2368-2394, 2379-2394, 2381-2396, 2368-2397, 2368-2396, 2420-2439, 2458-2476, 2459-2478, 2819-2838, 2818-2838, 2873-2892, and 3161-3182.
In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 60% inhibition: 1-20, 10-29, 10-53, 13-38, 25-50, 43-68, 55-74, 58-84, 58-77, 58-74, 58-73, 58-79, 59-80, 59-74, 59-75, 60-75, 60-76, 61-77, 61-76, 61-80, 62-77, 68-114, 98-123, 101-123, 113-138, 116-138, 131-150, 137-162, 152-186, 191-215, 196-224, 196-215, 199-228, 199-218, 200-223, 199-218, 205-224, 206-228, 218-237, 224-243, 233-263, 244-263, 245-264, 247-266, 250-265, 251-266, 252-267, 253-272, 253-269, 251-267, 253-274, 254-270, 255-276, 256-279, 256-276, 256-274, 256-272, 256-271, 258-273, 259-274, 265-388, 265-284, 266-291, 266-288, 260-279, 281-321, 281-303, 290-321, 290-312, 293-312, 296-315, 302-321, 324-343, 339-367, 339-361, 342-367, 348-367, 358-392, 358-378, 360-392, 360-379, 366-392, 366-385, 369-388, 370-392, 382-401, 405-428, 405-424, 409-428, 411-436, 411-433, 411-431, 411-426, 411-430, 411-427, 412-431, 412-428, 412-427, 413-428, 413-429, 413-433, 414-433, 414-430, 414-429, 414-433, 415-430, 415-431, 415-434, 415-435, 415-436, 416-429, 416-434, 416-431, 416-432, 416-436, 416-435, 417-436, 417-433, 417-432, 418-434, 418-433, 418-437, 419-434, 420-435, 421-436, 422-437, 423-436, 425-465, 454-472, 455-472, 457-476, 457-472, 457-473, 458-485, 458-473, 458-483, 463-498, 467-498, 463-482, 470-493, 472-491, 485-519, 485-513, 500-519, 512-534, 524-546, 536-558, 548-567, 554-573, 548-576, 560-594, 584-606, 608-648, 639-654, 640-656, 641-656, 642-657, 642-658, 643-658, 653-672, 662-685, 665-685, 670-706, 670-689, 670-685, 670-686, 671-690, 671-686, 671-687, 672-707, 672-697, 672-693, 672-687, 672-688, 673-688, 679-707, 679-698, 679-694, 680-695, 681-696, 682-697, 682-701, 683-698, 684-699, 685-700, 686-701, 687-754, 687-702, 687-705, 687-703, 687-706, 688-704, 688-703, 688-704, 688-705, 688-707, 689-710, 689-709, 689-705, 689-704, 690-754, 690-705, 690-706, 691-706, 691-710, 692-711, 697-716, 724-758, 724-754, 724-752, 724-746, 738-754, 738-753, 739-754, 742-785, 757-785, 790-815, 811-906, 811-844, 811-833, 822-867, 822-844, 823-842, 845-867, 854-906, 854-873, 878-897, 899-958, 899-933, 936-958, 945-964, 951-1044, 951-1024, 951-985, 951-997, 963-1044, 963-1024, 963-997, 966-985, 978-997, 1031-1056, 1046-1083, 1070-1095, 1081-1143, 1081-1134, 1082-1101, 1088-1146, 1088-1134, 1118-1146, 1118-1143, 1127-1193, 1170-1189, 1176-1192, 1176-1191, 1177-1192, 1203-1297, 1206-1255, 1209-1228, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1251-1270, 1251-1285, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1278, 1260-1279, 1261-1276, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1281, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1279, 1263-1282, 1264-1297, 1264-1279, 1264-1280, 1264-1283, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1287, 1269-1284, 1269-1285, 1270-1285, 1281-1336, 1281-1324, 1281-1306, 1286-1305, 1290-1324, 1311-1336, 1326-1345, 1353-1381, 1395-1414, 1498-1535, 1498-1532, 1515-1535, 1515-1534, 1521-1540, 1550-1655, 1553-1599, 1553-1590, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1579-1594, 1579-1595, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1642-1664, 1651-1720, 1716-1738, 1743-1763, 1764-1783, 1773-1792, 1777-1800, 1777-1797, 1655-1674, 1778-1794, 1778-1800, 1781-1800, 1781-1797, 1784-1800, 1779-1799, 1778-1794, 1778-1797, 1779-1795, 1779-1798, 1780-1795, 1780-1796, 1780-1799, 1781-1796, 1781-1797, 1781-1800, 1782-1797, 1794-1813, 1806-1837, 1806-1825, 1812-1837, 1812-1831, 1815-1844, 1815-1834, 1818-1837, 1821-1837, 1822-1838, 1827-1846, 1861-1884, 1821-1840, 1866-1885, 1867-1886, 1888-1914, 1888-1907, 1891-1914, 1895-1938, 1895-1935, 1919-1938, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2278-2297, 2281-2300, 2284-2303, 2368-2397, 2368-2396, 2368-2394, 2368-2393, 2379-2394, 2381-2396, 2420-2439, 2458-2476, 2819-2838, 2873-2892, and 3161-3182.
In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 65% inhibition: 1-20, 10-29, 10-53, 13-38, 25-50, 43-68, 55-74, 58-84, 58-79, 58-74, 58-73, 58-77, 59-75, 59-80, 58-77, 60-75, 60-76, 61-77, 61-76, 61-80, 62-77, 68-114, 98-123, 101-123, 113-138, 116-138, 131-150, 137-162, 152-186, 191-215, 196-215, 199-228, 199-218, 200-223, 199-218, 205-224, 206-228, 218-237, 224-243, 233-263, 244-263, 245-264, 250-265, 251-266, 253-269, 253-274, 255-276, 256-279, 256-276, 256-274, 256-272, 256-271, 247-266, 253-272, 258-273, 266-291, 266-288, 260-279, 281-321, 281-303, 290-321, 290-312, 296-315, 293-312, 302-321, 324-343, 339-367, 339-361, 342-367, 348-367, 358-392, 358-378, 360-392, 360-379, 366-392, 366-385, 369-388, 370-392, 382-401, 405-428, 405-424, 409-428, 411-433, 411-431, 411-430, 411-427, 411-426, 412-431, 412-428, 412-427, 413-433, 413-428, 413-429, 413-432, 414-433, 414-430, 414-429, 415-430, 415-431, 415-434, 415-435, 415-436, 416-434, 416-436, 416-435, 416-432, 416-431, 417-436, 417-433, 417-432, 418-433, 418-434, 418-437, 420-435, 422-437, 423-436, 425-465, 454-472, 455-472, 457-472, 458-485, 458-483, 458-473, 463-498, 467-498, 457-476, 470-493, 472-491, 485-519, 485-513, 500-519, 512-534, 524-546, 536-558, 548-567, 554-573, 548-576, 560-594, 584-606, 608-648, 639-654, 640-656, 641-656, 642-657, 642-658, 643-658, 653-672, 662-685, 665-685, 670-685, 670-706, 670-689, 670-686, 670-685, 671-686, 671-687, 671-690, 672-688, 672-687, 672-707, 672-697, 672-693, 673-688, 679-698, 680-695, 681-696, 682-697, 682-701, 683-698, 684-699, 685-700, 686-701, 687-702, 688-703, 688-707, 687-754, 690-754, 690-706, 690-705, 687-705, 687-703, 687-706, 687-702, 688-705, 688-703, 688-704, 689-705, 691-706, 692-711, 697-716, 724-758, 724-754, 724-752, 724-746, 738-754, 739-754, 742-785, 757-785, 790-815, 811-906, 811-844, 811-833, 822-867, 822-844, 823-842, 845-867, 854-906, 854-873, 878-897, 899-958, 899-933, 936-958, 945-964, 951-1044, 951-1024, 951-985, 951-997, 963-1044, 963-1024, 963-997, 966-985, 978-997, 1031-1056, 1046-1083, 1070-1095, 1081-1143, 1081-1134, 1082-1101, 1088-1146, 1088-1134, 1118-1146, 1118-1143, 1127-1193, 1170-1189, 1176-1192, 1177-1192, 1203-1297, 1206-1255, 1209-1228, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1251-1270, 1251-1285, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1278, 1260-1279, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1281, 1262-1277, 1262-1278, 1263-1278, 1263-1279, 1263-1282, 1264-1297, 1264-1279, 1264-1280, 1264-1283, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1287, 1269-1284, 1269-1285, 1270-1285, 1281-1336, 1281-1324, 1281-1306, 1290-1324, 1311-1336, 1326-1345, 1353-1381, 1395-1414, 1498-1535, 1498-1532, 1515-1535, 1515-1534, 1550-1655, 1553-1599, 1553-1590, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1579-1594, 1579-1595, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1642-1664, 1651-1720, 1655-1674, 1716-1738, 1743-1763, 1764-1783, 1773-1792, 1777-1800, 1777-1797, 1778-1800, 1778-1797, 1779-1799, 1778-1794, 1779-1794, 1779-1795, 1779-1798, 1780-1796, 1780-1799, 1780-1795, 1781-1796, 1781-1797, 1781-1800, 1782-1797, 1794-1813, 1806-1837, 1806-1825, 1812-1837, 1812-1831, 1815-1844, 1815-1834, 1818-1837, 1821-1837, 1822-1838, 1827-1846, 1861-1884, 1866-1885, 1867-1886, 1888-1914, 1888-1907, 1891-1914, 1895-1938, 1895-1935, 1919-1938, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2278-2297, 2281-2300, 2284-2303, 2368-2397, 2368-2396, 2368-2394, 2368-2393, 2379-2394, 2381-2396, 2420-2439, 2458-2476, 2819-2838, 2873-2892, and 3161-3182.
In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 70% inhibition: 1-20, 10-29, 10-53, 13-38, 25-50, 43-68, 55-74, 58-84, 58-79, 58-74, 59-75, 59-80, 58-77, 60-75, 60-76, 61-77, 68-114, 98-123, 101-123, 113-138, 116-138, 131-150, 137-162, 152-186, 191-215, 199-228, 199-218, 200-223, 205-224, 206-228, 218-237, 224-243, 233-263, 244-263, 245-264, 253-269, 253-274, 255-276, 256-279, 256-276, 256-274, 256-272, 247-266, 250-265, 251-266, 253-272, 256-271, 266-291, 266-288, 260-279, 281-321, 281-303, 290-321, 290-312, 293-312, 302-321, 324-343, 339-367, 339-361, 342-367, 348-367, 358-392, 358-378, 360-392, 360-379, 366-392, 366-385, 370-392, 382-401, 405-428, 405-424, 409-428, 411-433, 411-431, 411-430, 411-427, 411-426, 412-431, 412-428, 412-427, 413-428, 413-429, 413-432, 414-433, 414-430, 414-429, 415-430, 414-433, 415-434, 415-435, 415-436, 416-431, 416-434, 416-436, 416-435, 416-432, 417-436, 417-433, 418-433, 418-437, 423-436, 425-465, 454-472, 455-472, 457-472, 457-476, 458-473, 458-485, 458-483, 463-498, 467-498, 457-476, 470-493, 470-493, 472-491, 485-519, 485-513, 485-519, 485-513, 500-519, 512-534, 524-546, 536-558, 548-567, 554-573, 548-576, 560-594, 584-606, 608-648, 639-654, 640-656, 641-656, 642-657, 642-658, 643-658, 653-672, 662-685, 665-685, 670-706, 670-689, 670-685, 670-686, 671-690, 671-686, 671-687, 672-687, 672-688, 672-707, 672-697, 672-693, 673-688, 679-698, 681-696, 682-697, 682-701, 683-698, 684-699, 686-701, 687-702, 687-754, 687-702, 688-703, 690-754, 690-706, 687-705, 687-703, 687-706, 692-711, 697-716, 724-758, 724-754, 724-752, 724-746, 738-754, 739-754, 738-754, 742-785, 757-785, 790-815, 811-906, 811-844, 811-833, 822-867, 822-844, 845-867, 854-906, 854-873, 878-897, 899-958, 899-933, 936-958, 945-964, 951-1044, 951-1024, 951-985, 951-997, 963-1044, 963-1024, 963-997, 966-985, 978-997, 1031-1056, 1046-1083, 1070-1095, 1081-1143, 1081-1134, 1082-1101, 1088-1146, 1088-1134, 1118-1146, 1118-1143, 1127-1193, 1170-1189, 1176-1192, 1177-1192, 1203-1297, 1206-1255, 1209-1228, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1251-1285, 1251-1270, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1278, 1260-1279, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1281, 1262-1277, 1262-1278, 1263-1278, 1263-1279, 1263-1282, 1264-1297, 1264-1279, 1264-1280, 1264-1283, 1265-1281, 1265-1284, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1287, 1269-1284, 1269-1285, 1270-1285, 1281-1336, 1281-1324, 1281-1306, 1290-1324, 1311-1336, 1326-1345, 1353-1381, 1395-1414, 1498-1535, 1498-1532, 1515-1535, 1550-1655, 1553-1599, 1553-1590, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1579-1594, 1579-1595, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1642-1664, 1651-1720, 1716-1738, 1743-1763, 1764-1783, 1773-1792, 1777-1800, 1777-1797, 1778-1800, 1778-1797, 1779-1799, 1778-1794, 1779-1795, 1779-1798, 1780-1795, 1780-1796, 1780-1799, 1781-1800, 1782-1797, 1794-1813, 1806-1837, 1806-1825, 1812-1837, 1812-1831, 1815-1844, 1815-1834, 1818-1837, 1821-1837, 1822-1838, 1827-1846, 1861-1884, 1866-1885, 1867-1886, 1888-1914, 1888-1907, 1891-1914, 1895-1938, 1895-1935, 1919-1938, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2278-2297, 2281-2300, 2284-2303, 2368-2397, 2368-2396, 2368-2394, 2368-2393, 2379-2394, 2381-2396, 2420-2439, 2458-2476, 2819-2838, 2873-2892, and 3161-3182.
In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 75% inhibition: 13-32, 16-35, 19-38, 25-44, 28-47, 31-50, 43-62, 46-65, 49-68, 55-74, 58-82, 58-74, 58-77, 59-75, 60-75, 60-76, 61-77, 65-84, 98-117, 101-120, 104-123, 116-135, 119-138, 131-150, 137-156, 140-159, 143-162, 158-177, 161-180, 164-183, 167-186, 200-219, 203-226, 209-228, 218-237, 233-252, 236-255, 239-258, 242-264, 247-266, 251-266, 253-272, 255-276, 266-285, 269-288, 281-300, 284-303, 290-313, 298-317, 302-321, 324-343, 339-358, 342-361, 348-367, 358-381, 364-383, 366-386, 370-389, 373-392, 382-401, 405-424, 409-428, 411-430, 411-426, 411-427, 412-427, 412-431, 413-428, 413-429, 413-432, 414-436, 414-430, 414-429, 415-430, 416-431, 416-432, 417-433, 418-437, 422-441, 425-444, 428-447, 434-453, 440-459, 443-462, 446-465, 456-477, 458-473, 464-483, 470-493, 476-495, 479-498, 488-507, 491-510, 494-513, 500-519, 512-531, 515-534, 524-543, 527-546, 536-555, 539-558, 560-579, 566-585, 569-588, 572-591, 575-594, 584-603, 587-606, 608-627, 614-633, 617-636, 620-639, 623-642, 626-645, 629-648, 639-654, 641-656, 642-657, 643-658, 653-672, 665-684, 668-688, 670-706, 670-686, 670-685, 671-691, 671-687, 671-686, 672-688, 673-688, 679-703, 681-696, 682-697, 686-701, 686-706, 687-702, 687-703, 688-703, 689-708, 693-712, 695-714, 696-715, 697-716, 727-746, 739-754, 742-761, 748-767, 751-770, 754-773, 757-776, 760-779, 763-782, 766-785, 790-809, 793-812, 796-815, 811-830, 814-833, 817-836, 820-839, 822-844, 845-864, 854-873, 857-876, 863-882, 866-885, 872-891, 875-894, 878-897, 881-900, 884-903, 887-906, 899-918, 902-921, 905-924, 908-927, 911-930, 914-933, 936-955, 939-958, 951-970, 954-973, 957-976, 960-979, 963-982, 966-985, 969-988, 972-991, 975-994, 978-997, 996-1015, 1002-1021, 1025-1044, 1031-1050, 1034-1053, 1037-1056, 1046-1065, 1049-1068, 1052-1071, 1055-1074, 1058-1077, 1061-1080, 1064-1083, 1070-1089, 1073-1092, 1076-1095, 1082-1101, 1088-1107, 1094-1113, 1097-1116, 1100-1119, 1103-1122, 1106-1125, 1109-1128, 1112-1131, 1115-1134, 1121-1140, 1127-1146, 1153-1172, 1156-1175, 1159-1178, 1162-1181, 1165-1184, 1168-1191, 1174-1193, 1206-1225, 1209-1228, 1212-1231, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1239-1258, 1242-1261, 1245-1264, 1251-1270, 1254-1273, 1254-1279, 1257-1283, 1257-1276, 1258-1277, 1260-1279, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1277, 1262-1278, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1287, 1269-1284, 1269-1285, 1270-1285, 1272-1291, 1275-1294, 1282-1303, 1286-1306, 1290-1309, 1293-1312, 1296-1315, 1299-1318, 1305-1324, 1311-1330, 1314-1333, 1317-1336, 1353-1381, 1356-1375, 1359-1378, 1498-1517, 1501-1520, 1504-1523, 1510-1529, 1553-1572, 1556-1575, 1559-1578, 1562-1581, 1565-1584, 1571-1590, 1574-1599, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1579-1594, 1579-1595, 1579-1598, 1580-1595, 1580-1596, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1582-1602, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1605, 1586-1602, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1604-1623, 1607-1626, 1630-1649, 1633-1652, 1645-1664, 1651-1670, 1654-1674, 1657-1676, 1660-1679, 1663-1682, 1666-1685, 1689-1708, 1695-1714, 1698-1717, 1701-1720, 1716-1735, 1778-1797, 1778-1794, 1778-1797, 1779-1795, 1779-1798, 1780-1795, 1780-1796, 1780-1799, 1781-1800, 1794-1813, 1895-1914, 1898-1917, 1901-1920, 1907-1926, 1910-1929, 1913-1932, 1916-1935, 1919-1938, 2278-2297, 2281-2300, and 2284-2303.
In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 80% inhibition: 13-32, 16-35, 19-38, 25-44, 28-47, 46-65, 49-68, 58-77, 59-80, 63-82, 98-120, 116-135, 137-159, 158-177, 167-186, 203-224, 205-224, 209-228, 218-237, 233-252, 236-263, 245-264, 253-272, 256-275, 257-276, 266-288, 281-300, 290-312, 293-312, 324-343, 339-358, 348-367, 358-378, 360-379, 361-383, 366-385, 373-392, 382-401, 405-424, 411-431, 411-426, 411-427, 411-430, 413-428, 414-433, 414-434, 415-430, 415-434, 416-431, 416-435, 417-436, 418-437, 422-441, 425-444, 434-453, 456-476, 458-473, 458-477, 464-483, 471-493, 488-507, 494-513, 512-531, 524-543, 527-546, 536-558, 560-579, 566-585, 572-591, 575-594, 584-603, 587-606, 608-627, 614-633, 617-636, 620-639, 623-642, 626-645, 629-648, 639-654, 641-656, 642-657, 643-658, 665-688, 670-687, 670-686, 671-686, 671-687, 671-691, 673-688, 679-699, 682-697, 682-706, 686-701, 687-702, 687-706, 687-703, 693-715, 727-746, 742-761, 748-767, 757-776, 766-785, 790-815, 814-833, 820-839, 822-844, 845-864, 854-873, 854-876, 863-885, 872-906, 878-897, 899-918, 905-933, 936-955, 951-979, 963-985, 966-985, 972-1015, 978-997, 1002-1021, 1025-1044, 1031-1056, 1049-1074, 1061-1083, 1070-1089, 1082-1101, 1088-11107, 1094-1119, 1109-1134, 1121-1140, 1127-1146, 1159-1187, 1171-1191, 1206-1228, 1209-1228, 1215-1255, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1245-1264, 1251-1279, 1251-1270, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1278, 1260-1279, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1282, 1264-1279, 1264-1283, 1265-1284, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1269-1284, 1269-1285, 1269-1288, 1270-1285, 1275-1294, 1282-1301, 1286-1306, 1293-1318, 1311-1333, 1326-1345, 1359-1378, 1553-1578, 1565-1584, 1571-1590, 1574-1599, 1577-1592, 1577-1596, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1579-1595, 1579-1598, 1580-1596, 1580-1599, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1601, 1582-1602, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1603, 1585-1601, 1585-1604, 1586-1605, 1587-1602, 1587-1606, 1588-1603, 1589-1604, 1589-1605, 1657-1679, 1780-1795, 1780-1796, 1780-1799, 1913-1935, 2278-2297, 2281-2300, and 2284-2303.
In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 85% inhibition: 13-32, 16-35, 19-38, 25-44, 46-65, 59-80, 101-120, 140-159, 158-177, 167-186, 200-219, 205-224, 209-228, 233-252, 242-263, 253-272, 266-285, 281-300, 290-311, 293-312, 359-379, 361-381, 370-389, 382-401, 411-426, 411-430, 411-427, 413-428, 414-433, 415-430, 416-435, 417-436, 422-441, 456-476, 458-473, 470-493, 512-531, 524-543, 536-558, 566-585, 575-594, 587-606, 608-627, 614-636, 623-645, 639-654, 665-687, 671-686, 671-687, 680-699, 682-703, 687-706, 687-703, 727-746, 742-761, 757-776, 793-812, 822-843, 854-876, 854-873, 863-885, 878-900, 878-897, 887-906, 899-918, 905-927, 914-933, 936-955, 951-985, 966-985, 972-1015, 978-997, 1002-1021, 1025-1044, 1037-1056, 1049-1074, 1064-1083, 1070-1089, 1088-1107, 1094-1119, 1109-1128, 1121-1140, 1156-1175, 1162-1187, 1172-1191, 1206-1228, 1209-1228, 1215-1255, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1245-1264, 1251-1279, 1251-1270, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1278, 1260-1279, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1282, 1264-1279, 1264-1283, 1265-1284, 1266-1285, 1267-1282, 1267-1283, 1268-1284, 1269-1284, 1269-1285, 1269-1288, 1270-1285, 1275-1294, 1282-1301, 1293-1315, 1311-1330, 1359-1378, 1574-1593, 1577-1592, 1577-1593, 1577-1596, 1577-1606, 1578-1593, 1578-1594, 1578-1597, 1579-1598, 1580-1596, 1580-1599, 1581-1597, 1581-1600, 1582-1601, 1583-1598, 1583-1602, 1584-1603, 1585-1601, 1585-1604, 1586-1605, 1587-1602, 1588-1603, 1780-1799, 1780-1796, and 2278-2297, 2281-2300, and 2284-2303.
In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 90% inhibition: 13-32, 16-35, 60-80, 140-159, 158-177, 167-186, 242-261, 292-311, 362-381, 370-389, 382-401, 411-427, 411-426, 413-428, 415-430, 416-435, 422-441, 473-492, 617-636, 623-642, 639-654, 668-687, 680-699, 682-701, 684-703, 687-706, 727-746, 757-776, 824-843, 854-873, 854-876, 863-882, 878-897, 878-900, 887-906, 899-918, 905-927, 914-933, 936-955, 951-970, 960-985, 966-985, 972-1015, 978-997, 1025-1044, 1037-1056, 1070-1089, 1097-1119, 1109-1128, 1121-1140, 1165-1187, 1172-1191, 1206-1228, 1209-1228, 1215-1234, 1215-1234, 1215-1255, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1245-1264, 1251-1279, 1251-1270, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1278, 1260-1279, 1261-1285, 1261-1280, 1262-1278, 1261-1276, 1262-1281, 1262-1277, 1263-1282, 1263-1278, 1264-1283, 1265-1284, 1266-1285, 1268-1284, 1269-1284, 1269-1285, 1269-1288, 1296-1315, 1577-1605, 1577-1596, 1577-1593, 1577-1592, 1578-1597, 1581-1600, 1582-1601, 1583-1602, 1583-1598, 1585-1601, 1585-1604, 1586-1605, 1588-1603, 1780-1799, 1780-1796, 2278-2297, 2281-2300, and 2284-2303.
In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 95% inhibition: 411-426, 411-427, 413-428, 617-636, 623-642, 668-687, 680-699, 682-701, 854-873, 878-897, 887-906, 914-933, 966-985, 978-997, 1209-1228, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1245-1264, 1251-1270, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1278, 1260-1279, 1261-1285, 1261-1280, 1262-1281, 1263-1282, 1263-1278, 1264-1283, 1265-1284, 1266-1285, 1268-1284, 1269-1288, 1577-1592, 1577-1596, 1577-1601, 1583-1598, 1585-1601, 1588-1603, 1780-1799, 2278-2297, 2281-2300, and 2284-2303.
In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 50% inhibition of a HBV mRNA, ISIS IDs: 510088, 510089, 510090, 510092, 510096, 510097, 510098, 510099, 510100, 510101, 510102, 505330, 509928, 510104, 509929, 510105, 509930, 510106, 510107, 510108, 510111, 510115, 509931, 510116, 510117, 510118, 510119, 510120, 510121, 509932, 510122, 509933, 510123, 509934, 510124, 509935, 510125, 510126, 510127, 510128, 510140, 146779, 505314, 505315, 505316, 505317, 146821, 505318, 509922, 505319, 509925, 505320, 509952, 505321, 505322, 505323, 505324, 505325, 505326, 505327, 505328, 505329, 509956, 509957, 509927, 509958, 510038, 505330, 509959, 510039, 509960, 510040, 509961, 510041, 509962, 509963, 505331, 505332, 509968, 509969, 510050, 510052, 505333, 505334, 505335, 505336, 509972, 146823, 509974, 505338, 505339, 509975, 505340, 509978, 505341, 509979, 510058, 505342, 509981, 510061, 505344, 505345, 509983, 505346, 509984, 505347, 505348, 505350, 505352, 505353, 505354, 505355, 505356, 146786, 505357, 505358, 505359, 505360, 509985, 509986, 509987, 509988, 505363, 505364, 505365, 505366, 146787, 510079, 524410, 524411, 524413, 524414, 524415, 524416, 524417, 524418, 524419, 524420, 524421, 524422, 524424, 524425, 524426, 524427, 524428, 524429, 524431, 524432, 524433, 524434, 524435, 524436, 524439, 524440, 524442, 524444, 524446, 524447, 524448, 524450, 524451, 524452, 524453, 524454, 524455, 524456, 524457, 524458, 524459, 524460, 524461, 524462, 524464, 524466, 524467, 524468, 524469, 524470, 524471, 524472, 524473, 524474, 524475, 524477, 524478, 524479, 524480, 524481, 524482, 524483, 524484, 524485, 524486, 524487, 524489, 524490, 524491, 524492, 524493, 524494, 524495, 524496, 524498, 524499, 524500, 524501, 524502, 524503, 524504, 524506, 524507, 524508, 524509, 524510, 524511, 524512, 524513, 524514, 524515, 524516, 524517, 524518, 524519, 524520, 524521, 524522, 524523, 524524, 524525, 524526, 524527, 524528, 524529, 524530, 524531, 524532, 524533, 524534, 524535, 524536, 524537, 524538, 524539, 524540, 524541, 524543, 524544, 524546, 524547, 524548, 524549, 524550, 524551, 524552, 524553, 524554, 524555, 524556, 524557, 524558, 524559, 524560, 524561, 524562, 524563, 524564, 524565, 524568, 524569, 524570, 524571, 524572, 524573, 524574, 524575, 524576, 524577, 524578, 524579, 524580, 524581, 524582, 524584, 524585, 524586, 524587, 524588, 524589, 524590, 524591, 524592, 524593, 524594, 524595, 524598, 524599, 524600, 524601, 524602, 524603, 524604, 524605, 524606, 524607, 524608, 524609, 524610, 524611, 524614, 524615, 524616, 524617, 524618, 524619, 524620, 524621, 524622, 524623, 524624, 524625, 524626, 524627, 524629, 524632, 524633, 524634, 524635, 524636, 524637, 524638, 524639, 524640, 524641, 524642, 524643, 524644, 524646, 524647, 524648, 524649, 524650, 524651, 524652, 524654, 524656, 524657, 524658, 524659, 524660, 524661, 524662, 524663, 524664, 524665, 524666, 524667, 524668, 524669, 524670, 524672, 524673, 524675, 524676, 524678, 524679, 524680, 524682, 524683, 524684, 524685, 524686, 524687, 524688, 524689, 524690, 524691, 524692, 524693, 524694, 524695, 524696, 524697, 524698, 524699, 524700, 524701, 524702, 524703, 524704, 524705, 524706, 524707, 524708, 524709, 524710, 524712, 524713, 524714, 524715, 524716, 524717, 524718, 524719, 524721, 524722, 524723, 524724, 524726, 524727, 524728, 524729, 524730, 524731, 524732, 524733, 524734, 524735, 524736, 524737, 524738, 524739, 524740, 524741, 524742, 524743, 524744, 524745, 524746, 524747, 524748, 524749, 524750, 524751, 524752, 524753, 524754, 524755, 524756, 524757, 524758, 524759, 524760, 524761, 524762, 524763, 524764, 524765, 524766, 524767, 524768, 524769, 524770, 524771, 524772, 524773, 524774, 524775, 524776, 524777, 524778, 524779, 524780, 524781, 524782, 524783, 524784, 524785, 524786, 524787, 524788, 524789, 524790, 524791, 524792, 524793, 524794, 524795, 524796, 524797, 524798, 524799, 524800, 524801, 524802, 524803, 524804, 524805, 524806, 524807, 524808, 524809, 524810, 524811, 524812, 524813, 524814, 524815, 524816, 524817, 524818, 524819, 524820, 524821, 524822, 524823, 524824, 524825, 524826, 524827, 524828, 524829, 524830, 524831, 524632, 524833, 524834, 524835, 524842, 524843, 524844, 524845, 524847, 524848, 524856, 524857, 524861, 524866, 524867, 524868, 524869, 524870, 524871, 524872, 524873, 524875, 524876, 524877, 524878, 524879, 524880, 524881, 524882, 524883, 524884, 524885, 524886, 524887, 524888, 524889, 524890, 524891, 524892, 524893, 524894, 524895, 524896, 524897, 524898, 524899, 524900, 524901, 524902, 524903, 524904, 524905, 524906, 524907, 524908, 524909, 524910, 524911, 524912, 524913, 524914, 524915, 524916, 524917, 524918, 524919, 524921, 524922, 524923, 524924, 524925, 524926, 524927, 524928, 524929, 524930, 524931, 524932, 524933, 524934, 524935, 524936, 524937, 524938, 524939, 524940, 524941, 524942, 524943, 524944, 524945, 524946, 524947, 524948, 524949, 524950, 524951, 524952, 524953, 524954, 524955, 524956, 524957, 524958, 524959, 524960, 524961, 524962, 524964, 524965, 524976, 524977, 524978, 524979, 524980, 524981, 524982, 524983, 524984, 524985, 524986, 524987, 524988, 524989, 524991, 524992, 524993, 524994, 524997, 524998, 525021, 525022, 525037, 525039, 525043, 525050, 525052, 525086, 525090, 525100, 551909, 551910, 551911, 551912, 551913, 551916, 551917, 551918, 551919, 551920, 551921, 551922, 551923, 551924, 551925, 551926, 551927, 551928, 551929, 551930, 551932, 551933, 551934, 551935, 551936, 551937, 551939, 551940, 551941, 551942, 551943, 551944, 551945, 551946, 551947, 551948, 551949, 551950, 551951, 551952, 551953, 551954, 551955, 551956, 551957, 551958, 551959, 551960, 551962, 551963, 551964, 551965, 551966, 551967, 551968, 551971, 551972, 551973, 551974, 551975, 551976, 551977, 551978, 551979, 551980, 551981, 551982, 551983, 551984, 551985, 551986, 551987, 551988, 551989, 551990, 551992, 551993, 551994, 551995, 551996, 551997, 551998, 551999, 552000, 552001, 552002, 552003, 552004, 552005, 552006, 552007, 552009, 552010, 552011, 552012, 552013, 552014, 552015, 552016, 552017, 552018, 552019, 552020, 552021, 552022, 552023, 552024, 552025, 552026, 552027, 552028, 552029, 552030, 552031, 552032, 552033, 552034, 552035, 552036, 552037, 552038, 552039, 552040, 552041, 552042, 552043, 552044, 552045, 552046, 552047, 552048, 552049, 552050, 552051, 552052, 552053, 552054, 552055, 552056, 552057, 552058, 552059, 552060, 552061, 552062, 552063, 552064, 552065, 552067, 552068, 552069, 552070, 552071, 552072, 552073, 552074, 552075, 552076, 552077, 552078, 552079, 552080, 552081, 552082, 552083, 552084, 552085, 552086, 552087, 552088, 552089, 552090, 552091, 552092, 552093, 552094, 552095, 552096, 552097, 552098, 552099, 552100, 552101, 552102, 552114, 552115, 552116, 552117, 552118, 552119, 552122, 552123, 552124, 552125, 552126, 552127, 552128, 552129, 552131, 552132, 552133, 552134, 552135, 552136, 552137, 552138, 552139, 552140, 552141, 552142, 552143, 552144, 552145, 552146, 552147, 552148, 552149, 552150, 552151, 552152, 552153, 552154, 552155, 552158, 552159, 552160, 552161, 552162, 552163, 552164, 552165, 552167, 552168, 552169, 552170, 552171, 552175, 552176, 552177, 552178, 552179, 552180, 552181, 552182, 552183, 552185, 552186, 552187, 552188, 552189, 552191, 552192, 552193, 552194, 552195, 552196, 552197, 552198, 552199, 552200, 552201, 552202, 552203, 552204, 552205, 552206, 552207, 552208, 552209, 552210, 552211, 552212, 552213, 552214, 552215, 552216, 552217, 552218, 552220, 552222, 552224, 552225, 552230, 552239, 552240, 552241, 552242, 552243, 552246, 552247, 552248, 552249, 552250, 552251, 552252, 552253, 552254, 552255, 552256, 552257, 552258, 552259, 552260, 552261, 552262, 552263, 552264, 552265, 552266, 552267, 552268, 552269, 552270, 552271, 552279, 552285, 552288, 552293, 552294, 552295, 552296, 552297, 552300, 552301, 552302, 552303, 552304, 552305, 552306, 552307, 552308, 552309, 552310, 552312, 552313, 552314, 552315, 552316, 552317, 552318, 552319, 552320, 552321, 552322, 552323, 552325, 552326, 552330, 552331, 552332, 552333, 552337, 552338, 552339, 552340, 552341, 552342, 552343, 552344, 552345, 552347, 552348, 552349, 552350, 552351, 552352, 552354, 552355, 552356, 552357, 552358, 552359, 552360, 552361, 552362, 552363, 552364, 552365, 552366, 552367, 552368, 552369, 552370, 552371, 552372, 552373, 552374, 552375, 552376, 552377, 552378, 552379, 552380, 552385, 552386, 552390, 552391, 552393, 552394, 552395, 552396, 552397, 552398, 552399, 552400, 552401, 552402, 552403, 552408, 552409, 552410, 552411, 552412, 552413, 552414, 552415, 552416, 552417, 552418, 552419, 552420, 552421, 552422, 552423, 552424, 552425, 552428, 552430, 552431, 552432, 552433, 552440, 552442, 552443, 552444, 552445, 552446, 552447, 552448, 552449, 552450, 552452, 552453, 552455, 552456, 552458, 552459, 552464, 552465, 552466, 552467, 552468, 552469, 552470, 552471, 552472, 552473, 552474, 552475, 552476, 552477, 552478, 552479, 552480, 552481, 552482, 552484, 552485, 552486, 552487, 552488, 552490, 552491, 552493, 552497, 552499, 552500, 552501, 552502, 552503, 552504, 552505, 552506, 552508, 552509, 552510, 552511, 552512, 552513, 552514, 552515, 552516, 552517, 552520, 552521, 552522, 552523, 552525, 552526, 552527, 552528, 552529, 552530, 552531, 552532, 552533, 552534, 552535, 552538, 552539, 552540, 552541, 552542, 552544, 552547, 552548, 552553, 552554, 552555, 552557, 552558, 552559, 552561, 552562, 552565, 552566, 552567, 552568, 552569, 552570, 552571, 552572, 552576, 552577, 552578, 552579, 552580, 552581, 552582, 552583, 552584, 552585, 552586, 552587, 552588, 552589, 552590, 552591, 552592, 552594, 552595, 552596, 552597, 552598, 552600, 552606, 552608, 552787, 552788, 552789, 552790, 552791, 552794, 552795, 552796, 552797, 552798, 552799, 552800, 552801, 552802, 552803, 552804, 552805, 552806, 552807, 552808, 552809, 552810, 552811, 552812, 552813, 552814, 552815, 552816, 552817, 552818, 552819, 552820, 552821, 552822, 552823, 552824, 552825, 552826, 552827, 552828, 552829, 552830, 552831, 552832, 552833, 552834, 552835, 552836, 552837, 552838, 552839, 552840, 552841, 552842, 552843, 552844, 552845, 552846, 552847, 552848, 552849, 552850, 552851, 552852, 552853, 552854, 552855, 552856, 552857, 552858, 552859, 552860, 552861, 552862, 552863, 552864, 552865, 552866, 552868, 552870, 552871, 552872, 552876, 552889, 552890, 552891, 552892, 552893, 552894, 552895, 552896, 552898, 552899, 552901, 552902, 552903, 552904, 552905, 552907, 552908, 552909, 552910, 552911, 552912, 552913, 552914, 552915, 552916, 552917, 552918, 552919, 552922, 552923, 552925, 552926, 552927, 552928, 552929, 552930, 552931, 552932, 552933, 552934, 552935, 552936, 552937, 552938, 552939, 552940, 552941, 552942, 552943, 552944, 552945, 552946, 552947, 552948, 552950, 552951, 552953, 552954, 552955, 552956, 552957, 552958, 552959, 552960, 552961, 552965, 552966, 552969, 552970, 552971, 552972, 552973, 552974, 552975, 552976, 552977, 552979, 552980, 552981, 552982, 552983, 552984, 552987, 552988, 552989, 552990, 552991, 552992, 552993, 552994, 552995, 552996, 552997, 552998, 552999, 553000, 553001, 553002, 553003, 553004, 553005, 553006, 553007, 553008, 553009, 553010, 553011, 553012, 553014, 553015, 553016, 566828, 566829, 566830, 566831, 566832, 577120, 577121, 577122, 577123, 577124, 577125, 577126, 577127, 577128, 577129, 577130, 577131, 577132, 577133, 577134, 577135, 577136, 582665, and 582666.
In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 50% inhibition of a HBV mRNA, SEQ ID NOs: 5, 6, 7, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 33, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 74, 83, 85, 86, 87, 88, 89, 92, 96, 98, 99, 100, 102, 103, 104, 106, 108, 109, 111, 112, 115, 117, 121, 122, 123, 124, 125, 126, 127, 128, 136, 137, 139, 140, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 155, 157, 159, 161, 165, 166, 167, 168, 169, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 186, 187, 188, 189, 190, 191, 192, 193, 194, 197, 198, 199, 201, 203, 206, 207, 208, 209, 210, 211, 212, 213, 215, 217, 218, 220, 221, 222, 224, 225, 226, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 240, 241, 242, 243, 244, 250, 283, 321, 322, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 335, 336, 337, 338, 339, 340, 342, 343, 344, 345, 346, 347, 350, 351, 353, 355, 357, 358, 359, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 399, 400, 401, 402, 403, 404, 405, 406, 408, 409, 410, 411, 412, 413, 414, 416, 417, 418, 419, 420 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 453, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 473, 474, 475, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 539, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 564, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 582, 583, 585, 586, 588, 589, 590, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 622, 623, 624, 625, 626, 627, 628, 629, 631, 632, 633, 634, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 754, 755, 756, 757, 759, 760, 768, 769, 773, 777, 778, 779, 780, 781, 782, 783, 784, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 876, 877, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 903, 904, 905, 906, 909, 910, 933, 934, 949, 951, 955, 962, 964, 998, 1002, 1013, 1052, 1267, 1271, 1272, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1375, and 1376.
In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 60% inhibition of a HBV mRNA, ISIS IDs: 510090, 510100, 510102, 505330, 509928, 510104, 509929, 510105, 509930, 510106, 510107, 510111, 509931, 510116, 510117, 510118, 510119, 510120, 510121, 509932, 510122, 509933, 510123, 509934, 510124, 509935, 510125, 510128, 146779, 505314, 505315, 505316, 505317, 146821, 505318, 505319, 505322, 505323, 505324, 505325, 505326, 505327, 505328, 505329, 509956, 509957, 509958, 505330, 509959, 510041, 505332, 509968, 505333, 505335, 146823, 509974, 505338, 505339, 509975, 505340, 505341, 509979, 505342, 509981, 505344, 505345, 509983, 505346, 509984, 505347, 505348, 505353, 505354, 505356, 146786, 505357, 505358, 505359, 505360, 509985, 509986, 505363, 505366, 524410, 524413, 524414, 524415, 524416, 524417, 524418, 524419, 524420, 524421, 524422, 524424, 524425, 524426, 524428, 524431, 524432, 524433, 524434, 524435, 524439, 524440, 524446, 524447, 524448, 524451, 524452, 524453, 524454, 524455, 524456, 524457, 524459, 524460, 524461, 524464, 524466, 524467, 524468, 524469, 524471, 524472, 524473, 524474, 524475, 524477, 524478, 524479, 524480, 524481, 524482, 524485, 524486, 524487, 524489, 524490, 524491, 524492, 524493, 524494, 524495, 524496, 524499, 524500, 524501, 524502, 524503, 524504, 524506, 524507, 524508, 524509, 524510, 524511, 524512, 524513, 524514, 524515, 524516, 524517, 524519, 524520, 524521, 524523, 524525, 524526, 524527, 524528, 524529, 524532, 524533, 524534, 524535, 524536, 524537, 524538, 524539, 524540, 524541, 524543, 524546, 524547, 524549, 524550, 524552, 524553, 524554, 524555, 524556, 524557, 524558, 524559, 524560, 524561, 524562, 524563, 524564, 524565, 524568, 524569, 524570, 524571, 524572, 524573, 524574, 524575, 524576, 524577, 524578, 524579, 524580, 524581, 524582, 524585, 524586, 524587, 524588, 524589, 524590, 524591, 524593, 524594, 524595, 524598, 524599, 524600, 524602, 524603, 524604, 524605, 524606, 524607, 524610, 524611, 524614, 524615, 524616, 524617, 524618, 524619, 524620, 524621, 524622, 524623, 524625, 524627, 524629, 524632, 524633, 524634, 524635, 524636, 524637, 524638, 524639, 524640, 524641, 524642, 524643, 524644, 524646, 524647, 524648, 524649, 524650, 524651, 524654, 524656, 524657, 524658, 524659, 524661, 524662, 524663, 524664, 524665, 524666, 524667, 524668, 524669, 524670, 524673, 524675, 524676, 524678, 524679, 524680, 524683, 524684, 524685, 524686, 524687, 524688, 524689, 524690, 524691, 524692, 524694, 524695, 524696, 524697, 524698, 524699, 524700, 524701, 524702, 524703, 524704, 524705, 524706, 524707, 524708, 524709, 524710, 524713, 524714, 524715, 524716, 524717, 524718, 524719, 524721, 524722, 524724, 524726, 524727, 524728, 524729, 524730, 524731, 524732, 524733, 524734, 524735, 524736, 524737, 524738, 524739, 524741, 524742, 524743, 524744, 524746, 524747, 524748, 524749, 524750, 524751, 524752, 524753, 524754, 524755, 524756, 524757, 524758, 524759, 524760, 524761, 524762, 524763, 524764, 524765, 524766, 524767, 524768, 524769, 524770, 524771, 524772, 524773, 524774, 524775, 524776, 524777, 524778, 524779, 524780, 524781, 524782, 524783, 524784, 524785, 524787, 524788, 524789, 524790, 524791, 524792, 524793, 524794, 524795, 524796, 524797, 524798, 524799, 524800, 524801, 524802, 524803, 524804, 524805, 524806, 524807, 524808, 524809, 524810, 524811, 524812, 524813, 524814, 524815, 524816, 524817, 524818, 524819, 524820, 524821, 524822, 524823, 524824, 524825, 524826, 524827, 524828, 524829, 524830, 524632, 524833, 524842, 524843, 524844, 524845, 524847, 524856, 524866, 524867, 524868, 524869, 524870, 524871, 524872, 524873, 524876, 524878, 524879, 524880, 524881, 524882, 524883, 524884, 524885, 524886, 524887, 524888, 524889, 524890, 524891, 524892, 524893, 524894, 524895, 524896, 524897, 524898, 524899, 524900, 524901, 524902, 524903, 524904, 524905, 524906, 524907, 524908, 524909, 524910, 524911, 524912, 524913, 524914, 524915, 524916, 524921, 524922, 524923, 524924, 524925, 524926, 524928, 524929, 524930, 524931, 524932, 524933, 524936, 524937, 524938, 524939, 524940, 524941, 524942, 524944, 524946, 524947, 524948, 524949, 524950, 524952, 524953, 524954, 524955, 524961, 524977, 524978, 524979, 524980, 524981, 524982, 524983, 524984, 524985, 524986, 524987, 524988, 524991, 524992, 524993, 524994, 525037, 525052, 551909, 551911, 551919, 551920, 551921, 551922, 551924, 551925, 551926, 551927, 551928, 551932, 551933, 551934, 551935, 551936, 551941, 551943, 551944, 551948, 551949, 551950, 551951, 551952, 551953, 551954, 551955, 551956, 551957, 551958, 551959, 551960, 551962, 551963, 551965, 551966, 551967, 551968, 551973, 551975, 551979, 551981, 551982, 551983, 551984, 551985, 551986, 551987, 551989, 551990, 551992, 551993, 551994, 551995, 551996, 551997, 551998, 551999, 552000, 552001, 552002, 552003, 552005, 552006, 552007, 552009, 552010, 552012, 552013, 552014, 552015, 552016, 552017, 552018, 552019, 552020, 552021, 552022, 552023, 552024, 552025, 552026, 552027, 552028, 552029, 552030, 552031, 552032, 552033, 552034, 552035, 552036, 552038, 552039, 552041, 552042, 552044, 552045, 552046, 552047, 552048, 552049, 552050, 552051, 552052, 552053, 552054, 552055, 552056, 552057, 552058, 552059, 552060, 552061, 552062, 552063, 552064, 552065, 552068, 552069, 552070, 552071, 552073, 552074, 552075, 552076, 552077, 552078, 552079, 552080, 552081, 552082, 552083, 552084, 552085, 552086, 552087, 552088, 552089, 552090, 552091, 552092, 552093, 552094, 552095, 552096, 552097, 552098, 552099, 552100, 552101, 552102, 552114, 552115, 552116, 552117, 552118, 552119, 552123, 552124, 552125, 552126, 552127, 552128, 552129, 552131, 552132, 552133, 552134, 552135, 552136, 552138, 552139, 552140, 552141, 552143, 552144, 552145, 552146, 552147, 552148, 552149, 552150, 552151, 552152, 552153, 552155, 552158, 552159, 552160, 552162, 552163, 552168, 552169, 552170, 552171, 552176, 552178, 552179, 552180, 552182, 552183, 552185, 552187, 552188, 552191, 552192, 552193, 552194, 552195, 552196, 552197, 552198, 552199, 552200, 552201, 552202, 552203, 552204, 552205, 552206, 552207, 552208, 552209, 552210, 552211, 552212, 552213, 552214, 552215, 552216, 552222, 552224, 552225, 552239, 552240, 552242, 552246, 552247, 552248, 552252, 552253, 552254, 552255, 552256, 552257, 552258, 552259, 552261, 552263, 552265, 552266, 552268, 552285, 552293, 552294, 552295, 552296, 552301, 552302, 552303, 552306, 552307, 552308, 552309, 552310, 552312, 552313, 552314, 552315, 552316, 552317, 552318, 552320, 552321, 552322, 552323, 552325, 552326, 552331, 552332, 552337, 552338, 552339, 552340, 552343, 552345, 552347, 552348, 552349, 552351, 552354, 552355, 552356, 552358, 552359, 552360, 552361, 552362, 552363, 552364, 552365, 552366, 552367, 552368, 552369, 552370, 552371, 552372, 552373, 552374, 552375, 552376, 552377, 552378, 552379, 552396, 552397, 552398, 552403, 552408, 552409, 552410, 552411, 552412, 552414, 552416, 552418, 552419, 552420, 552421, 552422, 552423, 552424, 552431, 552442, 552445, 552449, 552455, 552456, 552459, 552464, 552465, 552466, 552467, 552469, 552472, 552473, 552474, 552475, 552477, 552478, 552479, 552480, 552484, 552487, 552497, 552508, 552509, 552511, 552512, 552515, 552516, 552520, 552521, 552522, 552523, 552526, 552527, 552528, 552529, 552530, 552531, 552534, 552540, 552541, 552542, 552559, 552567, 552568, 552569, 552570, 552572, 552576, 552577, 552578, 552579, 552582, 552583, 552584, 552585, 552586, 552587, 552588, 552590, 552595, 552596, and 552597, 552788, 552789, 552790, 552791, 552796, 552800, 552801, 552803, 552804, 552805, 552806, 552807, 552808, 552809, 552811, 552812, 552813, 552814, 552815, 552816, 552817, 552818, 552819, 552820, 552821, 552822, 552823, 552824, 552826, 552827, 552828, 552829, 552830, 552831, 552832, 552833, 552834, 552835, 552836, 552837, 552838, 552839, 552841, 552842, 552843, 552844, 552845, 552846, 552847, 552848, 552849, 552850, 552851, 552852, 552853, 552854, 552855, 552856, 552857, 552858, 552859, 552860, 552861, 552862, 552863, 552864, 552865, 552866, 552872, 552891, 552892, 552893, 552894, 552902, 552903, 552904, 552905, 552907, 552908, 552909, 552910, 552911, 552912, 552913, 552914, 552915, 552916, 552917, 552918, 552922, 552923, 552925, 552927, 552928, 552929, 552930, 552931, 552932, 552933, 552934, 552935, 552936, 552937, 552938, 552939, 552940, 552941, 552942, 552943, 552944, 552945, 552946, 552951, 552955, 552956, 552957, 552958, 552960, 552961, 552966, 552969, 552971, 552972, 552973, 552974, 552975, 552976, 552977, 552979, 552980, 552981, 552982, 552983, 552984, 552988, 552989, 552990, 552991, 552992, 552993, 552994, 552995, 552996, 552998, 552999, 553000, 553001, 553002, 553003, 553004, 553005, 553006, 553007, 553008, 553009, 553010, 553011, 553012, 553016, 566828, 566829, 566830, 566831, 566832, 577120, 577121, 577122, 577123, 577124, 577125, 577126, 577127, 577128, 577129, 577130, 577131, 577132, 577133, 577134, 577135, 577136, and 582666.
In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 60% inhibition of a HBV mRNA, SEQ ID NOs: 7, 9, 10, 12, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 33, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 56, 83, 85, 86, 87, 88, 89, 92, 96, 98, 100, 102, 103, 112, 115, 117, 122, 123, 124, 125, 126, 127, 128, 136, 137, 139, 140, 142, 143, 145, 147, 149, 150, 151, 153, 155, 157, 159, 161, 166, 167, 168, 172, 174, 176, 177, 178, 179, 180, 181, 186, 187, 188, 189, 190, 191, 192, 193, 194, 198, 199, 201, 206, 207, 208, 209, 210, 211, 212, 213, 218, 220, 222, 224, 225, 226, 227, 228, 230, 231, 232, 233, 234, 240, 243, 321, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 342, 343, 344, 345, 346, 350, 351, 357, 358, 359, 362, 363, 364, 365, 366, 367, 368, 370, 371, 372, 375, 376, 377, 378, 379, 381, 382, 383, 384, 387, 388, 389, 390, 391, 392, 395, 396, 397, 399, 400, 401, 402, 403, 404, 405, 406, 409, 410, 411, 412, 413, 414, 416, 417, 418, 419, 420 421, 422, 423, 424, 425, 426, 427, 429, 430, 431, 433, 435, 436, 437, 438, 439, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 453, 456, 457, 459, 460, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 473, 474, 475, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 495, 496, 497, 498, 499, 500, 501, 503, 504, 505, 508, 509, 510, 512, 513, 514, 515, 516, 517, 520, 521, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 535, 537, 539, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 564, 566, 567, 568, 569, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 583, 585, 586, 588, 589, 590, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 623, 624, 625, 626, 627, 628, 629, 631, 632, 634, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 652, 653, 654, 655, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 740, 741, 742, 744, 745, 754, 755, 756, 757, 759, 768, 777, 778, 779, 780, 781, 782, 783, 784, 787, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 833, 834, 835, 836, 837, 838, 840, 841, 842, 843, 844, 845, 848, 849, 850, 851, 852, 853, 854, 856, 858, 859, 860, 861, 862, 864, 865, 866, 867, 873, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 903, 904, 905, 906, 949, 964, 1271, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, and 1376.
In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 70% inhibition of a HBV mRNA, ISIS IDs: 510100, 505330, 509928, 509929, 509930, 510106, 509931, 510116, 510119, 510120, 510121, 509932, 510122, 509933, 510123, 509934, 510124, 509935, 146779, 505317, 146821, 505318, 505319, 505323, 505325, 505326, 505327, 509957, 505330, 505332, 505335, 509974, 505338, 505339, 509975, 505342, 509981, 505345, 505346, 505347, 505348, 146786, 505357, 505358, 505359, 505363, 524410, 524413, 524414, 524415, 524416, 524418, 524419, 524420, 524421, 524424, 524425, 524426, 524428, 524431, 524432, 524433, 524434, 524435, 524446, 524447, 524448, 524452, 524453, 524457, 524459, 524460, 524461, 524464, 524466, 524467, 524468, 524469, 524472, 524473, 524474, 524475, 524477, 524478, 524479, 524480, 524481, 524482, 524485, 524487, 524490, 524491, 524492, 524493, 524494, 524495, 524499, 524500, 524502, 524503, 524507, 524508, 524510, 524511, 524512, 524513, 524514, 524515, 524516, 524517, 524520, 524525, 524526, 524528, 524532, 524533, 524534, 524535, 524536, 524537, 524538, 524539, 524540, 524541, 524547, 524549, 524552, 524553, 524554, 524555, 524556, 524557, 524558, 524559, 524560, 524561, 524563, 524564, 524565, 524568, 524569, 524570, 524571, 524572, 524573, 524574, 524575, 524577, 524578, 524579, 524580, 524582, 524586, 524587, 524590, 524591, 524594, 524595, 524598, 524600, 524602, 524603, 524604, 524605, 524606, 524607, 524610, 524611, 524614, 524615, 524616, 524617, 524618, 524619, 524620, 524621, 524629, 524633, 524634, 524635, 524636, 524637, 524638, 524641, 524642, 524643, 524644, 524646, 524647, 524648, 524649, 524650, 524651, 524656, 524657, 524659, 524661, 524662, 524663, 524664, 524665, 524666, 524667, 524668, 524669, 524670, 524678, 524679, 524680, 524685, 524686, 524687, 524688, 524689, 524690, 524691, 524692, 524695, 524696, 524698, 524699, 524700, 524701, 524702, 524703, 524704, 524705, 524706, 524707, 524708, 524709, 524713, 524714, 524715, 524716, 524717, 524718, 524721, 524722, 524724, 524726, 524727, 524728, 524729, 524730, 524731, 524732, 524733, 524734, 524735, 524736, 524737, 524738, 524739, 524741, 524742, 524743, 524746, 524747, 524748, 524749, 524750, 524751, 524752, 524754, 524755, 524756, 524758, 524760, 524761, 524762, 524763, 524764, 524765, 524766, 524767, 524768, 524769, 524771, 524773, 524775, 524776, 524777, 524778, 524779, 524780, 524781, 524782, 524783, 524784, 524785, 524787, 524788, 524789, 524790, 524791, 524792, 524793, 524794, 524795, 524796, 524797, 524798, 524799, 524800, 524801, 524802, 524803, 524804, 524805, 524806, 524807, 524808, 524809, 524810, 524811, 524812, 524813, 524814, 524815, 524816, 524817, 524818, 524819, 524821, 524822, 524823, 524824, 524825, 524826, 524827, 524828, 524829, 524830, 524833, 524842, 524843, 524844, 524845, 524856, 524866, 524867, 524868, 524869, 524870, 524871, 524873, 524879, 524880, 524881, 524882, 524883, 524884, 524885, 524886, 524887, 524888, 524889, 524890, 524891, 524892, 524893, 524894, 524895, 524896, 524897, 524898, 524899, 524900, 524902, 524903, 524905, 524906, 524907, 524908, 524909, 524910, 524911, 524912, 524913, 524914, 524915, 524916, 524921, 524922, 524930, 524931, 524932, 524937, 524940, 524942, 524948, 524980, 524981, 524982, 524983, 524984, 524985, 524986, 524987, 524988, 551919, 551921, 551922, 551924, 551925, 551926, 551933, 551941, 551950, 551951, 551952, 551953, 551955, 551956, 551957, 551958, 551966, 551983, 551984, 551985, 551986, 551987, 551989, 551990, 551992, 551993, 551994, 551995, 551996, 551997, 551998, 551999, 552000, 552005, 552006, 552009, 552012, 552013, 552014, 552015, 552017, 552018, 552019, 552020, 552021, 552022, 552023, 552024, 552025, 552026, 552027, 552028, 552029, 552030, 552031, 552032, 552033, 552034, 552038, 552039, 552041, 552044, 552046, 552047, 552049, 552050, 552051, 552052, 552053, 552054, 552055, 552056, 552057, 552058, 552059, 552060, 552061, 552062, 552063, 552064, 552065, 552068, 552069, 552070, 552071, 552073, 552074, 552075, 552076, 552077, 552078, 552079, 552080, 552081, 552082, 552083, 552084, 552085, 552086, 552087, 552088, 552089, 552090, 552091, 552092, 552093, 552094, 552095, 552096, 552097, 552098, 552099, 552100, 552101, 552115, 552117, 552123, 552125, 552127, 552128, 552129, 552132, 552133, 552138, 552139, 552140, 552141, 552143, 552144, 552145, 552146, 552147, 552148, 552149, 552150, 552151, 552152, 552158, 552159, 552160, 552163, 552168, 552179, 552187, 552188, 552192, 552193, 552195, 552199, 552200, 552201, 552202, 552203, 552204, 552205, 552206, 552207, 552208, 552210, 552211, 552213, 552214, 552222, 552246, 552247, 552248, 552253, 552254, 552255, 552258, 552294, 552301, 552302, 552306, 552307, 552308, 552309, 552310, 552312, 552314, 552315, 552317, 552318, 552321, 552322, 552323, 552325, 552332, 552337, 552339, 552347, 552348, 552349, 552354, 552355, 552358, 552359, 552360, 552361, 552362, 552363, 552364, 552365, 552366, 552367, 552368, 552369, 552371, 552373, 552374, 552375, 552376, 552377, 552378, 552379, 552403, 552408, 552409, 552411, 552418, 552419, 552420, 552424, 552442, 552464, 552465, 552466, 552467, 552472, 552474, 552475, 552477, 552478, 552521, 552522, 552523, 552527, 552528, 552529, 552530, 552534, 552567, 552578, 552579, 552584, 552586, 552587, 552588, 552590, 552789, 552803, 552804, 552805, 552808, 552816, 552817, 552818, 552819, 552820, 552821, 552822, 552823, 552824, 552828, 552829, 552830, 552833, 552834, 552835, 552842, 552843, 552844, 552846, 552848, 552849, 552850, 552851, 552852, 552853, 552854, 552855, 552856, 552857, 552858, 552859, 552860, 552861, 552863, 552864, 552865, 552872, 552894, 552903, 552904, 552907, 552909, 552910, 552911, 552913, 552914, 552915, 552916, 552917, 552918, 552922, 552923, 552925, 552927, 552928, 552929, 552930, 552931, 552932, 552933, 552934, 552935, 552936, 552937, 552938, 552939, 552940, 552941, 552942, 552943, 552944, 552945, 552946, 552957, 552961, 552966, 552969, 552971, 552972, 552974, 552976, 552979, 552980, 552981, 552983, 552984, 552988, 552989, 552990, 552991, 552995, 552996, 552998, 552999, 553001, 553002, 553003, 553004, 553006, 553008, 553009, 553010, 553011, 553012, 566828, 566829, 566830, 566831, 566832, 577120, 577121, 577122, 577123, 577124, 577125, 577126, 577127, 577128, 577129, 577130, 577131, 577132, 577133, 577134, 577135, 577136, and 582666.
In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 70% inhibition of a HBV mRNA, SEQ ID NOs: 12, 17, 18, 20, 21, 22, 24, 25, 26, 27, 28, 29, 39, 40, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 83, 89, 92, 96, 98, 100, 103, 112, 123, 125, 126, 127, 136, 137, 139, 140, 142, 143, 145, 147, 149, 151, 153, 166, 167, 168, 174, 176, 177, 178, 179, 181, 186, 187, 188, 190, 198, 201, 207, 209, 210, 211, 212, 213, 224, 225, 226, 227, 232, 234, 240, 321, 324, 325, 326, 327, 329, 330, 331, 332, 335, 336, 337, 339, 342, 343, 344, 345, 346, 357, 358, 359, 363, 364, 368, 370, 371, 372, 375, 376, 377, 378, 379, 382, 383, 384, 387, 388, 389, 390, 391, 392, 395, 397, 400, 401, 402, 403, 404, 405, 409, 410, 412, 413, 417, 418, 420 421, 422, 423, 424, 425, 426, 427, 430, 435, 436, 438, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 457, 459, 462, 463, 464, 465, 466, 467, 468, 469, 470, 473, 474, 475, 478, 479, 480, 481, 482, 483, 484, 485, 487, 488, 489, 490, 492, 496, 497, 500, 501, 504, 505, 508, 510, 512, 513, 514, 515, 516, 517, 520, 521, 524, 525, 526, 527, 528, 529, 530, 531, 539, 543, 544, 545, 546, 547, 548, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 566, 567, 569, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 588, 589, 590, 595, 596, 597, 598, 599, 600, 601, 602, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 623, 624, 625, 626, 627, 628, 631, 632, 634, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 652, 653, 654, 657, 658, 659, 660, 661, 662, 663, 665, 666, 667, 669, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 682, 684, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 733, 734, 735, 736, 737, 738, 740, 741, 742, 745, 754, 755, 756, 757, 768, 777, 778, 779, 780, 781, 782, 784, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 814, 815, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 833, 834, 842, 843, 844, 849, 852, 854, 860, 892, 893, 894, 895, 896, 897, 898, 899, 900, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1320, 1322, 1323, 1324, 1325, 1326, 1327, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, and 1350, 1367, 1368, 1369, 1370, 1372, and 1376.
In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 80% inhibition of a HBV mRNA, ISIS IDs: 510100, 509931, 510116, 505317, 505319, 505323, 505326, 505327, 505330, 505339, 505346, 505347, 505358, 509934, 146786, 524414, 524415, 524416, 524418, 524419, 524425, 524426, 524431, 524432, 524434, 524446, 524447, 524452, 524459, 524460, 524466, 524469, 524475, 524477, 524478, 524479, 524482, 524485, 524490, 524491, 524492, 524493, 524494, 524495, 524499, 524502, 524503, 524507, 524510, 524511, 524512, 524520, 524525, 524528, 524532, 524533, 524534, 524535, 524536, 524540, 524541, 524547, 524552, 524553, 524556, 524561, 524564, 524565, 524568, 524570, 524571, 524572, 524573, 524578, 524580, 524586, 524590, 524591, 524594, 524595, 524602, 524604, 524606, 524607, 524610, 524611, 524614, 524616, 524617, 524618, 524619, 524620, 524621, 524633, 524634, 524635, 524636, 524637, 524641, 524643, 524644, 524646, 524649, 524650, 524651, 524657, 524662, 524664, 524667, 524670, 524678, 524679, 524680, 524686, 524688, 524690, 524691, 524692, 524695, 524698, 524699, 524701, 524702, 524704, 524705, 524706, 524707, 524708, 524709, 524713, 524715, 524716, 524717, 524718, 524721, 524726, 524727, 524728, 524729, 524730, 524731, 524733, 524734, 524735, 524737, 524739, 524741, 524742, 524743, 524747, 524748, 524749, 524751, 524752, 524754, 524758, 524760, 524762, 524763, 524764, 524767, 524768, 524769, 524771, 524773, 524777, 524778, 524779, 524780, 524781, 524783, 524784, 524788, 524789, 524791, 524792, 524793, 524794, 524795, 524796, 524797, 524798, 524801, 524803, 524804, 524805, 524806, 524807, 524808, 524809, 524810, 524811, 524813, 524816, 524819, 524822, 524823, 524824, 524827, 524828, 524829, 524833, 524842, 524844, 524880, 524881, 524882, 524884, 524886, 524887, 524888, 524889, 524890, 524891, 524893, 524907, 524908, 524980, 524986, 524987, 551921, 551924, 551925, 551953, 551956, 551957, 551984, 551986, 551987, 551989, 551990, 551993, 551994, 551995, 551996, 551997, 551998, 551999, 552000, 552005, 552006, 552018, 552019, 552020, 552021, 552022, 552023, 552024, 552025, 552026, 552027, 552028, 552029, 552030, 552031, 552032, 552033, 552034, 552039, 552044, 552046, 552050, 552051, 552052, 552053, 552054, 552055, 552056, 552057, 552058, 552059, 552060, 552061, 552062, 552063, 552064, 552065, 552073, 552077, 552078, 552079, 552080, 552082, 552083, 552084, 552085, 552086, 552087, 552088, 552089, 552090, 552091, 552092, 552093, 552094, 552095, 552096, 552097, 552098, 552138, 552139, 552145, 552146, 552147, 552149, 552192, 552193, 552199, 552200, 552201, 552207, 552246, 552247, 552253, 552301, 552307, 552308, 552310, 552317, 552347, 552348, 552354, 552355, 552360, 552361, 552362, 552363, 552364, 552365, 552366, 552367, 552371, 552375, 552464, 552465, 552521, 552808, 552816, 552817, 552818, 552819, 552820, 552822, 552824, 552834, 552844, 552849, 552850, 552851, 552852, 552853, 552854, 552916, 552922, 552923, 552925, 552930, 552931, 552932, 552933, 552936, 552937, 552938, 552939, 552942, 552943, 552944, 552980, 552988, 552989, 552996, 552998, 553002, 553003, 566828, 566829, 566830, 566831, 566832, 577120, 577121, 577122, 577123, 577124, 577125, 577126, 577127, 577128, 577130, 577131, 577132, 577133, 577134, 577135, 577136, and 582666.
In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 80% inhibition of a HBV mRNA, SEQ ID NOs: 17, 20, 22, 24, 26, 28, 39, 40, 50, 51, 83, 89, 103, 123, 126, 127, 136, 137, 143, 147, 149, 168, 176, 177, 178, 179, 187, 188, 210, 211, 212, 224, 225, 226, 227, 232, 325, 326, 327, 329, 330, 336, 337, 342, 343, 345, 357, 358, 363, 370, 371, 376, 379, 387, 388, 389, 392, 395, 400, 401, 402, 403, 404, 405, 409, 412, 413, 417, 420 421, 422, 430, 435, 438, 442, 443, 444, 445, 446, 450, 451, 457, 462, 463, 466, 474, 475, 478, 480, 481, 482, 483, 488, 490, 496, 500, 501, 504, 505, 512, 514, 516, 517, 520, 521, 524, 526, 527, 528, 529, 530, 531, 543, 544, 545, 546, 547, 551, 553, 554, 555, 559, 560, 561, 567, 572, 574, 577, 580, 588, 589, 590, 596, 598, 600, 601, 602, 605, 608, 609, 611, 612, 614, 615, 616, 617, 618, 619, 623, 625, 626, 627, 628, 631, 636, 637, 638, 639, 640, 641, 643, 644, 645, 646, 648, 650, 652, 653, 654, 658, 659, 660, 662, 663, 665, 669, 671, 673, 674, 675, 678, 679, 680, 682, 684, 688, 689, 690, 691, 692, 694, 695, 699, 700, 702, 703, 704, 705, 706, 707, 708, 709, 712, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 725, 728, 731, 734, 735, 736, 740, 741, 745, 756, 791, 792, 793, 795, 797, 798, 799, 800, 801, 802, 804, 805, 806, 807, 819, 820, 892, 898, 899, 1292, 1293, 1295, 1296, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1310, 1312, 1316, 1322, 1324, 1325, 1326, 1327, 1330, 1331, 1332, 1333, 1334, 1335, 1338, 1339, 1340, 1341, 1344, 1345, 1349, 1350, 1368, 1372, and 1376.
In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 90% inhibition of a HBV mRNA, ISIS IDs: 524414, 524415, 524432, 524460, 524466, 524469, 524475, 524477, 524493, 524512, 524535, 524540, 524552, 524561, 524572, 524617, 524619, 524634, 524641, 524644, 524657, 524667, 524691, 524698, 524699, 524701, 524706, 524707, 524709, 524713, 524715, 524716, 524718, 524721, 524726, 524729, 524730, 524731, 524733, 524734, 524735, 524739, 524743, 524754, 524763, 524764, 524767, 524771, 524780, 524781, 524784, 524788, 524789, 524791, 524792, 524793, 524794, 524795, 524796, 524797, 524798, 524801, 524803, 524804, 524805, 524806, 524807, 524808, 524809, 524810, 524811, 524822, 524827, 524842, 551986, 551987, 551989, 552005, 552018, 552019, 552020, 552021, 552022, 552023, 552025, 552046, 552050, 552051, 552052, 552053, 552054, 552055, 552057, 552082, 552083, 552084, 552085, 552086, 552087, 552088, 552089, 552092, 552093, 552096, 552097, 552307, 552317, 552355, 552361, 552362, 552363, 552817, 552851, 552922, 552923, 566828, 566829, 566830, 566831, 566832, 577120, 577121, 577122, 577123, 577124, 577125, 577126, 577127, 577128, 577130, 577131, 577132, 577134, 577135, 577136, and 582666.
In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 90% inhibition of a HBV mRNA, SEQ ID NOs: 17, 24, 50, 51, 137, 143, 147, 176, 211, 212, 224, 226, 227, 325, 326, 343, 371, 376, 379, 403, 422, 445, 450, 462, 482, 527, 529, 544, 551, 554, 567, 577, 601, 608, 609, 611, 616, 617, 619, 623, 625, 626, 628, 631, 636, 639, 640, 641, 643, 644, 645, 646, 650, 654, 665, 674, 675, 678, 682, 691, 692, 695, 699, 700, 702, 703, 704, 705, 706, 707, 708, 709, 712, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 735, 801, 804, 805, 807, 1296, 1302, 1303, 1304, 1312, 1325, 1326, 1332, 1334, 1340, 1345, 1349, and 1376.
In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 95% inhibition of a HBV mRNA, ISIS IDs: 524619, 524634, 524641, 505339, 524698, 524709, 524718, 524731, 524734, 524789, 524791, 524792, 524793, 524794, 524795, 524796, 524797, 524798, 524801, 524803, 524804, 524805, 524806, 505346, 146785, 524807, 505347, 524808, 524809, 524810, 524811, 146786, 525101, 525102, 525103, 525107, 525108, 525109, 525110, 525111, 525112, 525113, 525114, 525115, 525116, 525117, 525118, 525119, 525120, 552018, 552050, 552019, 552051, 552020, 552052, 551987, 552021, 552053, 552005, 552022, 552054, 551989, 552023, 552055, 552084, 552085, 552086, 552087, 552088, 552361, 552317, 566831, 577123, 577124, 566830, 566828, 566829, 577127, 577135, 577132, 577136, 566832, and 577122.
In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 95% inhibition of a HBV mRNA, SEQ ID NOs: 17, 50, 137, 143, 187, 210, 212, 224, 529, 544, 551, 608, 619, 628, 641, 645, 700, 702, 703, 704, 705, 706, 707, 708, 709, 712, 715, 716, 717, 718, 719, 720, 721, 722, 723, 1014, 1015, 1016, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1236, 1302, 1312, 1334, 1340, 1345, 1349.
Certain embodiments provide methods of treating HBV related disease, disorder, or condition in an animal, comprising administering to an animal in need thereof a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5-310, 321-802, 804-1272, 1288-1350, 1364-1372, 1375, 1376, and 1379.
Certain embodiments provide methods of treating HBV related disease, disorder, or condition in an animal, comprising administering to an animal in need thereof a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17, 51, 86, 93, 95, 98, 100, 102, 104, 106, 109, 112, 115, 117, 137, 140, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 167, 168, 176, 177-179, 181, 188, 190-192, 194, 199, 201, 208, 209, 211, 226, 230-237, 244, 245, 247, 252, 254, 256, 258, 260, 262, 264, 266, 271, 1318-1347, 1364-1372, 1375, 1376, and 1379, wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar and/or constrained ethyl (cEt) sugar. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, or 4 sugar modified nucleosides.
Certain embodiments provide a method of reducing HBV expression in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5-310, 321-802, 804-1272, 1288-1350, 1364-1372, 1375, 1376, and 1379.
Certain embodiments provide a method of reducing HBV expression in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17, 51, 86, 93, 95, 98, 100, 102, 104, 106, 109, 112, 115, 117, 137, 140, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 167, 168, 176, 177-179, 181, 188, 190-192, 194, 199, 201, 208, 209, 211, 226, 230-237, 244, 245, 247, 252, 254, 256, 258, 260, 262, 264, 266, 271, 1318-1347, 1364-1372, 1375, and 1376, wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar and/or constrained ethyl (cEt) sugar. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, or 5 sugar modified nucleosides.
Certain embodiments provide a method of preventing, ameliorating or treating an HBV-related disease, disorder or condition in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17, 51, 86, 93, 95, 98, 100, 102, 104, 106, 109, 112, 115, 117, 137, 140, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 167, 168, 176, 177-179, 181, 188, 190-192, 194, 199, 201, 208, 209, 211, 226, 230-237, 244, 245, 247, 252, 254, 256, 258, 260, 262, 264, 266, 271, 1318-1347, 1364-1372, 1375, and 1376, wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar and/or constrained ethyl (cEt) sugar. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, or 5 sugar modified nucleosides.
Certain embodiments provide methods of treating HBV related disease, disorder, or condition in an animal, comprising administering to an animal in need thereof a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5, 15, 16, 33, 39-95, 123-135, 163-175, 180-310, 321-406, 413-455, 461-802, or 804-1272.
Certain embodiments provide methods of treating HBV related disease, disorder, or condition in an animal, comprising administering to an animal in need thereof a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 6-14, 17-32, 34-38, 96-122, 136-162, 176-179, 407-412, 456-462, 523-538 wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar.
In certain embodiments, the modified oligonucleotide is 14 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-3 or 2 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, or 5 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 17 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3-4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, or 6 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 18 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 3-5, or 4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 20 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, or 5 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, 6, 7, or 8 sugar modified nucleosides.
Certain embodiments provide a method of reducing HBV expression in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5-310, 321-802, 804-1272, or 1288-1350.
Certain embodiments provide a method of reducing HBV expression in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5, 15, 16, 33, 39-95, 123-135, 163-175, 180-310, 321-406, 413-455, 461-802, or 804-1272.
Certain embodiments provide a method of reducing HBV expression in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 6-14, 17-32, 34-38, 96-122, 136-162, 176-179, 407-412, 456-462, 523-538 wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar.
In certain embodiments, the modified oligonucleotide is 14 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-3 or 2 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, or 5 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 17 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, or 6 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 17 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3-4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 18 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 3-5, or 4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 20 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, 6, 7, or 8 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 20 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, or 5 sugar modified nucleosides.
Certain embodiments provide a method of preventing, ameliorating or treating an HBV-related disease, disorder or condition in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5-310, 321-802, 804-1272, or 1288-1350, wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar. In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5-310, 321-802, or 804-1272. In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5, 15, 16, 33, 39-95, 123-135, 163-175, 180-310, 321-406, 413-455, 461-802, or 804-1272. In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 6-14, 17-32, 34-38, 96-122, 136-162, 176-179, 407-412, 456-462, 523-538 wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar. In certain embodiments, the modified oligonucleotide is 14 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-3 or 2 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, or 5 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 17 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, or 6 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 17 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3-4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 18 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 3-5, or 4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 20 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, 6, 7, or 8 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 20 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, or 5 sugar modified nucleosides.
Examples of HBV-related diseases, disorders or conditions include, but are not limited to chronic HBV infection, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, and conditions having symptoms which may include any or all of the following: flu-like illness, weakness, aches, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain over the liver area of the body, clay- or grey-colored stool, itching all over, and dark-colored urine, when coupled with a positive test for presence of a hepatitis B virus, a hepatitis B viral antigen, or a positive test for the presence of an antibody specific for a hepatitis B viral antigen.
Certain embodiments provide a method of reducing HBV mRNA expression in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. In certain embodiments, reduction of HBV mRNA expression in an animal prevents, ameliorates or treats an HBV-related disease, disorder or condition. In certain embodiments, reduction of HBV mRNA expression in an animal prevents, ameliorates or treats liver disease. In certain embodiments, the HBV mRNA expression is reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.
Certain embodiments provide a method of reducing HBV protein levels in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. In certain embodiments, reduction of HBV protein levels in an animal prevents, ameliorates or treats an HBV-related disease, disorder or condition. In certain embodiments, reduction of HBV protein levels in an animal prevents, ameliorates or treats liver disease. In certain embodiments, the HBV protein level is reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.
Certain embodiments provide a method of reducing HBV DNA levels in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. In certain embodiments, reduction of HBV DNA levels in an animal prevents, ameliorates or treats an HBV-related disease, disorder or condition. In certain embodiments, the mammal may be human, and the hepatitis B virus may be a human hepatitis B virus. More particularly, the human hepatitis B virus may be any of the human geographical genotypes: A (Northwest Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, Korea, China, Japan, Polynesia, Vietnam); D (Mediterranean area, Middle East, India); E (Africa); F (Native Americans, Polynesia); G (United States, France); or H (Central America). In certain embodiments, reduction of HBV DNA levels in an animal prevents, ameliorates or treats liver disease. In certain embodiments, the HBV DNA level is reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.
Certain embodiments provide a method of reducing HBV antigen levels in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. In certain embodiments, the antigen is HBsAG or HBeAG. In certain embodiments, reduction of HBV antigen levels in an animal prevents, ameliorates or treats an HBV-related disease, disorder or condition. In certain embodiments, reduction of HBV antigen levels in an animal prevents, ameliorates or treats liver disease. In certain embodiments, the HBV antigen levels are reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.
Certain embodiments provide a method of reducing HBV DNA and HBV antigen in a animal infected with a hepatitis B virus, comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. In certain embodiments, the antigen is HBsAG or HBeAG. In certain embodiments, the amount of HBV antigen may be sufficiently reduced to result in seroconversion, defined as serum HBeAg absence plus serum HBeAb presence if monitoring HBeAg as the determinant for seroconversion, or defined as serum HBsAg absence if monitoring HBsAg as the determinant for seroconversion, as determined by currently available detection limits of commercial ELISA systems.
Certain embodiments provide a method for treating an animal with a HBV related disease, disorder or condition comprising: a) identifying said animal with the HBV related disease, disorder or condition, and b) administering to said animal a therapeutically effective amount of a compound or composition comprising a modified oligonucleotide consisting of 14 to 20 linked nucleosides and having a nucleobase sequence at least 90% complementary to any of SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, as measured over the entirety of said modified oligonucleotide. In certain embodiments, the therapeutically effective amount of the compound or composition administered to the animal treats or reduces the HBV related disease, disorder or condition, or a symptom thereof, in the animal. In certain embodiments, the HBV related disease, disorder or condition is a liver disease. In certain embodiments, the related disease, disorder or condition is chronic HBV infection, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, or liver disease-related to transplantation.
Certain embodiments provide a method for treating an animal with a HBV related disease, disorder or condition comprising: a) identifying said animal with the HBV related disease, disorder or condition, and b) administering to said animal a therapeutically effective amount of a compound or composition comprising a modified oligonucleotide consisting of 14 to 20 linked nucleosides and having a nucleobase sequence at least 90% complementary to SEQ ID NO: 1, as measured over the entirety of said modified oligonucleotide. In certain embodiments, the therapeutically effective amount of the compound or composition administered to the animal treats or reduces the HBV related disease, disorder or condition, or a symptom thereof, in the animal. In certain embodiments, the HBV related disease, disorder or condition is a liver disease. In certain embodiments, the related disease, disorder or condition is chronic HBV infection, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, or liver disease-related to transplantation.
In certain embodiments, HBV has the sequence as set forth in GenBank Accession Numbers U95551.1 (incorporated herein as SEQ ID NO: 1) or any variant or fragment thereof. In certain embodiments, HBV has truncated portions of the human sequence as set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363.
In certain embodiments, the animal is a human.
In certain embodiments, the compounds or compositions are designated as a first agent. In certain embodiments, the methods comprise administering a first agent and one or more second agents. In certain embodiments, the methods comprise administering a first agent and one or more second agents. In certain embodiments, the first agent and one or more second agents are co-administered. In certain embodiments the first agent and one or more second agents are co-administered sequentially or concomitantly.
In certain embodiments, the one or more second agents are also a compound or composition described herein. In certain embodiments, the one or more second agents are different from a compound or composition described herein. Examples of one or more second agents include, but are not limited to, an anti-inflammatory agent, chemotherapeutic agent or anti-infection agent.
In other related embodiments, the additional therapeutic agent may be an HBV agent, an HCV agent, a chemotherapeutic agent, an antibiotic, an analgesic, a non-steroidal anti-inflammatory (NSAID) agent, an antifungal agent, an antiparasitic agent, an anti-nausea agent, an anti-diarrheal agent, or an immunosuppressant agent.
In certain embodiments, the one or more second agents are an HBV agent. In certain embodiments the HBV agent can include, but is not limited to, interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1 (pegylated and unpegylated), ribavirin; an HBV RNA replication inhibitor; a second antisense oligomer; an HBV therapeutic vaccine; an HBV prophylactic vaccine; lamivudine (3TC); entecavir (ETV); tenofovir diisoproxil fumarate (TDF); telbivudine (LdT); adefovir; or an HBV antibody therapy (monoclonal or polyclonal).
In certain embodiments, the one or more second agents are an HCV agent. In certain embodiments the HBV agent can include, but is not limited to interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1 (pegylated and unpegylated); ribavirin; an HCV RNA replication inhibitor (e.g., ViroPharma's VP50406 series); an HCV antisense agent; an HCV therapeutic vaccine; an HCV protease inhibitor; an HCV helicase inhibitor; or an HCV monoclonal or polyclonal antibody therapy.
In certain embodiments, the one or more second agents are an anti-inflammatory agent (i.e., an inflammation lowering therapy). In certain embodiments the inflammation lowering therapy can include, but is not limited to, a therapeutic lifestyle change, a steroid, a NSAID or a DMARD. The steroid can be a corticosteroid. The NSAID can be an aspirin, acetaminophen, ibuprofen, naproxen, COX inhibitors, indomethacin and the like. The DMARD can be a TNF inhibitor, purine synthesis inhibitor, calcineurin inhibitor, pyrimidine synthesis inhibitor, a sulfasalazine, methotrexate and the like.
In certain embodiments, the one or more second agents are a chemotherapeutic agent (i.e., a cancer treating agent). Chemotherapeutic agents can include, but are not limited to, daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin, gemcitabine and diethylstilbestrol (DES).
In certain embodiments, the one or more second agents are an anti-infection agent. Examples of anti-infection agents include, but are not limited to, antibiotics, antifungal drugs and antiviral drugs.
In certain embodiments, administration comprises parenteral administration.
Certain embodiment provides a method for reducing an amount of HBV mRNA, DNA, protein and/or an amount of HBV antigen in a mammal infected with a hepatitis B virus, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal in need thereof so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of HBV mRNA, protein and an amount of HBV antigen in the mammal before treatment. In some embodiments, the mammal may be human, and the hepatitis B virus may be a human hepatitis B virus. More particularly, the human hepatitis B virus may be any of the human geographical genotypes: A (Northwest Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, Korea, China, Japan, Polynesia, Vietnam); D (Mediterranean area, Middle East, India); E (Africa); F (Native Americans, Polynesia); G (United States, France); or H (Central America).
In certain embodiments, a method is provided for reducing an amount of HBV mRNA, DNA, protein and/or an amount of HBV antigen in a mammal infected with a hepatitis B virus, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal in need thereof so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of HBV mRNA, protein and an amount of HBV antigen in the mammal before treatment, wherein the amount of mRNA is reduced at least 70% compared to the amount before administration of the modified antisense oligonucleotide. In certain embodiments, a method is provided for reducing an amount of HBV mRNA, DNA, protein and/or an amount of HBV antigen in a mammal infected with a hepatitis B virus, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal in need thereof so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of HBV mRNA, protein and an amount of HBV antigen in the mammal before treatment, wherein the amount of mRNA is reduced at least 75% compared to the amount before administration of the modified antisense oligonucleotide. In certain embodiments, a method is provided for reducing an amount of HBV mRNA, DNA, protein and/or an amount of HBV antigen in a mammal infected with a hepatitis B virus, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal in need thereof so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of HBV mRNA, protein and an amount of HBV antigen in the mammal before treatment, wherein the amount of mRNA is reduced at least 80% compared to the amount before administration of the modified antisense oligonucleotide. In certain embodiments, a method is provided for reducing an amount of HBV mRNA, DNA, protein and/or an amount of HBV antigen in a mammal infected with a hepatitis B virus, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal in need thereof so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of HBV mRNA, protein and an amount of HBV antigen in the mammal before treatment, wherein the amount of mRNA is reduced at least 85% compared to the amount before administration of the modified antisense oligonucleotide. In certain embodiments, a method is provided for reducing an amount of HBV mRNA, DNA, protein and/or an amount of HBV antigen in a mammal infected with a hepatitis B virus, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal in need thereof so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of HBV mRNA, protein and an amount of HBV antigen in the mammal before treatment, wherein the amount of mRNA is reduced at least 90% compared to the amount before administration of the modified antisense oligonucleotide. In certain embodiments, a method is provided for reducing an amount of HBV mRNA, DNA, protein and/or an amount of HBV antigen in a mammal infected with a hepatitis B virus, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal in need thereof so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of HBV mRNA, protein and an amount of HBV antigen in the mammal before treatment, wherein the amount of mRNA is reduced at least 95% compared to the amount before administration of the modified antisense oligonucleotide. In related methods, the HBV antigen may be HBsAg or may be HBeAg, and more particularly, the amount of HBV antigen may be sufficiently reduced to result in seroconversion, defined as serum HBeAg absence plus serum HBeAb presence if monitoring HBeAg as the determinant for seroconversion, or defined as serum HBsAg absence if monitoring HBsAg as the determinant for seroconversion, as determined by currently available detection limits of commercial ELISA systems.
Certain embodiment provides a method for promoting seroconversion of a hepatitis B virus in a mammal infected with HBV, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal infected with hepatitis B; monitoring for presence of HBeAg plus HBeAb in a serum sample of the mammal, or monitoring for presence of HBsAg in a serum sample of the mammal, such that the absence of HBeAg plus the presence of HBeAb in the serum sample if monitoring HBeAg as the determinant for seroconversion, or the absence of HBsAg in the serum sample if monitoring HBsAg as the determinant for seroconversion, as determined by current detection limits of commercial ELISA systems, is indication of seroconversion in the mammal.
Certain embodiments provide the use of a compound or composition as described herein for preventing, ameliorating or treating liver disease, or symptom thereof, in an animal. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17, 51, 86, 93, 95, 98, 100, 102, 104, 106, 109, 112, 115, 117, 137, 140, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 167, 168, 176, 177-179, 181, 188, 190-192, 194, 199, 201, 208, 209, 211, 226, 230-237, 244, 245, 247, 252, 254, 256, 258, 260, 262, 264, 266, 271, 1318-1347, 1364-1372, 1375, 1376, and 1379.
In certain embodiments, the compounds or compositions as described herein are efficacious by virtue of having at least one of an in vitro IC50 of less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 65 nM, less than 60 nM, less than 55 nM, less than 50 nM, less than 49 nM, less than 47 nM, less than 46 nM, when delivered to HepG2.2.1 cells. In certain embodiments inhibition is measured with primer probe set RTS3370, as described herein.
In certain embodiments, the compounds or compositions as described herein are efficacious by virtue of having at least one of an in vitro IC50 of less than 250 nM, less than 200 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 35 nM, less than 34 nM, less than 33 nM, less than 32 nM, less than 31 nM, when delivered to HepG2.2.1 cells. In certain embodiments inhibition is measured with primer probe set RTS3371, as described herein.
In certain embodiments, the compounds or compositions as described herein are efficacious by virtue of having at least one of an in vitro IC50 of less than 20 μM, less than 10 μM, less than 9.5 μM, less than 9.0 μM, less than 8.5 μM, less than 8.0 μM, less than 7.5 μM, less than 7.0 μM, less than 6.5 μM, less than 6.0 μM, less than 5.5 μM, less than 5.0 μM, less than 4.5 μM, less than 4.0 μM, less than 3.5 μM, less than 3.0 μM, less than 2.5 μM, when delivered to HepG2.2.1 cells as described herein.
In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having at least one of an increase an ALT or AST value of no more than 4 fold, 3 fold, or 2 fold over saline treated animals or an increase in liver, spleen, or kidney weight of no more than 30%, 20%, 15%, 12%, 10%, 5%, or 2%. In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having no increase of ALT or AST over saline treated animals. In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having no increase in liver, spleen, or kidney weight over saline treated animals. In certain embodiments, these compounds or compositions include ISIS 146779, ISIS 146786, ISIS 505317, ISIS 505329, ISIS 505332, ISIS 505346, ISIS 505347, ISIS 505358, ISIS 509926, ISIS 509927, ISIS 509932, ISIS 509934, ISIS 509960, ISIS 509974, ISIS 510038, ISIS 510039, ISIS 510040, ISIS 510041, ISIS 510050 ISIS 509975, ISIS 510100, ISIS 510106, and ISIS 510116. In certain embodiments, such compounds or compositions include compounds comprising the nucleobase sequence of any one of SEQ ID NOs: 5-310, 321-802, or 804-1272. In certain embodiments, such compounds or compositions include compounds comprising the nucleobase sequence of any one of SEQ ID NOs: 5, 15, 16, 33, 39-95, 123-135, 163-175, 180-310, 321-406, 413-455, 461-802, or 804-1272. In certain embodiments, such compounds or compositions include compounds comprising the nucleobase sequence of any one of SEQ ID NOs: 6-14, 17-32, 34-38, 96-122, 136-162, 176-179, 407-412, 456-462, 523-538 (update SEQ ID NOs) wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar. In certain embodiments, the modified oligonucleotide is 14 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-3 or 2 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 17 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3-4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 18 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 3-5, or 4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 20 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, or 5 sugar modified nucleosides.
Certain embodiments provide the use of a compound or composition as described herein in the manufacture of a medicament for treating, ameliorating, delaying or preventing an HBV-related disease, disorder or condition in an animal.
Certain embodiments provide the use of a compound or composition as described herein in the manufacture of a medicament for treating, ameliorating, delaying or preventing liver disease in an animal.
Certain embodiments provide a kit for treating, preventing, or ameliorating an HBV-related disease, disorder or condition, or a symptom thereof, as described herein wherein the kit comprises: a) a compound or compositions as described herein; and optionally b) an additional agent or therapy as described herein. The kit can further include instructions or a label for using the kit to treat, prevent, or ameliorate the HBV-related disease, disorder or condition.
Antisense Compounds
Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be “antisense” to a target nucleic acid, meaning that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 10-30 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 12 to 30 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 12 to 22 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 14 to 30 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 14 to 20 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 15 to 30 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 15 to 20 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 16 to 30 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 16 to 20 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 17 to 30 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 17 to 20 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 18 to 30 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 18 to 21 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 18 to 20 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 20 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, 18 to 21 subunits, 20 to 30 subunits, or 12 to 22 linked subunits, respectively. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 14 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 16 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 17 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 18 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 20 subunits in length. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such embodiments, the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides.
In certain embodiments antisense oligonucleotides targeted to a HBV nucleic acid may be shortened or truncated. For example, a single subunit may be deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to a HBV nucleic acid may have two subunits deleted from the 5′ end, or alternatively may have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end.
When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5′ end and one subunit added to the 3′ end.
It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.
Gautschi et al. (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.
Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.
Antisense Compound Motifs
In certain embodiments, antisense compounds targeted to a HBV nucleic acid have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.
Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.
Antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE and 2′-O—CH3, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a constrained ethyl). In certain embodiments, nucleosides in the wings may include several modified sugar moieties, including, for example 2′-MOE and bicyclic sugar moieties such as constrained ethyl or LNA. In certain embodiments, wings may include several modified and unmodified sugar moieties. In certain embodiments, wings may include various combinations of 2′-MOE nucleosides, bicyclic sugar moieties such as constrained ethyl nucleosides or LNA nucleosides, and 2′-deoxynucleosides.
Each distinct region may comprise uniform sugar moieties, variant, or alternating sugar moieties. The wing-gap-wing motif is frequently described as “X-Y-Z”, where “X” represents the length of the 5′-wing, “Y” represents the length of the gap, and “Z” represents the length of the 3′-wing. “X” and “Z” may comprise uniform, variant, or alternating sugar moieties. In certain embodiments, “X” and “Y” may include one or more 2′-deoxynucleosides. “Y” may comprise 2′-deoxynucleosides. As used herein, a gapmer described as “X-Y-Z” has a configuration such that the gap is positioned immediately adjacent to each of the 5′-wing and the 3′ wing. Thus, no intervening nucleotides exist between the 5′-wing and gap, or the gap and the 3′-wing. Any of the antisense compounds described herein can have a gapmer motif. In certain embodiments, “X” and “Z” are the same; in other embodiments they are different. In certain embodiments, “Y” is between 8 and 15 nucleosides. X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleosides.
In certain embodiments, gapmers provided herein include, for example, 11-mers having a motif of 1-9-1.
In certain embodiments, gapmers provided herein include, for example, 12-mers having a motif of 1-9-2, 2-9-1, or 1-10-1.
In certain embodiments, gapmers provided herein include, for example, 13-mers having a motif of 1-9-3, 2-9-2, 3-9-1, 1-10-2, or 2-10-1.
In certain embodiments, gapmers provided herein include, for example, 14-mers having a motif of 1-9-4, 2-9-3, 3-9-2, 4-9-1, 1-10-3, 2-10-2, or 3-10-1.
In certain embodiments, gapmers provided herein include, for example, 15-mers having a motif of 1-9-5, 2-9-4, 3-9-3, 4-9-2, 5-9-1, 1-10-4, 2-10-3, 3-10-2, or 4-10-1.
In certain embodiments, gapmers provided herein include, for example, 16-mers having a motif of 4-8-4, 2-9-5, 3-9-4, 4-9-3, 5-9-2, 1-10-5, 2-10-4, 3-10-3, 4-10-2, 3-8-5, or 5-10-1.
In certain embodiments, gapmers provided herein include, for example, 17-mers having a motif of 3-9-5, 3-10-4, 4-9-4, 5-9-3, 2-10-5, 3-10-4, 4-10-3, 5-10-2, 2-9-6, 5-8-4, 5-7-5, 6-7-4, or 6-9-2.
In certain embodiments, gapmers provided herein include, for example, 18-mers having a motif of 4-9-5, 5-9-4, 3-10-5, 4-10-4, or 5-10-3.
In certain embodiments, gapmers provided herein include, for example, 19-mers having a motif of 5-9-5, 4-10-5, or 5-10-4.
In certain embodiments, gapmers provided herein include, for example, 20-mers having a motif of 5-10-5, 2-10-8, 8-10-2, 3-10-7, 7-10-3, 4-10-6, or 6-10-4.
In certain embodiments, the antisense compound has a “wingmer” motif, having a wing-gap or gap-wing configuration, i.e. an X-Y or Y-Z configuration as described above for the gapmer configuration. Thus, wingmer configurations provided herein include, but are not limited to, for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13, 5-13, 5-8, or 6-8.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 2-10-2 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 3-10-3 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 4-10-4 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 5-10-5 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 3-10-4 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 2-10-4 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 2-10-8 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 8-10-2 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 3-10-7 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 7-10-3 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 4-10-6 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 6-10-4 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 2-9-6 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 6-9-2 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 4-9-4 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 5-9-3 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 3-9-5 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 5-9-2 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 2-9-5 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 4-9-3 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 3-9-4 gapmer motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a gap-widened motif.
In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a gapmer motif in which the gap consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 linked nucleosides.
In certain embodiments, the antisense compounds targeted to a HBV nucleic acid has any of the following sugar motifs:
k-d(10)-k
e-d(10)-k
k-d(10)-e
k-k-d(10)-k-k
k-k-d(10)-e-e
e-e-d(10)-k-k
k-k-k-d(10)-k-k-k
e-e-e-d(10)-k-k-k
k-k-k-d(10)-e-e-e
k-k-k-d(10)-k-k-k
e-k-k-d(10)-k-k-e
e-e-k-d(10)-k-k-e
e-d-k-d(10)-k-k-e
e-k-d(10)-k-e-k-e
k-d(10)-k-e-k-e-e
e-e-k-d(10)-k-e-k-e
e-d-d-k-d(9)-k-k-e
e-e-e-e-d(9)-k-k-e
e-e-e-e-e-d(10)-e-e-e-e-e
k-d-k-d-k-d(9)-e-e
e-e-k-k-d(9)-e-k-e-e
k-d-k-d-k-d(10)-e-e-e-e-e
k-e-k-d(10)-k-e-k
e-e-e-k-k-d(8)-e-e-e-e
e-e-e-k-k-d(7)-k-k-e-e-e
e-e-e-k-d(9)-k-e-e-e
e-e-e-k-k-d(7)-k-k-e-e-e
e-e-e-e-k-k-d(7)-e-e-e-e
e-k-e-k-d(9)-e-e-e-e
e-k-e-k-d-k-d(7)-e-e-e-e
e-e-e-k-k-d(7)-k-k-e-e-e
k-d-k-d-k-d(8)-e-e-e-e-e
wherein, k is a constrained ethyl nucleoside, e is a 2′-MOE substituted nucleoside, and d is a 2′-deoxynucleoside.
In certain embodiments, the antisense oligonucleotide has a sugar motif described by Formula A as follows: (J)m-(B)n-(J)p-(B)r-(A)t-(D)g-(A)v-(B)w-(J)x-(B)y-(J)z 
wherein:
each A is independently a 2′-substituted nucleoside;
each B is independently a bicyclic nucleoside;
each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;
each D is a 2′-deoxynucleoside;
m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14;
provided that:
at least one of m, n, and r is other than 0;
at least one of w and y is other than 0;
the sum of m, n, p, r, and t is from 2 to 5; and
the sum of v, w, x, y, and z is from 2 to 5.
Target Nucleic Acids, Target Regions and Nucleotide Sequences
Nucleotide sequences that encode HBV include, without limitation, the following: GENBANK Accession U95551.1 (incorporated herein as SEQ ID NO: 1).
It is understood that the sequence set forth in each SEQ ID NO in the Examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No) indicate a combination of nucleobase sequence and motif.
In certain embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region may encompass a 3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region. The structurally defined regions for HBV can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference. In certain embodiments, a target region may encompass the sequence from a 5′ target site of one target segment within the target region to a 3′ target site of another target segment within the same target region.
Targeting includes determination of at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs. In certain embodiments, the desired effect is a reduction in mRNA target nucleic acid levels. In certain embodiments, the desired effect is reduction of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.
A target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain embodiments, target segments within a target region are separated by a number of nucleotides that is, is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceeding values. In certain embodiments, target segments within a target region are separated by no more than, or no more than about, 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous. Contemplated are target regions defined by a range having a starting nucleic acid that is any of the 5′ target sites or 3′ target sites listed herein.
Suitable target segments may be found within a 5′ UTR, a coding region, a 3′ UTR, an intron, an exon, or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment may specifically exclude a certain structurally defined region such as the start codon or stop codon.
The determination of suitable target segments may include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that may hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).
There may be variation in activity (e.g., as defined by percent reduction of target nucleic acid levels) of the antisense compounds within an active target region. In certain embodiments, reductions in HBV mRNA levels are indicative of inhibition of HBV expression. Reductions in levels of a HBV protein are also indicative of inhibition of target mRNA expression. Further, phenotypic changes are indicative of inhibition of HBV expression. In certain embodiments, reduced fatigue, reduced flu-like symptoms, increase in appetite, reduced nausea, reduced joint pain, reduced jaundice, reduced pain in the abdomen, reduced weakness, reduced weight loss, reduction in breast enlargement in men, reduced rash on the palms, reduced difficulty with blood clotting, reduced cirrhosis, reduced spider-like blood vessels on the skin, increased Vitamins A and D absorption, reduced tumor growth, reduced tumor volume, reduced headache, reduced fever, reduced diarrhea, reduced pain over the liver area of the body, reduced clay- or grey-colored stool, reduced itching, reduced dark-colored urine, and reduced nausea and vomiting can be indicative of inhibition of HBV expression, In certain embodiments, amelioration of symptoms associated with HBV-related conditions, disease, and disorders can be indicative of inhibition of HBV expression. In certain embodiments, reduction of cirrhosis is indicative of inhibition of HBV expression. In certain embodiments, reduction of liver cancer markers can be indicative of inhibition of HBV expression.
Hybridization
In some embodiments, hybridization occurs between an antisense compound disclosed herein and a HBV nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.
Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.
Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a HBV nucleic acid.
Complementarity
An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a HBV nucleic acid).
Non-complementary nucleobases between an antisense compound and a HBV nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a HBV nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).
In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a HBV nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.
For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having four noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).
In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense compound may be fully complementary to a HBV nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.
The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.
In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a HBV nucleic acid, or specified portion thereof.
In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a HBV nucleic acid, or specified portion thereof.
The antisense compounds provided also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.
Identity
The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.
In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.
In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.
In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.
Modifications
A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.
Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.
Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.
Modified Internucleoside Linkages
The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.
In certain embodiments, antisense compounds targeted to a HBV nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.
Modified Sugar Moieties
Antisense compounds provided herein can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, without limitation, addition of substitutent groups (including 5′ and 2′ substituent groups); bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA); replacement of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R)2 (R═H, C1-C12 alkyl or a protecting group); and combinations thereof. Examples of chemically modified sugars include, 2′-F-5′-methyl substituted nucleoside (see, PCT International Application WO 2008/101157, published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides), replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see, published U.S. Patent Application US2005/0130923, published on Jun. 16, 2005), or, alternatively, 5′-substitution of a BNA (see, PCT International Application WO 2007/134181, published on Nov. 22, 2007, wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinyl group).
Examples of nucleosides having modified sugar moieties include, without limitation, nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH3, and 2′-O(CH2)2OCH3 substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, OCF3, O(CH2)2SCH3, O(CH2)2—O—N(Rm)(Rn), and O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl.
As used herein, “bicyclic nucleosides” refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include, without limitation, nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more bicyclic nucleosides wherein the bridge comprises a 4′ to 2′ bicyclic nucleoside. Examples of such 4′ to 2′ bicyclic nucleosides, include, but are not limited to, one of the formulae: 4′-(CH2)—O-2′ (LNA); 4′-(CH2)—S-2; 4′-(CH2)2—O-2′ (ENA); 4′-CH(CH3)—O-2′ (cEt) and 4′-CH(CH2OCH3)—O-2′, and analogs thereof (see, U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH3)(CH3)—O-2′, and analogs thereof (see, published PCT International Application WO2009/006478, published Jan. 8, 2009); 4′-CH2—N(OCH3)-2′, and analogs thereof (see, published PCT International Application WO2008/150729, published Dec. 11, 2008); 4′-CH2—O—N(CH3)-2′ (see, published U.S. Patent Application US2004/0171570, published Sep. 2, 2004); 4′-CH2—N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH2—C(H)(CH3)-2′ (see, Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2—C(═CH2)-2′, and analogs thereof (see, published PCT International Application WO 2008/154401, published on Dec. 8, 2008). Also see, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 6,670,461, 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 7,399,845; published PCT International applications WO 2004/106356, WO 94/14226, WO 2005/021570, and WO 2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; and U.S. patent Ser. Nos. 12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and PCT International Application Nos. PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).
In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the pentofuranosyl sugar moiety wherein such bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(Ra)(Rb)]n—, —C(Ra)═C(Rb)—, —C(Ra)═N—, —C(═NRa)—, —C(═O)—, —C(═S)—, —O—, —Si(Ra)2—, —S(═O)x—, and —N(Ra)—;
wherein:
x is 0, 1, or 2;
n is 1, 2, 3, or 4;
each Ra and Rb is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)2-J1), or sulfoxyl (S(═O)-J1); and
each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.
In certain embodiments, the bridge of a bicyclic sugar moiety is, —[C(Ra)(Rb)]n—, —[C(Ra)(Rb)]n—O—, —C(RaRb)—N(R)—O— or, —C(RaRb)—O—N(R)—. In certain embodiments, the bridge is 4′-CH2-2′,4′-(CH2)2-2′,4′-(CH2)3-2′,4′-CH2—O-2′,4′-(CH2)2—O-2′,4′-CH2—O—N(R)-2′, and 4′-CH2—N(R)—O-2′-, wherein each R is, independently, H, a protecting group, or C1-C12 alkyl.
In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-2′ methylene-oxy bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH2—O-2′) BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).
In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) α-L-Methyleneoxy (4′-CH2—O-2′) BNA, (B) β-D-Methyleneoxy (4′-CH2—O-2′) BNA, (C) Ethyleneoxy (4′-(CH2)2—O-2′) BNA, (D) Aminooxy (4′-CH2—O—N(R)-2′) BNA, (E) Oxyamino (4′-CH2—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy) (4′-CH(CH3)—O-2′) BNA, (G) methylene-thio (4′-CH2—S-2′) BNA, (H) methylene-amino (4′-CH2—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH2—CH(CH3)-2′) BNA, and (J) propylene carbocyclic (4′-(CH2)3-2′) BNA as depicted below.







wherein Bx is the base moiety and R is, independently, H, a protecting group or C1-C12 alkyl.

In certain embodiments, bicyclic nucleoside having Formula I:




wherein:

Bx is a heterocyclic base moiety;
-Qa-Qb-Qc- is —CH2—N(Rc)—CH2—, —C(═O)—N(Rc)—CH2—, —CH2—O—N(Rc)—, —CH2—N(Rc)—O—, or —N(Rc)—O—CH2;
Rc is C1-C12 alkyl or an amino protecting group; and
Ta and Tb are each, independently, H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium.
In certain embodiments, bicyclic nucleoside having Formula II:




wherein:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently, H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium;
Za is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, substituted amide, thiol, or substituted thio.
In one embodiment, each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJc, NJcJd, SJc, N3, OC(═X)Jc, and NJeC(═X)NJcJd, wherein each Jc, Jd, and Je is, independently, H, C1-C6 alkyl, or substituted C1-C6 alkyl and X is O or NJc.
In certain embodiments, bicyclic nucleoside having Formula III:




wherein:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently, H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium;
Zb is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, or substituted acyl (C(═O)—).
In certain embodiments, bicyclic nucleoside having Formula IV:




wherein:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium;
Rd is C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl;
each qa, qb, qc and qd is, independently, H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl, C1-C6 alkoxyl, substituted C1-C6 alkoxyl, acyl, substituted acyl, C1-C6 aminoalkyl, or substituted C1-C6 aminoalkyl;
In certain embodiments, bicyclic nucleoside having Formula V:




wherein:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently, H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium;
qa, qb, qe and qf are each, independently, hydrogen, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, alkoxy, substituted C1-C12 alkoxy, OJj, SJj, SOJj, SO2Jj, NJjJk, N3, CN, C(═O)Jj, C(═O)NJjJk, C(═O)Jj, O—C(═O)NJjJk, N(H)C(═NH)NJjJk, N(H)C(═O)NJjJk or N(H)C(═S)NJjJk;
or qe and qf together are ═C(qg)(qh);
qg and qh are each, independently, H, halogen, C1-C12 alkyl, or substituted C1-C12 alkyl.
The synthesis and preparation of the methyleneoxy (4′-CH2—O-2′) BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (see, e.g., Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
Analogs of methyleneoxy (4′-CH2—O-2′) BNA, methyleneoxy (4′-CH2—O-2′) BNA, and 2′-thio-BNAs, have also been prepared (see, e.g., Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (see, e.g., Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-BNA, a novel conformationally restricted high-affinity oligonucleotide analog, has been described in the art (see, e.g., Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and 2′-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.
In certain embodiments, bicyclic nucleoside having Formula VI:




wherein:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently, H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium;
each qi, qj, qk and ql is, independently, H, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 alkoxyl, substituted C1-C12 alkoxyl, OJj, SJj, SOJj, SO2Jj, NJjJk, N3, CN, C(═O)OJj, C(═O)NJjJk, C(═O)Jj, —C(═O)NJjJk, N(H)C(═NH)NJjJk, N(H)C(═O)NJjJk, or N(H)C(═S)NJjJk; and
qi and qj or ql and qk together are ═C(qg)(qh), wherein qg and qh are each, independently, H, halogen, C1-C12 alkyl, or substituted C1-C12 alkyl.
One carbocyclic bicyclic nucleoside having a 4′-(CH2)3-2′ bridge and the alkenyl analog, bridge 4′-CH═CH—CH2-2′, have been described (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).
As used herein, “bicyclic nucleoside” refers to a nucleoside comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic sugar moiety. In certain embodiments, the bridge connects the 2′ carbon and another carbon of the sugar ring.
As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting the 2′ carbon atom and the 4′ carbon atom.
As used herein, “monocylic nucleosides” refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.
As used herein, “2′-modified sugar” means a furanosyl sugar modified at the 2′ position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2′ modifications are selected from substituents including, but not limited to: O[(CH2)nO]mCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, OCH2C(═O)N(H)CH3, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. Other 2′-substituent groups can also be selected from: C1-C12 alkyl; substituted alkyl; alkenyl; alkynyl; alkaryl; aralkyl; O-alkaryl or O-aralkyl; SH; SCH3; OCN; Cl; Br; CN; CF3; OCF3; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving pharmacokinetic properties; and a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modified nucleosides comprise a 2′-MOE side chain (see, e.g., Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2′-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2′-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2′-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (see, e.g., Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).
As used herein, a “modified tetrahydropyran nucleoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran “sugar” substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate). Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), or those compounds having Formula X:




wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula X:

Bx is a heterocyclic base moiety;
T3 and T4 are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T3 and T4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5′ or 3′-terminal group;
q1, q2, q3, q4, q5, q6 and q7 are each, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and
one of R1 and R2 is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2, and CN, wherein X is O, S, or NJ1, and each J1, J2, and J3 is, independently, H or C1-C6 alkyl.
In certain embodiments, the modified THP nucleosides of Formula X are provided wherein qm, qn, qp, qr, qs, qt, and qu are each H. In certain embodiments, at least one of qm, qn, qp, qr, qs, qt, and qu is other than H. In certain embodiments, at least one of qm, qn, qp, qr, qs, qt and qu is methyl. In certain embodiments, THP nucleosides of Formula X are provided wherein one of R1 and R2 is F. In certain embodiments, R1 is fluoro and R2 is H, R1 is methoxy and R2 is H, and R1 is methoxyethoxy and R2 is H.
As used herein, “2′-modified nucleoside” or “2′-substituted nucleoside” refers to a nucleoside comprising a sugar comprising a substituent at the 2′ position of a furanose ring other than H or OH. 2′-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2′ carbon and another carbon of the sugar ring and nucleosides with non-bridging 2′ substituents, such as allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. 2′-modified nucleosides may further comprise other modifications, for example, at other positions of the sugar and/or at the nucleobase.
As used herein, “2′-F” refers to a sugar comprising a fluoro group at the 2′ position.
As used herein, “2′-OMe” or “2′-OCH3” or “2′-O-methyl” each refers to a nucleoside comprising a sugar comprising an —OCH3 group at the 2′ position of the sugar ring.
As used herein, “oligonucleotide” refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).
Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see, e.g., review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.
Methods for the preparations of modified sugars are well known to those skilled in the art.
In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified, or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.
In certain embodiments, antisense compounds comprise one or more nucleotides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOE modified nucleotides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a cEt. In certain embodiments, the cEt modified nucleotides are arranged throughout the wings of a gapmer motif.
Compositions and Methods for Formulating Pharmaceutical Compositions
Antisense oligonucleotides may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
An antisense compound targeted to a HBV nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to a HBV nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide.
Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound.
Conjugated Antisense Compounds
Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.
Cell Culture and Antisense Compounds Treatment
The effects of antisense compounds on the level, activity or expression of HBV nucleic acids can be tested in vitro in a variety of cell types. Cell types used for such analyses are available from commercial vendors (e.g. American Type Culture Collection, Manassus, Va.; Zen-Bio, Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville, Md.) and are cultured according to the vendor's instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, Calif.). Illustrative cell types include, but are not limited to, HuVEC cells, b.END cells, HepG2 cells, Hep3B cells, and primary hepatocytes.
In Vitro Testing of Antisense Oligonucleotides
Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.
Cells may be treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.
One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.
Cells are treated with antisense oligonucleotides by routine methods. Cells may be harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.
The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.
RNA Isolation
RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocols.
Analysis of Inhibition of Target Levels or Expression
Inhibition of levels or expression of a HBV nucleic acid can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitative real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently accomplished using the commercially available ABI PRISM 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.
Quantitative Real-Time PCR Analysis of Target RNA Levels
Quantitation of target RNA levels may be accomplished by quantitative real-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.
Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents may be obtained from Invitrogen (Carlsbad, Calif.). RT real-time-PCR reactions are carried out by methods well known to those skilled in the art.
Gene (or RNA) target quantities obtained by real time PCR are normalized using either the expression level of a gene whose expression is constant, such as cyclophilin A, or by quantifying total RNA using RIBOGREEN (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, Oreg.). Methods of RNA quantification by RIBOGREEN are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN fluorescence.
Probes and primers are designed to hybridize to a HBV nucleic acid. Methods for designing real-time PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS Software (Applied Biosystems, Foster City, Calif.).
Quantitative Real-Time PCR Analysis of Target DNA Levels
Quantitation of target DNA levels may be accomplished by quantitative real-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.
Gene (or DNA) target quantities obtained by real time PCR are normalized using either the expression level of a gene whose expression is constant, such as cyclophilin A, or by quantifying total DNA using RIBOGREEN (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total DNA is quantified using RIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, Oreg.). Methods of DNA quantification by RIBOGREEN are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN fluorescence.
Probes and primers are designed to hybridize to a HBV nucleic acid. Methods for designing real-time PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS Software (Applied Biosystems, Foster City, Calif.).
Analysis of Protein Levels
Antisense inhibition of HBV nucleic acids can be assessed by measuring HBV protein levels. Protein levels of HBV can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.
In Vivo Testing of Antisense Compounds
Antisense compounds, for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of HBV and produce phenotypic changes. Testing may be performed in normal animals, or in experimental disease models. For administration to animals, antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, subcutaneous, intrathecal, and intracerebroventricular. Calculation of antisense oligonucleotide dosage and dosing frequency is within the abilities of those skilled in the art, and depends upon factors such as route of administration and animal body weight. Following a period of treatment with antisense oligonucleotides, RNA is isolated from liver tissue and changes in HBV nucleic acid expression are measured. Changes in HBV DNA levels are also measured. Changes in HBV protein levels are also measured. Changes in HBV HBeAg levels are also measured. Changes in HBV HBsAg levels are also measured.
Certain Indications
In certain embodiments, provided herein are methods, compounds, and compositions of treating an individual comprising administering one or more pharmaceutical compositions provided herein. In certain embodiments, the individual has an HBV-related condition. In certain embodiments, chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, and HBV viremia. In certain embodiments, the HBV-related condition may have which may include any or all of the following: flu-like illness, weakness, aches, headache, fever, loss of appetite, diarrhea, jaundice, nausea and vomiting, pain over the liver area of the body, clay- or grey-colored stool, itching all over, and dark-colored urine, when coupled with a positive test for presence of a hepatitis B virus, a hepatitis B viral antigen, or a positive test for the presence of an antibody specific for a hepatitis B viral antigen. In certain embodiments, the individual is at risk for an HBV-related condition. This includes individuals having one or more risk factors for developing an HBV-related condition, including sexual exposure to an individual infected with Hepatitis B virus, living in the same house as an individual with a lifelong hepatitis B virus infection, exposure to human blood infected with the hepatitis B virus, injection of illicit drugs, being a person who has hemophilia, and visiting an area where hepatitis B is common. In certain embodiments, the individual has been identified as in need of treatment for an HBV-related condition. In certain embodiments provided herein are methods for prophylactically reducing HBV expression in an individual. Certain embodiments include treating an individual in need thereof by administering to an individual a therapeutically effective amount of an antisense compound targeted to an HBV nucleic acid.
Due to overlapping transmission routes, many people have been exposed to both hepatitis B virus (HBV) and hepatitis C virus (HCV), and a smaller proportion are chronically infected with both viruses, especially in regions such as Asia where HBV is endemic. Estimates suggest that up to 10% of people with HCV may also have HBV, while perhaps 20% of people with HBV are co-infected with HCV. However, treatment of hepatitis B or hepatitis B in HBV-HCV co-infected individuals has not been well studied. Treatment is complicated by the fact that HCV and HBV appear to inhibit each other's replication (though not all studied have observed this interaction). Therefore, treatment that fully suppresses HBV could potentially allow HCV to re-emerge, or vice versa. Therefore, the compounds and compositions described herein may advantageously be used for treating patients infected with both HBV and HCV. Exemplary treatment options for hepatitis C(HCV) include interferons, e.g., interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1. Less frequent interferon dosing can be achieved using pegylated interferon (interferon attached to a polyethylene glycol moiety which improves its pharmacokinetic profile). Combination therapy with interferon alpha-2b (pegylated and unpegylated) and ribavirin has also been shown to be efficacious for some patient populations. Other agents currently being developed include HCV RNA replication inhibitors (e.g., ViroPharma's VP50406 series), HCV antisense agents, HCV therapeutic vaccines, HCV protease inhibitors, HCV helicase inhibitors and HCV antibody therapy (monoclonal or polyclonal).
In certain embodiments, treatment with the methods, compounds, and compositions described herein is useful for preventing an HBV-related condition associated with the presence of the hepatitis B virus. In certain embodiments, treatment with the methods, compounds, and compositions described herein is useful for preventing an HBV-related condition.
In one embodiment, administration of a therapeutically effective amount of an antisense compound targeted to an HBV nucleic acid is accompanied by monitoring of HBV mRNA levels in the serum of an individual to determine an individual's response to administration of the antisense compound. In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to an HBV nucleic acid is accompanied by monitoring of HBV DNA levels in the serum of an individual to determine an individual's response to administration of the antisense compound. In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to an HBV nucleic acid is accompanied by monitoring of HBV protein levels in the serum of an individual to determine an individual's response to administration of the antisense compound. In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to an HBV nucleic acid is accompanied by monitoring of HBV S antigen (HBsAg) levels in the serum of an individual to determine an individual's response to administration of the antisense compound. In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to an HBV nucleic acid is accompanied by monitoring of HBV E antigen (HBeAg) levels in the serum of an individual to determine an individual's response to administration of the antisense compound. An individual's response to administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.
In certain embodiments, administration of an antisense compound targeted to an HBV nucleic acid results in reduction of HBV expression by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values. In certain embodiments, administration of an antisense compound targeted to an HBV nucleic acid results in reduced symptoms associated with the HBV-related condition and reduced HBV-related markers in the blood. In certain embodiments, administration of an HBV antisense compound decreases HBV RNA levels, HBV DNA levels, HBV protein levels, HBsAg levels, or HBeAg levels by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.
In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to HBV are used for the preparation of a medicament for treating a patient suffering or susceptible to an HBV-related condition.
Certain Combination Therapies
In certain embodiments, one or more pharmaceutical compositions provided herein are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease, disorder, or condition as the one or more pharmaceutical compositions provided herein. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease, disorder, or condition as the one or more pharmaceutical compositions provided herein. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired side effect of one or more pharmaceutical compositions provided herein. In certain embodiments, one or more pharmaceutical compositions provided herein are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions provided herein are co-administered with another pharmaceutical agent to produce a combinational effect. In certain embodiments, one or more pharmaceutical compositions provided herein are co-administered with another pharmaceutical agent to produce a synergistic effect.
In certain embodiments, one or more pharmaceutical compositions provided herein and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions provided herein and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions provided herein and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions provided herein and one or more other pharmaceutical agents are prepared separately. In certain embodiments the antisense oligonucleotides disclosed is administered in combination with an HCV agent. In further embodiments, the HCV compound is administered simultaneously as the antisense compound; in other embodiments, the HCV compound is administered separately; so that a dose of each of the HCV agent and the antisense compound overlap, in time, within the patient's body. In related embodiments, the HCV agent may be selected from interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1 (pegylated and unpegylated); ribavirin; an HCV RNA replication inhibitor (e.g., ViroPharma's VP50406 series); an HCV antisense agent; an HCV therapeutic vaccine; an HCV protease inhibitor; an HCV helicase inhibitor; and an HCV antibody therapy (monoclonal or polyclonal).
In other embodiments, an HBV antisense compound of the present invention may be administered to a patient infected with HBV, in combination with one or more HBV therapeutic agents, wherein the one or more HBV therapeutic agents may be administered in the same drug formulation as the HBV ASO compound, or may be administered in a separate formulation. The one or more HBV therapeutic agents may be administered simultaneously with the ASO HBV compound, or may be administered separately, so that a dose of each of the HBV ASO compound and the HBV therapeutic agent overlap, in time, within the patient's body. In related embodiments, the one or more HBV therapeutic agent may be selected from interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1 (pegylated and unpegylated), ribavirin; an HBV RNA replication inhibitor; a second HBV antisense compound; an HBV therapeutic vaccine; an HBV prophylactic vaccine; lamivudine (3TC); entecavir; tenofovir; telbivudine (LdT); adefovir; and an HBV antibody therapy (monoclonal or polyclonal).
EXAMPLES
Non-Limiting Disclosure and Incorporation by Reference
While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.
Example 1
Antisense Inhibition of HBV Viral mRNA in HepG2.2.15 Cells by MOE Gapmers
Antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. Cultured HepG2.2.15 cells at a density of 25,000 cells per well were transfected using electroporation with 15,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 (forward sequence CTTGGTCATGGGCCATCAG, designated herein as SEQ ID NO: 2; reverse sequence CGGCTAGGAGTTCCGCAGTA, designated herein as SEQ ID NO: 3; probe sequence TGCGTGGAACCTTTTCGGCTCC, designated herein as SEQ ID NO: 4) was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in Table 1 were designed as either 5-10-5 MOE gapmers or 3-10-4 MOE gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleosides each. The 3-10-4 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three and 4 nucleosides respectively. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has an MOE sugar modification. Each nucleoside in the central gap segment has a deoxy sugar modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
“Viral Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. The ‘Motif’ column indicates the gap and wing structure of each gapmer. Each gapmer listed in Table 1 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1).







TABLE 1







Inhibition of viral HBV mRNA levels by MOE gapmers targeted to SEQ ID NO: 1













Viral
Viral







Start
Stop



%
SEQ ID


Site
Site
ISIS No
Sequence
Motif
inhibition
NO
















245
261
510088
CCACGAGTCTAGACTCT
3-10-4
55
5


 


250
266
510089
GTCCACCACGAGTCTAG
3-10-4
59
6


 


251
267
510090
AGTCCACCACGAGTCTA
3-10-4
60
7


 


252
268
510091
AAGTCCACCACGAGTCT
3-10-4
47
8


 


253
269
510092
GAAGTCCACCACGAGTC
3-10-4
59
9


 


254
270
510093
AGAAGTCCACCACGAGT
3-10-4
32
10


 


255
271
510094
GAGAAGTCCACCACGAG
3-10-4
41
11


 


256
272
510095
AGAGAAGTCCACCACGA
3-10-4
44
12


 


257
273
510096
GAGAGAAGTCCACCACG
3-10-4
54
13


 


258
274
510097
TGAGAGAAGTCCACCAC
3-10-4
57
14


 


384
400
510098
TGATAAAACGCCGCAGA
3-10-4
55
15


 


385
401
510099
ATGATAAAACGCCGCAG
3-10-4
59
16


 


411
427
510100
GGCATAGCAGCAGGATG
3-10-4
85
17


 


412
428
510101
AGGCATAGCAGCAGGAT
3-10-4
51
18


 


413
429
510102
GAGGCATAGCAGCAGGA
3-10-4
69
19


 


414
433
505330
AGATGAGGCATAGCAGCAGG
5-10-5
74
20


 


414
430
510103
TGAGGCATAGCAGCAGG
3-10-4
12
21


 


415
434
509928
AAGATGAGGCATAGCAGCAG
5-10-5
71
22


 


415
431
510104
ATGAGGCATAGCAGCAG
3-10-4
69
23


 


416
435
509929
GAAGATGAGGCATAGCAGCA
5-10-5
78
24


 


416
432
510105
GATGAGGCATAGCAGCA
3-10-4
69
25


 


417
436
509930
AGAAGATGAGGCATAGCAGC
5-10-5
72
26


 


417
433
510106
AGATGAGGCATAGCAGC
3-10-4
77
27


 


418
437
146783
AAGAAGATGAGGCATAGCAG
5-10-5
15
28


 


418
434
510107
AAGATGAGGCATAGCAG
3-10-4
69
29


 


419
435
510108
GAAGATGAGGCATAGCA
3-10-4
59
30


 


420
436
510109
AGAAGATGAGGCATAGC
3-10-4
0
31


 


421
437
510110
AAGAAGATGAGGCATAG
3-10-4
38
32


 


457
473
510111
ACGGGCAACATACCTTG
3-10-4
62
33


 


639
658
146784
CTGAGGCCCACTCCCATAGG
5-10-5
5
34


 


639
655
510112
AGGCCCACTCCCATAGG
3-10-4
44
35


 


640
656
510113
GAGGCCCACTCCCATAG
3-10-4
27
36


 


641
657
510114
TGAGGCCCACTCCCATA
3-10-4
44
37


 


642
658
510115
CTGAGGCCCACTCCCAT
3-10-4
52
38


 


687
706
509931? 
CGAACCACTGAACAAATGGC
5-10-5
89
39


 


687
703
510116
ACCACTGAACAAATGGC
3-10-4
89
40


 


688
704
510117
AACCACTGAACAAATGG
3-10-4
69
41


 


689
705
510118
GAACCACTGAACAAATG
3-10-4
63
42


 


690
706
510119
CGAACCACTGAACAAAT
3-10-4
74
43


 


738
754
510120
ACCACATCATCCATATA
3-10-4
71
44


 


1176
1192
510121
TCAGCAAACACTTGGCA
3-10-4
73
45


 


1778
1797
509932
AATTTATGCCTACAGCCTCC
5-10-5
76
46


 


1778
1794
510122
TTATGCCTACAGCCTCC
3-10-4
76
47


 


1779
1798
509933
CAATTTATGCCTACAGCCTC
5-10-5
72
48


 


1779
1795
510123
TTTATGCCTACAGCCTC
3-10-4
75
49


 


1780
1799
509934
CCAATTTATGCCTACAGCCT
5-10-5
75
50


 


1780
1796
510124
ATTTATGCCTACAGCCT
3-10-4
73
51


 


1781
1800
509935
ACCAATTTATGCCTACAGCC
5-10-5
72
52


 


1781
1797
510125
AATTTATGCCTACAGCC
3-10-4
69
53


 


1782
1798
510126
CAATTTATGCCTACAGC
3-10-4
59
54


 


1783
1799
510127
CCAATTTATGCCTACAG
3-10-4
58
55


 


1784
1800
510128
ACCAATTTATGCCTACA
3-10-4
60
56


 


1822
1838
510129
AGGCAGAGGTGAAAAAG
3-10-4
47
57


 


1823
1839
510130
TAGGCAGAGGTGAAAAA
3-10-4
30
58


 


1865
1884
509936
GCACAGCTTGGAGGCTTGAA
5-10-5
39
59


 


1865
1881
510131
CAGCTTGGAGGCTTGAA
3-10-4
4
60


 


1866
1885
509937
GGCACAGCTTGGAGGCTTGA
5-10-5
35
61


 


1866
1882
510132
ACAGCTTGGAGGCTTGA
3-10-4
0
62


 


1867
1886
505370
AGGCACAGCTTGGAGGCTTG
5-10-5
36
63


 


1867
1883
510133
CACAGCTTGGAGGCTTG
3-10-4
12
64


 


1868
1887
509938
AAGGCACAGCTTGGAGGCTT
5-10-5
7
65


 


1868
1884
510134
GCACAGCTTGGAGGCTT
3-10-4
20
66


 


1869
1888
509939
CAAGGCACAGCTTGGAGGCT
5-10-5
36
67


 


1869
1885
510135
GGCACAGCTTGGAGGCT
3-10-4
22
68


 


1870
1889
505371
CCAAGGCACAGCTTGGAGGC
5-10-5
35
69


 


1870
1886
510136
AGGCACAGCTTGGAGGC
3-10-4
14
70


 


1871
1887
510137
AAGGCACAGCTTGGAGG
3-10-4
0
71


 


1872
1888
510138
CAAGGCACAGCTTGGAG
3-10-4
6
72


 


1873
1889
510139
CCAAGGCACAGCTTGGA
3-10-4
17
73


 


1918
1934
510140
GCTCCAAATTCTTTATA
3-10-4
59
74


 


2378
2397
509940
TCTGCGAGGCGAGGGAGTTC
3-10-4
10
75


 


2378
2394
510141
GCGAGGCGAGGGAGTTC
3-10-4
5
76


 


2379
2395? 
510142
TGCGAGGCGAGGGAGTT  
3-10-4
0
77


 


2380
2396
510143
CTGCGAGGCGAGGGAGT
3-10-4
8
78


 


2381
2397
510144
TCTGCGAGGCGAGGGAG
3-10-4
17
79


 


2820
2836
510145
TTCCCAAGAATATGGTG
3-10-4
22
80


 


2821
2837
510146
GTTCCCAAGAATATGGT
3-10-4
11
81


 


2822
2838
510147
TGTTCCCAAGAATATGG
3-10-4
21
82









Example 2
Antisense Inhibition of HBV Viral mRNA in HepG2.2.15 Cells by MOE Gapmers
Additional antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. Cultured HepG2.2.15 cells at a density of 25,000 cells per well were transfected using electroporation with 15,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 was used to measure mRNA levels. RTS3370 detects the full length mRNA and the second portions of the pre-S1, pre-S2 and pre-C mRNA transcripts. The gapmers were also probed with additional primer probe sets. Viral primer probe set RTS3371 (forward sequence CCAAACCTTCGGACGGAAA, designated herein as SEQ ID NO: 311; reverse sequence TGAGGCCCACTCCCATAGG, designated herein as SEQ ID NO: 312; probe sequence CCCATCATCCTGGGCTTTCGGAAAAT, designated herein as SEQ ID NO: 313) was used also to measure mRNA levels. RTS3371 detects the full length mRNA and the second portions of the pre-S1, pre-S2 and pre-C mRNA transcripts, similar to RTS3370, but at different regions. Viral primer probe set RTS3372 (forward sequence ATCCTATCAACACTTCCGGAAACT, designated herein as SEQ ID NO: 314; reverse sequence CGACGCGGCGATTGAG, designated herein as SEQ ID NO: 315; probe sequence AAGAACTCCCTCGCCTCGCAGACG, designated herein as SEQ ID NO: 316) was used to measure mRNA levels. RTS3372 detects the full length genomic sequence. Viral primer probe set RTS3373MGB (forward sequence CCGACCTTGAGGCATACTTCA, designated herein as SEQ ID NO: 317; reverse sequence AATTTATGCCTACAGCCTCCTAGTACA, designated herein as SEQ ID NO: 318; probe sequence TTAAAGACTGGGAGGAGTTG, designated herein as SEQ ID NO: 319) was used to measure mRNA levels. RTS3373MGB detects the full length mRNA and the second portions of the pre-S1, pre-S2, pre-C, and pre-X mRNA transcripts.
HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in Table 2 were designed as either 5-10-5 MOE gapmers, 3-10-3 MOE gapmers, or 2-10-2 MOE gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleosides each. The 3-10-3 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three nucleosides each. The 2-10-2 MOE gapmers are 14 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising two nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has an MOE sugar modification. Each nucleoside in the central gap segment has a deoxy sugar modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5′-methylcytosines.
“Start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. The ‘Motif’ column indicates the gap and wing structure of each gapmer. Each gapmer listed in Table 2 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1).







TABLE 2







Inhibition of viral HBV mRNA levels by MOE gapmers targeted to SEQ ID NO: 1 (detected by RTS3370,


RTS3371, RTS3372, and RTS3373 MGB)




















RTS3370
RTS3371
RTS3372
RTS3373

SEQ


Start
Stop


%
%
%
MGB %

ID


Site
Site
ISIS No
Sequence
inhibition
inhibition
inhibition
inhibition
Motif
NO



















58
77
146779
GAACTGGA
76
80
82
81
5-10-5
83





GCCACCAG











CAGG








 


58
71
510019
GAGCCACC
38
32
45
31
2-10-2
84





AGCAGG








 


61
80
505314
CCTGAACT
68
71
67
66
5-10-5
85





GGAGCCAC











CAGC








 


62
77
509941
GAACTGGA
36
32
71
53
3-10-3
86





GCCACCAG








 


196
215
505315
AAAAACCC
69
74
80
88
5-10-5
87





CGCCTGTA











ACAC








 


199
218
505316
AAGAAAAA
60
60
64
64
5-10-5
88





CCCCGCCTG











TAA








 


205
224
505317
GTCAACAA
85
83
79
85
5-10-5
89





GAAAAACC











CCGC








 


228
241
510020
GTATTGTGA
28
18
0
16
2-10-2
90





GGATT








 


229
242
510021
GGTATTGTG
40
37
19
34
2-10-2
91





AGGAT








 


244
263
146821
CACCACGA
74
73
62
75
5-10-5
92





GTCTAGACT











CTG








 


245
260
509942
CACGAGTC
18
15
45
46
3-10-3
93





TAGACTCT








 


245
258
510022
CGAGTCTA
32
26
23
19
2-10-2
94





GACTCT








 


246
261
509943
CCACGAGT
34
35
63
60
3-10-3
95





CTAGACTC








 


247
266
505318
GTCCACCA
75
77
64
75
5-10-5
96





CGAGTCTA











GACT








 


250
269
509921
GAAGTCCA
46
46
39
40
5-10-5
97





CCACGAGT











CTAG








 


250
265
509944
TCCACCAC
38
39
65
59
3-10-3
98





GAGTCTAG








 


251
270
509922
AGAAGTCC
55
56
17
38
5-10-5
99





ACCACGAG











TCTA








 


251
266
509945
GTCCACCA
34
35
64
51
3-10-3
100





CGAGTCTA








 


252
271
509923
GAGAAGTC
39
38
39
33
5-10-5
101





CACCACGA











GTCT








 


252
267
509946
AGTCCACC
47
51
50
45
3-10-3
102





ACGAGTCT








 


253
272
505319
AGAGAAGT
88
83
80
78
5-10-5
103





CCACCACG











AGTC








 


253
268
509947
AAGTCCAC
46
50
56
46
3-10-3
104





CACGAGTC








 


254
273
509924
GAGAGAAG
43
40
49
44
5-10-5
105





TCCACCAC











GAGT








 


254
269
509948
GAAGTCCA
41
46
51
44
3-10-3
106





CCACGAGT








 


254
267
510023
AGTCCACC
41
32
47
48
2-10-2
107





ACGAGT








 


255
274
509925
TGAGAGAA
50
57
55
55
5-10-5
108





GTCCACCA











CGAG








 


255
270
509949
AGAAGTCC
40
41
52
34
3-10-3
109





ACCACGAG








 


255
268
510024
AAGTCCAC
26
29
19
23
2-10-2
110





CACGAG








 


256
275
505320
TTGAGAGA
51
57
55
66
5-10-5
111





AGTCCACC











ACGA








 


256
271
509950
GAGAAGTC
30
31
43
33
3-10-3
112





CACCACGA








 


256
269
510025
GAAGTCCA
44
38
53
54
2-10-2
113





CCACGA








 


257
270
510026
AGAAGTCC
39
42
32
25
2-10-2
114





ACCACG








 


258
273
509952
GAGAGAAG
54
52
60
48
3-10-3
115





TCCACCAC








 


258
271
510027
GAGAAGTC
29
30
25
19
2-10-2
116





CACCAC








 


259
274
509953
TGAGAGAA
39
44
47
38
3-10-3
117





GTCCACCA








 


259
272
510028
AGAGAAGT
31
29
3
15
2-10-2
118





CCACCA








 


260
273
510029
GAGAGAAG
21
19
23
18
2-10-2
119





TCCACC








 


261
274
510030
TGAGAGAA
16
22
21
20
2-10-2
120





GTCCAC








 


262
281
505321
AGAAAATT
53
58
52
56
5-10-5
121





GAGAGAAG











TCCA








 


265
284
505322
CCTAGAAA
62
65
69
67
5-10-5
122





ATTGAGAG











AAGT








 


293
312
505323
ATTTTGGCC
86
84
81
85
5-10-5
123





AAGACACA











CGG








 


296
315
505324
CGAATTTTG
67
67
69
64
5-10-5
124





GCCAAGAC











ACA








 


302
321
505325
GGACTGCG
77
75
73
76
5-10-5
125





AATTTTGGC











CAA








 


360
379
505326
TCCAGCGA
89
90
77
91
5-10-5
126





TAACCAGG











ACAA








 


366
385
505327
GACACATC
83
85
75
86
5-10-5
127





CAGCGATA











ACCA








 


369
388
505328
GCAGACAC
65
68
49
57
5-10-5
128





ATCCAGCG











ATAA








 


384
399
509954
GATAAAAC
37
46
53
35
3-10-3
129





GCCGCAGA








 


384
397
510031
TAAAACGC
36
36
33
33
2-10-2
130





CGCAGA








 


385
398
510032
ATAAAACG
12
7
19
15
2-10-2
131





CCGCAG








 


386
401
509955
ATGATAAA
49
55
57
53
3-10-3
132





ACGCCGCA








 


386
399
510033
GATAAAAC
39
39
45
37
2-10-2
133





GCCGCA








 


387
400
510034
TGATAAAA
40
37
29
39
2-10-2
134





CGCCGC








 


388
401
510035
ATGATAAA
22
24
9
22
2-10-2
135





ACGCCG








 


411
430
505329
TGAGGCAT
60
64
47
55
5-10-5
136





AGCAGCAG











GATG








 


411
426
509956
GCATAGCA
62
64
71
60
3-10-3
137





GCAGGATG








 


411
424
510036
ATAGCAGC
44
34
30
48
2-10-2
138





AGGATG








 


412
431
509926
ATGAGGCA
45
54
71
62
5-10-5






TAGCAGCA





139





GGAT








 


412
427
509957
GGCATAGC
72
75
80
71
3-10-3
140





AGCAGGAT








 


412
425
510037
CATAGCAG
29
24
24
20
2-10-2
141





CAGGAT








 


413
432
509927
GATGAGGC
54
58
54
49
5-10-5
142





ATAGCAGC











AGGA








 


413
428
509958
AGGCATAG
63
66
68
64
3-10-3
143





CAGCAGGA








 


413
426
510038
GCATAGCA
55
54
37
46
2-10-2
144





GCAGGA








 


414
433
505330
AGATGAGG
85
87
74
82
5-10-5
20





CATAGCAG











CAGG








 


414
429
509959
GAGGCATA
64
64
80
68
3-10-3
145





GCAGCAGG








 


414
427
510039
GGCATAGC
58
54
41
45
2-10-2
146





AGCAGG








 


415
430
509960
TGAGGCAT
59
59
66
64
3-10-3
147





AGCAGCAG








 


415
428
510040
AGGCATAG
58
55
38
41
2-10-2
148





CAGCAG








 


416
431
509961
ATGAGGCA
56
54
65
56
3-10-3
149





TAGCAGCA








 


416
429
510041
GAGGCATA
64
62
64
57
2-10-2
150





GCAGCA








 


417
432
509962
GATGAGGC
57
52
58
49
3-10-3
151





ATAGCAGC








 


417
430
510042
TGAGGCAT
48
50
55
48
2-10-2
152





AGCAGC








 


418
433
509963
AGATGAGG
50
52
64
51
3-10-3
153





CATAGCAG








 


418
431
510043
ATGAGGCA
36
31
36
26
2-10-2
154





TAGCAG








 


419
434
509964
AAGATGAG
48
47
72
65
3-10-3
155





GCATAGCA








 


419
432
510044
GATGAGGC
44
28
0
14
2-10-2
156





ATAGCA








 


420
435
509965
GAAGATGA
45
41
65
62
3-10-3
157





GGCATAGC








 


420
433
510045
AGATGAGG
41
43
37
29
2-10-2
158





CATAGC








 


421
436
509966
AGAAGATG
32
29
64
51
3-10-3
159





AGGCATAG








 


421
434
510046
AAGATGAG
21
18
26
27
2-10-2
160





GCATAG








 


422
437
509967
AAGAAGAT
21
17
55
46
3-10-3
161





GAGGCATA








 


422
435
510047
GAAGATGA
25
24
23
25
2-10-2
162





GGCATA








 


423
436
510048
AGAAGATG
21
17
25
19
2-10-2
163





AGGCAT








 


424
437
510049
AAGAAGAT
17
11
38
27
2-10-2
164





GAGGCA








 


454
473
505331
ACGGGCAA
55
57
65
60
5-10-5
165





CATACCTTG











ATA








 


457
476
505332
CAAACGGG
73
77
77
74
5-10-5
166





CAACATAC











CTTG








 


457
472
509968
CGGGCAAC
60
61
73
70
3-10-3
167





ATACCTTG








 


458
473
509969
ACGGGCAA
58
63
64
58
3-10-3
168





CATACCTT








 


458
471
510050
GGGCAACA
58
56
57
46
2-10-2
169





TACCTT








 


459
472
510051
CGGGCAAC
49
43
47
37
2-10-2
170





ATACCT








 


460
473
510052
ACGGGCAA
50
50
54
51
2-10-2
171





CATACC








 


463
482
505333
AGAGGACA
64
68
64
71
5-10-5
172





AACGGGCA











ACAT








 


466
485
505334
ATTAGAGG
59
62
42
69
5-10-5
173





ACAAACGG











GCAA








 


472
491
505335
CCTGGAATT
78
81
73
86
5-10-5
174





AGAGGACA











AAC








 


475
494
505336
GATCCTGG
56
65
61
72
5-10-5
175





AATTAGAG











GACA








 


639
654
509970
GGCCCACT
38
55
74
48
3-10-3
176





CCCATAGG








 


641
656
509971
GAGGCCCA
30
46
77
54
3-10-3
177





CTCCCATA








 


642
657
509972
TGAGGCCC
58
57
84
66
3-10-3
178





ACTCCCAT








 


643
658
509973
CTGAGGCC
38
53
70
66
3-10-3
179





CACTCCCA








 


670
689
146823
GGCACTAG
61
64
63
63
5-10-5
180





TAAACTGA











GCCA








 


670
685
509974
CTAGTAAA
71
71
78
80
3-10-3
181





CTGAGCCA








 


670
683
510053
AGTAAACT
49
48
52
53
2-10-2
182





GAGCCA








 


671
684
510054
TAGTAAAC
41
38
19
30
2-10-2
183





TGAGCC








 


672
685
510055
CTAGTAAA
25
27
42
47
2-10-2
184





CTGAGC








 


673
692
505337
AATGGCAC
34
46
49
52
5-10-5
185





TAGTAAAC











TGAG








 


679
698
505338
TGAACAAA
74
77
71
80
5-10-5
186





TGGCACTA











GTAA








 


682
701
505339
CACTGAAC
82
83
71
82
5-10-5
187





AAATGGCA











CTAG








 


687
702
509975
CCACTGAA
72
73
76
80
3-10-3
188





CAAATGGC








 


688
707
505340
ACGAACCA
69
69
78
76
5-10-5
189





CTGAACAA











ATGG








 


688
703
509976
ACCACTGA
47
48
67
65
3-10-3
190





ACAAATGG








 


689
704
509977
AACCACTG
33
33
39
41
3-10-3
191





AACAAATG








 


690
705
509978
GAACCACT
50
49
63
48
3-10-3
192





GAACAAAT








 


691
710
505341
CCTACGAA
64
70
70
72
5-10-5
193





CCACTGAA











CAAA








 


691
706
509979
CGAACCAC
67
66
78
77
3-10-3
194





TGAACAAA








 


691
704
510056
AACACTG
36
36
23
32
2-10-2
195





AACCAAA








 


692
705
510057
GAACCACT
45
44
51
43
2-10-2
196





GAACAA








 


693
706
510058
CGAACCAC
59
52
48
49
2-10-2
197





TGAACA








 


697
716
505342
GAAAGCCC
76
80
73
83
5-10-5
198





TACGAACC











ACTG








 


738
753
509980
CCACATCAT
40
33
62
54
3-10-3
199





CCATATA








 


738
751
510059
ACATCATCC
19
9
30
27
2-10-2
200





ATATA








 


739
754
509981
ACCACATC
76
78
93
85
3-10-3
201





ATCCATAT








 


739
752
510060
CACATCATC
45
35
24
17
2-10-2
202





CATAT








 


740
753
510061
CCACATCAT
52
49
43
40
2-10-2
203





CCATA








 


741
754
510062
ACCACATC
44
45
48
47
2-10-2
204





ATCCAT








 


756
775
505343
TGTACAGA
47
56
55
68
5-10-5
205





CTTGGCCCC











CAA








 


823
842
505344
AGGGTTTA
66
71
64
72
5-10-5
206





AATGTATA











CCCA








 


1170
1189
505345
GCAAACAC
76
80
35
70
5-10-5
207





TTGGCACA











GACC








 


1176
1191
509982
CAGCAAAC
42
44
56
54
3-10-3
208





ACTTGGCA








 


1177
1192
509983
TCAGCAAA
60
54
74
70
3-10-3
209





CACTTGGC








 


1259
1278
505346
CCGCAGTA
88
82
57
80
5-10-5
210





TGGATCGG











CAGA








 


1261
1276
509984
GCAGTATG
61
58
65
72
3-10-3
211





GATCGGCA








 


1262
1281
505347
GTTCCGCA
84
81
71
83
5-10-5
212





GTATGGAT











CGGC








 


1268
1287
505348
CTAGGAGT
78
68
70
79
5-10-5
213





TCCGCAGT











ATGG








 


1271
1290
505349
CGGCTAGG
47
54
59
61
5-10-5
214





AGTTCCGC











AGTA








 


1277
1296
505350
AACAAGCG
55
62
69
69
5-10-5
215





GCTAGGAG











TTCC








 


1280
1299
505351
CAAAACAA
20
49
49
54
5-10-5
216





GCGGCTAG











GAGT








 


1283
1302
505352
GAGCAAAA
53
83
73
87
5-10-5
217





CAAGCGGC











TAGG








 


1286
1305
505353
TGCGAGCA
64
73
68
78
5-10-5
218





AAACAAGC











GGCT








 


1413
1426
510063
ACAAAGGA
14
8
0
0
2-10-2
219





CGTCCC








 


1515
1534
505354
GAGGTGCG
68
81
61
80
5-10-5
220





CCCCGTGGT











CGG








 


1518
1537
505355
AGAGAGGT
59
75
75
84
5-10-5
221





GCGCCCCG











TGGT








 


1521
1540
505356
TAAAGAGA
63
76
83
78
5-10-5
222





GGTGCGCC











CCGT








 


1550
1563
510064
AAGGCACA
35
38
25
32
2-10-2
223





GACGGG








 


1577
1596
146786
GTGAAGCG
88
91
84
93
5-10-5
224





AAGTGCAC











ACGG








 


1580
1599
505357
GAGGTGAA
70
75
71
82
5-10-5
225





GCGAAGTG











CACA








 


1583
1602
505358
GCAGAGGT
77
82
72
84
5-10-5
226





GAAGCGAA











GTGC








 


1586
1605
505359
CGTGCAGA
72
73
67
80
5-10-5
227





GGTGAAGC











GAAG








 


1655
1674
505360
AGTCCAAG
66
68
54
68
5-10-5
228





AGTCCTCTT











ATG








 


1706
1719
510065
CAGTCTTTG
19
19
26
17
2-10-2
229





AAGTA








 


1778
1793
509985
TATGCCTAC
64
60
64
63
3-10-3
230





AGCCTCC








 


1779
1794
509986
TTATGCCTA
66
66
77
73
3-10-3
231





CAGCCTC








 


1780
1795
509987
TTTATGCCT
56
55
68
67
3-10-3
232





ACAGCCT








 


1781
1796
509988
ATTTATGCC
52
52
68
63
3-10-3
233





TACAGCC








 


1782
1797
509989
AATTTATGC
48
44
70
59
3-10-3
234





CTACAGC








 


1783
1798
509990
CAATTTATG
24
18
39
40
3-10-3
235





CCTACAG








 


1784
1799
509991
CCAATTTAT
37
37
55
55
3-10-3
236





GCCTACA








 


1785
1800
509992
ACCAATTTA
35
36
60
55
3-10-3
237





TGCCTAC








 


1806
1825
505361
AAAGTTGC
42
55
75
61
5-10-5
238





ATGGTGCT











GGTG








 


1809
1828
505362
GAAAAAGT
45
56
64
53
5-10-5
239





TGCATGGT











GCTG








 


1812
1831
505363
GGTGAAAA
71
70
80
72
5-10-5
240





AGTTGCAT











GGTG








 


1815
1834
505364
AGAGGTGA
51
57
77
82
5-10-5
241





AAAAGTTG











CATG








 


1818
1837
505365
GGCAGAGG
54
63
76
78
5-10-5
242





TGAAAAAG











TTGC








 


1821
1840
505366
TTAGGCAG
61
65
80
66
5-10-5
243





AGGTGAAA











AAGT








 


1822
1837
509993
GGCAGAGG
47
51
74
54
3-10-3
244





TGAAAAAG








 


1823
1838
509994
AGGCAGAG
47
40
76
54
3-10-3
245





GTGAAAAA








 


1824
1843
505367
TGATTAGG
41
39
62
29
5-10-5
246





CAGAGGTG











AAAA








 


1824
1839
509995
TAGGCAGA
46
42
79
59
3-10-3
247





GGTGAAAA








 


1826
1839
510066
TAGGCAGA
40
33
44
31
2-10-2
248





GGTGAA








 


1827
1846
505368
AGATGATT
27
46
62
51
5-10-5
249





AGGCAGAG











GTGA








 


1861
1880
146787
AGCTTGGA
59
61
65
72
5-10-5
250





GGCTTGAA











CAGT








 


1864
1883
505369
CACAGCTT
11
21
48
31
5-10-5
251





GGAGGCTT











GAAC








 


1865
1880
509996
AGCTTGGA
13
1
45
40
3-10-3
252





GGCTTGAA








 


1865
1878
510067
CTTGGAGG
22
17
20
14
2-10-2
253





CTTGAA








 


1866
1881
509997
CAGCTTGG
29
19
51
45
3-10-3
254





AGGCTTGA








 


1866
1879
510068
GCTTGGAG
24
25
37
32
2-10-2
255





GCTTGA








 


1867
1886
505370
AGGCACAG
32
36
58
33
5-10-5
63





CTTGGAGG











CTTG








 


1867
1882
509998
ACAGCTTG
1
4
23
12
3-10-3
256





GAGGCTTG








 


1867
1880
510069
AGCTTGGA
23
24
17
23
2-10-2
257





GGCTTG








 


1868
1883
509999
CACAGCTT
5
1
48
41
3-10-3
258





GGAGGCTT








 


1868
1881
510070
CAGCTTGG
21
20
0
18
2-10-2
259





AGGCTT








 


1869
1884
510000
GCACAGCT
14
10
50
37
3-10-3
260





TGGAGGCT








 


1869
1882
510071
ACAGCTTG
19
22
24
27
2-10-2
261





GAGGCT








 


1870
1889
505371
CCAAGGCA
27
40
68
38
5-10-5
69





CAGCTTGG











AGGC








 


1870
1885
510001
GGCACAGC
10
12
43
16
3-10-3
262





TTGGAGGC








 


1870
1883
510072
CACAGCTT
28
31
33
30
2-10-2
263





GGAGGC








 


1871
1886
510002
AGGCACAG
24
20
46
25
3-10-3
264





CTTGGAGG








 


1871
1884
510073
GCACAGCT
20
18
22
15
2-10-2
265





TGGAGG








 


1872
1887
510003
AAGGCACA
6
0
45
24
3-10-3
266





GCTTGGAG








 


1872
1885
510074
GGCACAGC
18
18
32
23
2-10-2
267





TTGGAG








 


1873
1892
505372
CACCCAAG
18
8
55
16
5-10-5
268





GCACAGCT











TGGA








 


1873
1888
510004
CAAGGCAC
9
0
31
15
3-10-3
269





AGCTTGGA








 


1873
1886
510075
AGGCACAG
23
9
27
10
2-10-2
270





CTTGGA








 


1874
1889
510005
CCAAGGCA
0
0
39
25
3-10-3
271





CAGCTTGG








 


1876
1895
505373
AGCCACCC
47
50
69
56
5-10-5
272





AAGGCACA











GCTT








 


1879
1898
505374
CAAAGCCA
27
27
55
30
5-10-5
273





CCCAAGGC











ACAG








 


1882
1901
505375
CCCCAAAG
34
40
54
39
5-10-5
274





CCACCCAA











GGCA








 


1885
1904
505376
ATGCCCCA
41
43
54
52
5-10-5
275





AAGCCACC











CAAG








 


1888
1907
505377
TCCATGCCC
40
42
72
40
5-10-5
276





CAAAGCCA











CCC








 


1891
1910
505378
ATGTCCATG
35
33
70
40
5-10-5
277





CCCCAAAG











CCA








 


1918
1933
510006
CTCCAAATT
9
2
53
41
3-10-3
278





CTTTATA








 


1918
1931
510076
CCAAATTCT
28
22
7
22
2-10-2
279





TTATA








 


1919
1934
510007
GCTCCAAA
43
39
72
57
3-10-3
280





TTCTTTAT








 


1919
1932
510077
TCCAAATTC
19
11
0
2
2-10-2
281





TTTAT








 


1920
1933
510078
CTCCAAATT
19
11
0
0
2-10-2
282





CTTTA








 


1921
1934
510079
GCTCCAAA
50
48
61
55
2-10-2
283





TTCTTT








 


1957
1976
505379
GGAAAGAA
17
14
81
39
5-10-5
284





GTCAGAAG











GCAA








 


2270
2285
510008
GTGCGAAT
21
4
36
11
3-10-3
285





CCACACTC








 


2270
2283
510080
GCGAATCC
32
29
41
33
2-10-2
286





ACACTC








 


2271
2284
510081
TGCGAATC
28
20
25
11
2-10-2
287





CACACT








 


2272
2285
510082
GTGCGAAT
28
20
32
22
2-10-2
288





CCACAC








 


2368
2387
505380
GAGGGAGT
24
22
90
48
5-10-5
289





TCTTCTTCT











AGG








 


2378
2393
510009
CGAGGCGA
12
1
65
10
3-10-3
290





GGGAGTTC








 


2378
2391
510083
AGGCGAGG
17
18
29
25
2-10-2
291





GAGTTC








 


2379
2394
510010
GCGAGGCG
18
13
82
37
3-10-3
292





AGGGAGTT








 


2379
2392
510084
GAGGCGAG
29
22
54
30
2-10-2
293





GGAGTT








 


2380
2395
510011
TGCGAGGC
13
11
69
44
3-10-3
294





GAGGGAGT








 


2380
2393
510085
CGAGGCGA
25
20
53
42
2-10-2
295





GGGAGT








 


2381
2396
510012
CTGCGAGG
17
14
79
53
3-10-3
296





CGAGGGAG








 


2381
2394
510086
GCGAGGCG
33
29
66
48
2-10-2
297





AGGGAG








 


2382
2397
510013
TCTGCGAG
18
4
77
47
3-10-3
298





GCGAGGGA








 


2420
2439
505381
CCGAGATT
12
18
83
28
5-10-5
299





GAGATCTTC











TGC








 


2459
2478
505382
CCCACCTTA
14
19
80
36
5-10-5
300





TGAGTCCA











AGG








 


2819
2838
505383
TGTTCCCAA
29
32
78
44
5-10-5
301





GAATATGG











TGA








 


2820
2835
510014
TCCCAAGA
10
10
68
40
3-10-3
302





ATATGGTG








 


2821
2836
510015
TTCCCAAG
5
0
62
24
3-10-3
303





AATATGGT








 


2822
2837
510016
GTTCCCAA
6
2
42
16
3-10-3
304





GAATATGG








 


2823
2838
510017
TGTTCCCAA
18
18
47
18
3-10-3
305





GAATATG








 


2824
2839
510018
TTGTTCCCA
7
5
57
19
3-10-3
306





AGAATAT








 


2825
2838
510087
TGTTCCCAA
25
20
44
25
2-10-2
307





GAATA








 


2873
2892
505384
GAAAGAAT
8
4
61
22
5-10-5
308





CCCAGAGG











ATTG








 


3161
3180
146833
ACTGCATG
47
46
82
54
5-10-5
309





GCCTGAGG











ATGA








 


3163
3182
505385
CCACTGCAT
25
34
69
19
5-10-5
310





GGCCTGAG











GAT









Example 3
Antisense Inhibition of HBV Viral mRNA in HepAD38 (Tet-HBV) Cells by MOE Gapmers
Certain antisense oligonucleotides selected from the study described in Example 2 were tested for their effects on HBV mRNA in another cell line, human hepatoma HepAD38 cells, in which HBV production is under the control of a tetracycline-regulated promoter. Cultured HepAD38 (Tet-HBV) cells at a density of 45,000 cells per well were transfected using electroporation with 15,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe sets RTS3372 and RTS3373MGB were used individually to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented in Table 3 as percent inhibition of HBV, relative to untreated control cells.







TABLE 3







Inhibition of viral HBV mRNA levels by MOE gapmersin HepAD38


(Tet-HBV) cells (detected by RTS3372 and RTS3373MGB)



















SEQ


Start
Stop
ISIS

RTS3373MGB
RTS3372 
ID


Site
Site
No
Motif
% inhibition
% inhibition
NO
















58
77
146779
5-10-5
76
82
83


58
71
510019
5-10-5
0
9
84


61
80
505314
5-10-5
65
75
85


196
215
505315
5-10-5
46
65
87


199
218
505316
5-10-5
57
71
88


205
224
505317
5-10-5
83
87
89


228
241
510020
2-10-2
6
0
90


229
242
510021
2-10-2
19
24
91


244
263
146821
5-10-5
72
71
92


245
258
510022
2-10-2
6
24
94


247
266
505318
5-10-5
68
77
96


250
269
509921
5-10-5
25
47
97


251
270
509922
5-10-5
28
46
99


252
271
509923
5-10-5
19
40
101


253
272
505319 
5-10-5
69
66
103


254
273
509924 
5-10-5
9
39
105


254
267
510023 
2-10-2
19
15
107


255
274
509925 
5-10-5
26
55
108


255
268
510024 
2-10-2
0
5
110


256
275
505320 
5-10-5
62
68
111


256
269
510025 
2-10-2
0
8
113


257
270
510026
2-10-2
7
21
114


258
271
510027 
2-10-2
0
0
116


259
272
510028 
2-10-2
0
0
118


260
273
510029
2-10-2
0
9
119


261
274
510030 
2-10-2
0
0
120


262
281
505321 
5-10-5
53
54
121


265
284
505322
5-10-5
59
60
122


293
312
505323
5-10-5
65
77
123


296
315
505324 
5-10-5
78
83
124


302
321
505325 
5-10-5
71
80
125


360
379
505326
5-10-5
76
84
126


366
385
505327 
5-10-5
77
83
127


369
388
505328
5-10-5
65
78
128


384
397
510031
2-10-2
0
16
130


385
398
510032 
2-10-2
0
0
131


386
399
510033
2-10-2
1
21
133


387
400
510034
2-10-2
8
28
134


388
401
510035
2-10-2
0
0
135


411
430
505329 
5-10-5
58
72
136


411
424
510036
2-10-2
6
11
138


412
431
509926 
5-10-5
20
54
139


412
425
510037
2-10-2
0
10
141


413
432
509927 
5-10-5
56
76
142


413
426
510038 
2-10-2
54
68
144


414
433
505330 
5-10-5
66
81
20


414
427
510039 
2-10-2
60
74
146


415
428
510040 
2-10-2
33
39
148


416
429
510041 
2-10-2
30
58
150


417
430
510042 
2-10-2
34
57
152


418
431
510043 
2-10-2
0
2
154


419
432
510044
2-10-2
0
29
156


420
433
510045
2-10-2
3
31
158


421
434
510046
2-10-2
0
0
160


422
435
510047
2-10-2
0
0
162


423
436
510048
2-10-2
0
0
163


424
437
510049
2-10-2
0
0
164


454
473
505331
5-10-5
60
77
165


457
476
505332
5-10-5
55
74
166


458
471
510050
2-10-2
47
47
169


459
472
510051
2-10-2
35
55
170


460
473
510052
2-10-2
27
41
171


463
482
505333
5-10-5
66
78
172


466
485
505334
5-10-5
53
63
173


472
491
505335
5-10-5
70
76
174


475
494
505336
5-10-5
64
77
175


670
689
146823
5-10-5
74
79
180


670
683
510053
2-10-2
18
20
182


671
684
510054
2-10-2
13
21
183


672
685
510055
2-10-2
4
2
184


673
692
505337
5-10-5
60
72
185


679
698
505338
5-10-5
62
75
186


682
701
505339
5-10-5
81
90
187


688
707
505340
5-10-5
67
81
189


691
710
505341
5-10-5
68
80
193


691
704
510056
2-10-2
0
0
195


692
705
510057
2-10-2
37
48
196


693
706
510058
2-10-2
44
59
197


697
716
505342
5-10-5
80
87
198


738
751
510059
2-10-2
0
0
200


739
752
510060
2-10-2
0
0
202


740
753
510061
2-10-2
23
19
203


741
754
510062
2-10-2
25
30
204


756
775
505343
5-10-5
62
71
205


823
842
505344
5-10-5
52
66
206


1170
1189
505345
5-10-5
83
81
207


1259
1278
505346
5-10-5
84
81
210


1262
1287
505347
5-10-5
89
84
212


1268
1287 
505348
5-10-5
78
78
213


1271
1290
505349
5-10-5
74
77
214


1277
1296
505350
5-10-5
75
77
215


1280
1299 
505351
5-10-5
49
62
216


1283
1302 
505352
5-10-5
70
66
217


1286
1305 
505353
5-10-5
62
60
218


1413 
1426 
510063
2-10-2
0
0
219


1515
1534 
505354
5-10-5
85
75
220


1518
1537 
505355
5-10-5
81
74
221


1521
1540 
505356
5-10-5
57
52
222


1550
1563
510064
2-10-2
0
0
223


1577
1596 
146786
5-10-5 
94
85
224


1580
1599
505357
5-10-5 
86
79
225


1583
1602
505358
5-10-5 
89
79
226


1586
1605
505359
5-10-5 
82
68
227


1655
1674 
505360
5-10-5 
84
74
228


1706
1719 
510065
2-10-2 
0
0
229


1806
1825
505361
5-10-5 
66
66
238


1809
1828
505362
5-10-5 
52
59
239


1812
1831 
505363
5-10-5
72
75
240


1815
1834 
505364
5-10-5 
73
80
241


1818
1837
505365
5-10-5 
68
82
242


1821 
1840
505366
5-10-5 
50
76
243


1824
1843
505367
5-10-5 
58
76
246


1826
1839
510066
2-10-2
0
31
248


1827 
1846 
505368
5-10-5 
71
84
249


1861
1880 
146787
5-10-5 
25
35
250


1864
1883
505369
5-10-5 
29
65
251


1865
1878
510067
2-10-2
0
0
253


1866
1879
510068
2-10-2
0
20
255


1867
1886 
505370
5-10-5 
45
70
63


1867
1880
510069
2-10-2
0
0
257


1868
1881
510070
2-10-2
0
0
259


1869
1882 
510071
2-10-2
0
0
261


1870
1889 
505371
5-10-5 
48
66
69


1870
1883 
510072
2-10-2
0
0
263


1871
1884
510073
2-10-2
0
0
265


1872
1885 
510074
2-10-2
0
2
267


1873
1892 
505372
5-10-5 
48
67
268


1873
1886
510075
2-10-2
0
0
270


1876
1895
505373
5-10-5 
23
48
272


1879
1898
505374
5-10-5
0
34
273


1882
1901 
505375
5-10-5 
39
66
274


1885
1904 
505376
5-10-5
0
40
275


1888
1907 
505377
5-10-5
4
47
276


1891
1910 
505378
5-10-5 
65
77
277


1918 
1931
510076
2-10-2
0
0
279


1919
1932 
510077
2-10-2
0
0
281


1920
1933 
510078
2-10-2
0
0
282


1921
1934
510079
2-10-2 
18
50
283


1957
1976
505379
5-10-5 
42
84
284


2270
2283 
510080
2-10-2
0
0
286


2271
2284
510081
2-10-2
0
0
287


2272
2285 
510082
2-10-2
0
10
288


2368
2387 
505380
5-10-5
29
79
289


2378
2391
510083
2-10-2
0
0
291


2379
2392
510084
2-10-2
31
17
293


2380
2393 
510085
2-10-2
0
8
295


2381 
2394 
510086
2-10-2
10
2
297


2420
2439 
505381
5-10-5
30
86
299


2459
2478 
505382
5-10-5
16
87
300


2819
2838
505383
5-10-5
26
81
301


2825
2838 
510087
2-10-2
0
0
307


2873
2892 
505384
5-10-5
31
59
308


3161
3180
146833
5-10-5
55
76
309


3163
3182
505385
5-10-5
58
83
310









Example 4
Antisense Inhibition of HBV Viral mRNA in HepAD38 (Tet-HBV) Cells by MOE Gapmers
Certain antisense oligonucleotides from the study described in Examples 1 and 2 were tested for their effects on HBV mRNA in vitro. Cultured HepAD38 (Tet-HBV) cells at a density of 45,000 cells per well were transfected using electroporation with 15,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3372 was used to measure mRNA levels. The mRNA levels were also measured using the RTS3373MGB primer probe set. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented in Table 4 as percent inhibition of HBV, relative to untreated control cells.







TABLE 4







Inhibition of viral HBV mRNA levels by MOE gapmers


(RTS3372 and RTS3373MGB)

















RTS3372
RTS3373MGB
SEQ


Start
Stop
ISIS 

%
% 
ID


Site
Site
No
Motif
inhibition
inhibition
NO
















62
77
509941
3-10-3
36
5
86


245
260
509942
3-10-3
3
0
93


245
261
510088
3-10-4
24
10
5


246
261
509943
3-10-3
27
13
95


250
265
509944
3-10-3
46
34
98


250
266
510089
3-10-4
61
33
6


251
266
509945
3-10-3
54
43
100


251
267
510090
3-10-4
58
32
7


252
267
509946
3-10-3
50
28
102


252
268
510091
3-10-4
60
42
8


253
268
509947
3-10-3
49
40
104


253
269
510092
3-10-4
40
9
9


254
269
509948
3-10-3
13
22
106


254
270
510093
3-10-4
39
2
10


255
270
509949
3-10-3
33
24
109


255
271
510094
3-10-4
40
16
11


256
271
509950
3-10-3
31
23
112


256
272
510095
3-10-4
24
6
12


257
273
510096
3-10-4
62
44
13


258
273 
509952
3-10-3
42
40
115


258
274
510097
3-10-4
65
48
14


259
274
509953
3-10-3
35
29
117


384
399
509954
3-10-3
35
18
129


384
400
510098
3-10-4
62
43
15


385
401
510099
3-10-4
67
50
16


386
401
509955
3-10-3
44
37
132


411
426
509956
3-10-3
67
53
137


411
427
510100
3-10-4
88
69
17


412
427 
509957
3-10-3
86
76
140


412
428
510101
3-10-4
71
46
18


413
428
509958
3-10-3
78
74
143


413
429 
510102
3-10-4
77
52
19


414
433 
505330
5-10-5
81
60
20


414
429 
509959
3-10-3
62
49
145


414
430
510103
3-10-4
9
5
21


415
434
509928
5-10-5
81
66
22


415
430
509960
3-10-3
67
57
147


415
431
510104
3-10-4
71
57
23


416
435
509929
5-10-5
82
69
24


416
431
509961
3-10-3
62
43
149


416
432
510105
3-10-4
81
64
25


417
436
509930
5-10-5
74
45
26


417
432
509962
3-10-3
59
48
151


417
433
510106
3-10-4
86
70
27


418
437
146783
5-10-5
19
3
28


418
433
509963
3-10-3
48
28
153


418
434
510107
3-10-4
74
51
29


419
434
509964
3-10-3
50
39
155


419
435
510108
3-10-4
67
50
30


420
435
509965
3-10-3
49
38
157


420
436
510109
3-10-4
12
13
31


421
436
509966
3-10-3
23
22
159


421
437
510110
3-10-4
34
16
32


422
437
509967
3-10-3
3
12
161


457
472 
509968
3-10-3
56
38
167


457
473 
510111
3-10-4
68
51
33


458
473
509969 
3-10-3
53
39
168


639
658
146784 
5-10-5
0
0
34


639
654
509970 
3-10-3
51
15
176


639
655
510112 
3-10-4
66
32
35


640
656
510113 
3-10-4
70
31
36


641
656
509971 
3-10-3
54
31
177


641
657
510114 
3-10-4
67
45
37


642
657
509972 
3-10-3
51
25
178


642
658
510115
3-10-4
73
50
38


643
658
509973 
3-10-3
49
32
179


670
685
509974
3-10-3
74
67
181


687
706
509931 
5-10-5
92
83
39


687
702
509975 
3-10-3
72
71
188


687
703
510116 
3-10-4
83
74
40


688
703
509976 
3-10-3
46
52
190


688
704
510117 
3-10-4
71
57
41


689
704
509977 
3-10-3
18
22
191


689
705
510118 
3-10-4
71
50
42


690
705
509978 
3-10-3
57
37
192


690
706
510119 
3-10-4
80
64
43


691
706
509979 
3-10-3
65
55
194


738
753
509980 
3-10-3
48
44
199


738
754
510120 
3-10-4
70
54
44


739
754
509981 
3-10-3
54
45
201


1176
1191 
509982 
3-10-3
44
36
208


1176
1192
510121 
3-10-4
74
69
45


1177
1192
509983 
3-10-3
57
53
209


1261
1276
509984 
3-10-3
57
50
211


1778
1797
509932 
5-10-5
30
76
46


1778
1793
509985 
3-10-3
0
46
230


1778
1794 
510122 
3-10-4
0
60
47


1779
1798
509933 
5-10-5
54
78
48


1779
1794
509986 
3-10-3
56
81
231


1779
1795
510123 
3-10-4
74
85
49


1780
1799
509934 
5-10-5
69
84
50


1780
1795 
509987 
3-10-3
52
78
232


1780
1796
510124 
3-10-4
75
84
51


1781
1800
509935 
5-10-5
72
85
52


1781
1796
509988 
3-10-3
57
68
232


1781
1797 
510125 
3-10-4
68
72
53


1782
1797 
509989 
3-10-3
46
41
234


1782
1798
510126 
3-10-4
56
51
54


1783
1798
509990
3-10-3
16
25
234


1783
1799
510127
3-10-4
61
69
55


1784
1799
509991
3-10-3
41
41
236


1784
1800
510128
3-10-4
61
68
56


1785
1800
509992
3-10-3
43
43
237


1822
1837
509993
3-10-3
72
44
244


1822
1838
510129
3-10-4
66
33
57


1823
1838
509994
3-10-3
79
32
245


1823
1839
510130
3-10-4
49
31
58


1824
1839
509995
3-10-3
63
30
247


1865
1884
509936
5-10-5
74
59
59


1865
1880
509996
3-10-3
36
0
252


1865
1881
510131
3-10-4
26
0
60


1866
1885
509937
5-10-5
78
63
61


1866
1881
509997
3-10-3
5
0
254


1866
1882 
510132
3-10-4
37
4
62


1867
1886
505370
5-10-5
54
17
63


1867
1882
509998
3-10-3
13
0
256


1867
1883
510133
3-10-4
42
25
64


1868
1887 
509938
5-10-5
9
6
65


1868
1883
509999
3-10-3
47
6
258


1868
1884
510134
3-10-4
56
27
66


1869
1888
509939
5-10-5
64
29
67


1869
1884
510000
3-10-3
24
1
260


1869
1885 
510135
3-10-4
70
43
68


1870
1889 
505371
5-10-5
63
46
69


1870
1885
510001
3-10-3
39
12
262


1870
1886 
510136
3-10-4
52
23
70


1871
1886 
510002
3-10-3
10
0
264


1871
1887
510137
3-10-4
28
0
71


1872
1887
510003
3-10-3
21
0
266


1872
1888
510138
3-10-4
25
7
72


1873
1888 
510004
3-10-3
21
38
269


1873
1889 
510139
3-10-4
18
0
73


1874
1889
510005
3-10-3
8
0
271


1918
1933 
510006
3-10-3
0
0
278


1918
1934 
510140
3-10-4
81
67
74


1919
1934 
510007
3-10-3
69
66
280


2270
2285
510008
3-10-3
23
0
285


2378
2397
509940
3-10-4
66
7
75


2378
2393
510009
3-10-3
23
0
290


2378
2394
510141
3-10-4
10
11
76


2379
2394
510010
3-10-3
39
6
292


2379
2395
510142
3-10-4
46
24
77


2380
2395
510011
3-10-3
33
23
294


2380
2396
510143
3-10-4
59
36
78


2381
2396
510012
3-10-3
38
22
296


2381
2397
510144
3-10-4
54
20
79


2382
2397
510013
3-10-3
42
0
298


2820
2835
510014
3-10-3
51
9
302


2820
2836
510145
3-10-4
68
19
80


2821
2836
510015
3-10-3
35
2
303


2821
2837
510146
3-10-4
65
15
81


2822
2837
510016
3-10-3
9
0
304


2822
2838
510147
3-10-4
30
0
85


2823
2838
510017
3-10-3
18
0
305


2824
2839
510018
3-10-3
24
5
306









Example 5
Dose-Dependent Inhibition of Viral HBV RNA in HepG2.2.15 Cells by MOE Gapmers
Certain gapmers from the study described in Examples 3 and 4 were tested at various doses in human HepG2.2.15 cells. Cells were plated at a density of 25,000 cells per well and transfected using electroporation with 2.5 μM, 5.0 μM, 10.0 μM, and 20.0 μM concentrations of antisense oligonucleotide, as specified in Table 5. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.
The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented in Table 5. As illustrated in Table 5, HBV mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.







TABLE 5







Dose-dependent antisense inhibition of HBV


RNA in HepG2.2.15 cells using RTS3370












ISIS No
2.5 μM
5.0 μM
10.0 μM
20.0 μM
IC50 (μM)















146786
33
50
54
81
5.7


505317
35
40
63
67
6.6


505323
16
33
48
63
11.1


505326
27
44
64
67
6.9


509929
21
44
60
62
8.4


509931
51
63
75
75
<2.5


509957
37
53
57
70
5.4


509974
25
35
54
63
9.5


509975
36
55
62
81
4.7


509981
7
23
35
52
18.8


510039
27
46
60
69
6.9


510040
10
28
43
59
13.4


510041
29
41
53
66
8.3


510058
9
34
42
63
11.9









Example 6
Dose-Dependent Inhibition of Viral HBV RNA in HepG2.2.15 Cells by MOE Gapmers
Additional gapmers from the study described in Examples 3 and 4 were further tested at various doses in human HepG2.2.15 cells. Cells were plated at a density of 28,000 cells per well and transfected using LipofectAMINE 2000® reagent with 15.625 nM, 31.25 nM, 62.5 nM, 125.0 nM, and 250.0 nM concentrations of antisense oligonucleotide, as specified in Table 6. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.
The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented in Table 6. As illustrated in Table 6, HBV mRNA levels were significantly reduced in a dose-dependent manner in some antisense oligonucleotide treated cells.







TABLE 6







Dose-dependent antisense inhibition of HBV RNA


in HepG2.2.15 cells using RTS3370













ISIS
15.625
31.25
62.5
125.0
250.0
IC50


No
nM
nM
nM
nM
nM
(nM)
















146779
14
25
44
70
78
73.1


146786
10
35
64
85
93
49.4


146833
12
16
32
62
72
99.8


505317
19
31
44
69
83
65.2


505319
5
11
24
39
69
152.8


505323
2
11
26
68
90
85.4


505326
1
15
45
72
89
73.7


505327
0
4
12
56
74
128.5


505329
3
16
33
51
64
130.4


505339
26
32
59
82
92
46.0


505342
10
4
34
69
74
95.7


505347
20
26
41
70
92
63.0


505356
0
0
0
38
69
182.0


505358
8
28
47
71
84
67.9


505382
5
0
3
26
19
>250.0


509926
0
6
18
42
67
159.3


509927
3
17
33
55
76
103.2


509929
7
19
36
60
69
102.9


509931
18
28
52
76
87
57.4


509934
14
14
40
61
76
89.3


509957
20
28
51
71
79
63.1


509958
12
17
37
56
76
96.4


509959
12
11
18
59
70
121.7


509960
9
19
30
57
74
103.4


509972
15
6
17
27
45
>250.0


509974
25
35
57
83
92
45.3


509975
33
44
45
61
80
53.1


509981
0
15
11
35
60
224.4


510007
0
0
15
31
45
>250.0


510038
12
19
48
73
84
68.9


510039
17
25
44
69
72
77.3


510040
17
20
23
59
72
108.6


510041
11
21
43
64
79
80.5


510050
3
21
16
51
70
132.4


510058
7
9
16
22
46
>250.0


510079
0
6
11
29
32
>250.0


510100
18
34
50
79
83
56.1


510106
23
25
35
69
74
78.4


510116
20
44
65
79
91
42.6


510140
7
28
30
55
58
136.5









The mRNA levels were also measured with primer probe set RTS3371. The results are presented in Table 7.







TABLE 7







Dose-dependent antisense inhibition of HBV RNA in 


HepG2.2.15 cells using RTS3371













ISIS
15.625
31.25
62.5
125.0
250.0
IC50


No
nM
nM
nM
nM
nM
(nM)
















146779
16
7
38
69
68
96.9


146786
28
39
65
86
93
35


146833
26
22
52
61
65
82.3


505317
18
33
40
77
84
61.4


505319
0
0
0
15
55
>250.0


505323
0
0
33
66
87
100.5


505326
0
21
7
57
85
114.6


505327
0
0
40
50
63
132.3


505329 
11
22
35
66
77
90.7


505342 
15
0
1
40
59
190.1


505347 
3
35
44
65
90
68.4


505356 
0
0
3
42
76
153.2


505358 
20
11
39
71
78
79.7


505382 
0
0
0
0
0
>250.0


509926 
0
4
14
55
72
130.6


509927 
11
25
31
61
78
88.4


509929 
11
26
41
70
77
75.8


509931 
25
39
55
79
85
46.6


509934 
0
25
32
54
65
119.9


509957 
25
44
48
74
80
50.6


509958 
24
18
20
57
72
114.5


509959
2
9
31
52
65
132.3


509960 
16
28
22
57
75
101.8


509972 
3
5
1
39
60
236.3


509974 
38
46
65
83
94
31.2


509975 
30
7
24
49
67
148.2


509981 
22
22
23
46
58
194.7


510007 
3
0
15
33
39
>250.0


510038 
16
22
50
76
84
62.9


510039 
23
36
32
70
68
79.7


510040
18
15
41
59
67
101.9


510041 
0
27
38
62
81
84.5


510050 
1
16
17
52
63
149


510058 
20
19
40
44
51
214.1


510079 
0
2
5
41
49
>250.0


510100 
35
52
61
86
90
30.7


510106 
27
23
5
75
81
87.9


510116 
11
44
70
72
94
46.5


510140 
0
18
26
45
41
>250.0









Example 7
Tolerability of MOE Gapmers Targeting HBV in BALB/c Mice
BALB/c mice (Charles River, Mass.) are a multipurpose model of mice, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various metabolic markers.
Study 1
Groups of four BALB/c mice each were injected subcutaneously twice a week for 3 weeks with 50 mg/kg of ISIS 146779, ISIS 146786, ISIS 505317, ISIS 505319, ISIS 505330, ISIS 505332, ISIS 505339, ISIS 505346, ISIS 505347, ISIS 505358, ISIS 509929, ISIS 509931, ISIS 509932, ISIS 509934, ISIS 509957, ISIS 510100, ISIS 510106, ISIS 510116, and ISIS 510140. A group of four BALB/c mice were injected subcutaneously twice a week for 3 weeks with 50 mg/kg of ISIS 141923 (CCTTCCCTGAAGGTTCCTCC (SEQ ID NO: 320)), a 5-10-5 MOE gapmer with no known homology to any human or mouse gene sequence. Another group of 4 BALB/c mice was injected subcutaneously twice a week for 3 weeks with PBS. This group of mice served as the control group. Three days after the last dose at each time point, body weights were taken, mice were euthanized and organs and plasma were harvested for further analysis.
Body and Organ Weights
The body weights of the mice were measured pre-dose and at the end of each treatment period. The body weights are presented in Table 8, and are expressed as percent change from the weight taken before the start of treatment. Liver, spleen and kidney weights were measured at the end of the study, and are presented in Table 9 as a percentage difference from the respective organ weights of the PBS control. The results indicate that most of the ISIS oligonucleotides did not cause any adverse effects on body or organ weights.







TABLE 8







Change in body weights of BALB/c mice


after antisense oligonucleotide treatment (%)









Body weight














PBS
9



ISIS 141923
9



ISIS 146779
11



ISIS 146786
9



ISIS 505317
10



ISIS 505319
14



ISIS 505330
11



ISIS 505332
10



ISIS 505339
14



ISIS 505346
12



ISIS 505347
16



ISIS 505358
12



ISIS 509929
8



ISIS 509931
9



ISIS 509932
21



ISIS 509934
14



ISIS 509957
10



ISIS 510100
10



ISIS 510106
15



ISIS 510116
16



ISIS 510140
19

















TABLE 9







Change in organ weights of BALB/c mice


after antisense oligonucleotide treatment (%)











Liver
Kidney
Spleen
















PBS
—
—
—



ISIS 141923
3
−3
−9



ISIS 146779
10
1
13



ISIS 146786
19
−3
4



ISIS 505317
−4
−7
9



ISIS 505319
1
−16
23



ISIS 505330
12
−4
9



ISIS 505332
7
−2
14



ISIS 505339
5
−6
7



ISIS 505346
7
−6
0



ISIS 505347
12
−7
5



ISIS 505358
8
0
3



ISIS 509929
17
14
200



ISIS 509931
−4
−9
3



ISIS 509932
18
−9
79



ISIS 509934
6
−6
2



ISIS 509957
0
−2
15



ISIS 510100
2
1
8



ISIS 510106
5
−2
58



ISIS 510116
12
−8
7



ISIS 510140
20
−8
49










Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in Table 10 expressed in IU/L. Plasma levels of cholesterol and triglycerides were also measured using the same clinical chemistry analyzer and the results are also presented in Table 10.







TABLE 10







Effect of antisense oligonucleotide treatment on


metabolic markers in the liver of BALB/c mice












ALT
AST
Cholesterol
Triglycerides



(IU/L)
(IU/L)
(mg/dL)
(mg/dL)















PBS
37
58
114
238


ISIS 141923
36
57
114
234


ISIS 146779
43
56
121
221


ISIS 146786
53
76
118
327


ISIS 505317
68
103
117
206


ISIS 505319
136
152
144
168


ISIS 505330
281
194
119
188


ISIS 505332
67
70
123
226


ISIS 505339
113
111
135
249


ISIS 505346
56
63
128
234


ISIS 505347
79
83
122
347


ISIS 505358
78
175
112
214


ISIS 509929
111
166
61
175


ISIS 509931
635
508
110
179


ISIS 509932
92
113
118
131


ISIS 509934
38
89
97
176


ISIS 509957
159
229
85
173


ISIS 510100
90
87
86
222


ISIS 510106
61
88
79
239


ISIS 510116
70
95
124
214


ISIS 510140
1247
996
161
167









Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma concentrations of blood urea nitrogen (BUN) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 11, expressed in mg/dL.







TABLE 11







Effect of antisense oligonucleotide treatment


on kidney markers of BALB/c mice









BUN (mg/dL)














PBS
29



ISIS 141923
29



ISIS 146779
28



ISIS 146786
30



ISIS 505317
30



ISIS 505319
30



ISIS 505330
29



ISIS 505332
28



ISIS 505339
29



ISIS 505346
27



ISIS 505347
26



ISIS 505358
26



ISIS 509929
25



ISIS 509931
23



ISIS 509932
28



ISIS 509934
25



ISIS 509957
24



ISIS 510100
27



ISIS 510106
27



ISIS 510116
25



ISIS 510140
22










Study 2

Groups of four BALB/c mice each were injected subcutaneously twice a week for 3 weeks with 50 mg/kg of ISIS 505329, ISIS 509926, ISIS 509927, ISIS 509958, ISIS 509959, ISIS 509960, ISIS 509974, ISIS 509975, ISIS 510038, ISIS 510039, ISIS 510040, ISIS 510041, and ISIS 510050. A group of 4 BALB/c mice was injected subcutaneously twice a week for 3 weeks with PBS. This group of mice served as the control group. Three days after the last dose at each time point, body weights were taken, mice were euthanized and organs and plasma were harvested for further analysis.
Organ Weights
Liver, spleen and kidney weights were measured at the end of the study, and are also presented in Table 12 as a percentage change over the respective organ weights of the PBS control.







TABLE 12







Change in organ weights of BALB/c mice


after antisense oligonucleotide treatment (%)












ISIS No
Liver
Kidney
Spleen
















505329
12
2
12



509926
23
3
30



509927
8
−4
27



509958
1
−4
9



509959
7
0
26



509960
16
6
30



509974
5
8
7



509975
1
−1
7



510038
6
4
23



510039
0
15
9



510040
3
1
2



510041
6
6
10



510050
5
5
18










Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in Table 13 expressed in IU/L.







TABLE 13







Effect of antisense oligonucleotide treatment on


transaminases (IU/L) in the liver of BALB/c mice










ALT
AST















PBS
37
78



ISIS 505329
48
65



ISIS 509926
77
120



ISIS 509927
71
92



ISIS 509958
106
105



ISIS 509959
119
122



ISIS 509960
40
66



ISIS 509974
38
43



ISIS 509975
33
45



ISIS 510038
69
66



ISIS 510039
32
61



ISIS 510040
83
113



ISIS 510041
32
45



ISIS 510050
26
47










Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma concentrations of blood urea nitrogen (BUN) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 14, expressed in mg/dL.







TABLE 14







Effect of antisense oligonucleotide treatment


on kidney markers of BALB/c mice









BUN














PBS
21



ISIS 505329
22



ISIS 509926
20



ISIS 509927
20



ISIS 509958
22



ISIS 509959
21



ISIS 509960
20



ISIS 509974
19



ISIS 509975
19



ISIS 510038
19



ISIS 510039
19



ISIS 510040
22



ISIS 510041
18



ISIS 510050
22










Example 8
Dose Response Confirmation of MOE Gapmers Targeting HBV in HepG2.2.15 Cells
Gapmers were chosen based on sequence conservation, activity and tolerability, as measured in the study described in Examples 7 and 8, and tested at various doses in HepG2.2.15 cells. Cells were plated at a density of 28,000 cells per well and transfected using LipofectAMINE 2000 reagent with 15.625 nM, 31.25 nM, 62.5 nM, 125.0 nM and 250.0 nM concentrations of antisense oligonucleotide. Two days post-transfection, the media was replaced with fresh media. Samples were collected 4 days post-transfection. DNA, RNA, HBsAg and HBeAg levels were measured in the supernatant.
HBV mRNA levels were measured by quantitative real-time PCR. HBV primer probe set RTS3370 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells. As illustrated in Table 15, HBV mRNA levels were reduced in a dose-dependent manner in most of the antisense oligonucleotide treated cells.
HBV antigens in the supernatants were detected with the ELISA technique. HBs antigen (HBsAg) levels were detected by ELISA from Abazyme LLC, Mass. As presented in Table 16, treatment with ISIS oligonucleotides 146779, 146786, 505329, 505330, 505339, 505347, 505358, 509927, 509934, 509958, 509959, 509960, 509974, 5100038, 510039, 510040, 510041, 510100, 510106, and 510116 caused significant reduction in HBsAg levels. HBe antigen (HBeAg) levels were detected by ELISA from International Immuno-diagnostics, CA. As presented in Table 17, treatment with ISIS oligonucleotides 146779, 146786, 505329, 505330, 505339, 505347, 505358, 509927, 509934, 509958, 509959, 509960, 509974, 5100038, 510039, 510040, 510041, 510100, 510106, and 510116 caused significant reduction in HBeAg levels. HBV DNA levels were measured using primer probe set RTS3370. As presented in Table 18, treatment with ISIS oligonucleotides 146779, 146786, 505329, 505330, 505339, 505347, 505358, 509927, 509934, 509958, 509959, 509960, 509974, 5100038, 510039, 510040, 510041, 510100, 510106, and 510116 caused significant reduction in HBV DNA levels. The total protein in the supernatants was measured by a DC protein assay (BioRad), as presented in Table 19.







TABLE 15







Dose-dependent antisense inhibition


of HBV RNA in HepG2.2.15cells












ISIS No
15.625 nM
31.25 nM
62.5 nM
125 nM
250 nM















146779
10
25
42
64
95


146786
23
59
78
84
90


505329
45
49
57
69
83


505330
31
61
65
80
93


505339
31
56
78
89
97


505347
30
50
72
87
96


505358
28
52
75
86
95


509927
41
61
67
61
76


509934
38
61
64
82
58


509958
50
67
72
79
89


509959
50
63
73
80
86


509960
63
61
72
82
74


509974
29
44
75
91
96


510038
29
40
85
89
93


510039
32
34
63
84
84


510040
18
0
51
71
77


510041
34
53
67
76
71


510100
29
64
70
89
93


510106
28
65
64
81
85


510116
13
34
78
89
95
















TABLE 16







Dose-dependent reduction of S antigen


in HepG2.2.15 cell supernatant











ISIS No
15.625 nM
31.25 nM
62.5 nM
125 nM














146779
40
58
80
92


146786
47
75
92
98


505329
37
58
71
89


505330
45
66
84
95


505339
62
79
93
96


505347
68
71
89
97


505358
69
83
92
96


509927
54
74
88
94


509934
40
59
78
89


509958
57
77
91
93


509959
54
72
84
100


509960
44
72
91
91


509974
58
77
92
95


510038
58
78
94
98


510039
53
74
89
95


510040
39
70
80
90


510041
47
65
82
92


510100
74
83
95
96


510106
54
75
86
92


510116
61
74
91
94
















TABLE 17







Dose-dependent reduction of E antigen


in HepG2.2.15 cell supernatant











ISIS No
15.625 nM
31.25 nM
62.5 nM
125 nM














146779
14
45
66
76


146786
26
58
75
80


505329
19
26
60
73


505330
28
70
69
80


505339
31
57
77
82


505347
24
33
64
77


505358
26
45
72
81


509927
34
54
72
79


509934
21
42
59
73


509958
29
45
72
77


509959
60
64
77
80


509960
19
36
67
77


509974
16
48
72
80


510038
20
35
79
80


510039
14
41
64
78


510040
0
8
37
69


510041
9
34
63
76


510100
26
52
73
81


510106
7
42
62
76


510116
27
56
76
81
















TABLE 18







Dose-dependent antisense inhibition


of HBV DNA in HepG2.2.15 cells











ISIS No
15.625 nM
31.25 nM
62.5 nM
125 nM














146779
71
71
84
85


146786
67
81
82
75


505329
53
65
72
67


505330
72
76
86
90


505339
83
85
89
88


505347
76
78
81
87


505358
79
82
90
87


509927
51
75
78
69


509934
61
60
64
75


509958
57
73
69
71


509959
59
54
73
73


509960
48
66
63
54


509974
76
90
84
85


510038
69
76
90
87


510039
70
79
81
86


510040
40
67
68
68


510041
53
71
62
68


510100
76
81
87
87


510106
46
74
73
76


510116
79
84
89
86
















TABLE 19







Total protein levels in HepG2.2.15 cell supernatant












15.625 nM
31.25 nM
62.5 nM
125 nM















PBS
5601
5601
5601
5601


146779
6491
6631
6027
5067


146786
5408
5328
4839
3518


505329
5719
5285
5384
4994


505330
7514
7262
6627
5179


505339
6572
6343
5349
4550


505347
7315
6602
6378
5908


505358
6357
6871
5798
5720


509927
5581
5487
5145
3601


509934
5476
5610
5394
4127


509958
5193
5492
5071
3957


509959
5051
5312
5144
3893


509960
4726
5160
5071
3305


509974
6913
7624
5798
5389


510038
5707
6381
5772
6733


510039
5981
7629
4802
6156


510040
4302
5209
5049
4188


510041
5565
5607
5205
3757


510100
8466
8378
7985
6402


510106
5703
5940
5231
4005


510116
5880
5380
4797
4757









Example 9
In Vivo Inhibition of HBV mRNA by MOE Gapmers in HBV-Transgenic Mice
ISIS 146786, a 5-10-5 MOE gapmer, and ISIS 510100, a 3-10-4 MOE gapmer, both demonstrating significant inhibition of HBV mRNA, were tested in transgenic mice containing the HBV gene (Chisari 1.3.32 line) (Guidotti, L. G. et al., J. Virol. 1995, 69, 6158-6169) and the efficacy of the gapmers was evaluated.
Treatment
Two groups of ten-eleven HBV-transgenic male and female mice each were administered subcutaneously twice a week for four weeks with 25 mg/kg of ISIS 146786 or ISIS 510100. Another group of 14 male and female HBV-transgenic mice was administered Entecavir, an oral antiviral drug used to treat Hepatitis B infection, at 1 mg/kg daily for two weeks. Another group of 10 male and female HBV-transgenic female mice were injected subcutaneously with PBS twice a week for four weeks. The mice injected with PBS served as a control group. Liver HBV mRNA and DNA levels, plasma ALT, and body and organ weights were measured.
RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of HBV using primer probe sets RTS3370, RTS3371, and RTS3372. Results are presented as percent inhibition of HBV mRNA, relative to PBS control. As shown in Table 20, treatment with ISIS antisense oligonucleotides resulted in significant reduction of HBV mRNA in comparison to the PBS control, irrespective of the primer probe set used for measurement. Entecavir did not decrease HBV mRNA expression.







TABLE 20







Inhibition of HBV mRNA in HBV-transgenic


mice liver relative to the PBS control












ISIS No
RTS3370
RTS3371
RTS3372
















146786
82
75
81



510100
93
83
89










DNA Analysis

DNA was extracted from liver tissue for real-time PCR analysis of HBV using primer probe sets RTS3370 and RTS3371. The levels were normalized to RIBOGREEN®. Results are presented as percent inhibition of HBV DNA, relative to PBS control. As shown in Table 21, treatment with ISIS antisense oligonucleotides resulted in significant reduction of HBV DNA in comparison to the PBS control, irrespective of the primer probe set used for measurement. Treatment with Entecavir also reduced DNA levels, as expected.







TABLE 21







Inhibition of HBV DNA in HBV-transgenic


mice liver relative to the PBS control









ISIS No
RTS3370
RTS3371





146786
65
69


510100
67
73


Entecavir
75
96









Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminase was measured using a manual clinical chemistry analyzer (Teco Diagnostics, Anaheim, Calif.) Plasma concentrations of ALT (alanine transaminase) were measured and the results are presented in Table 22, expressed in IU/L. The results indicate that antisense inhibition of HBV had no adverse effects on the liver function of the mice.







TABLE 22







Effect of antisense oligonucleotide treatment


on liver ALT of transgenic mice









IU/mL














PBS
12.7



ISIS 146786
24.1



ISIS 510100
25.8



Entecavir
23.7










The data from the study indicates that both ISIS 146786 and ISIS 510100 caused robust reductions in liver HBV RNA and DNA and treatment with these oligonucleotides were well tolerated in the transgenic mice.
Example 10
Antisense Inhibition of HBV Viral mRNA in HepG2.2.15 Cells by MOE Gapmers
Additional antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. Several of the antisense oligonucleotides from the studies described above were also included in the assay. Cultured HepG2.2.15 cells at a density of 28,000 cells per well were transfected using LipofectAMINE 2000® reagent with 100 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in Table 23 were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has an MOE sugar modification. Each nucleoside in the central gap segment has a deoxy sugar modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
“Viral Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. Each gapmer listed in Table 23 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1).







TABLE 23







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides targeted to


SEQ ID NO: 1 (RTS3370)












Viral
Viral






Start
Stop


%
SEQ ID


Site
Site
ISIS No
Sequence
inhibition
NO















1
20
524410
TGGTGAAAGGTTGTGGAATT
70
321


 


4
23
524411
GTTTGGTGAAAGGTTGTGGA
51
322


 


7
26
524412
AGAGTTTGGTGAAAGGTTGT
47
323


 


10
29
524413
TGCAGAGTTTGGTGAAAGGT
74
324


 


13
32
524414
TCTTGCAGAGTTTGGTGAAA
91
325


 


16
35
524415
GGATCTTGCAGAGTTTGGTG
93
326


 


19
38
524416
CTGGGATCTTGCAGAGTTTG
85
327


 


22
41
524417
ACTCTGGGATCTTGCAGAGT
66
328


 


25
44
524418
CTCACTCTGGGATCTTGCAG
86
329


 


28
47
524419
CCTCTCACTCTGGGATCTTG
81
330


 


31
50
524420
AGGCCTCTCACTCTGGGATC
77
331


 


34
53
524421
TACAGGCCTCTCACTCTGGG
71
332


 


37
56
524422
AAATACAGGCCTCTCACTCT
68
333


 


40
59
524423
GGGAAATACAGGCCTCTCAC
43
334


 


43
62
524424
GCAGGGAAATACAGGCCTCT
76
335


 


46
65
524425
CCAGCAGGGAAATACAGGCC
89
336


 


49
68
524426
CCACCAGCAGGGAAATACAG
82
337


 


52
71
524427
GAGCCACCAGCAGGGAAATA
53
338


 


55
74
524428
CTGGAGCCACCAGCAGGGAA
76
339


 


56
75
524429
ACTGGAGCCACCAGCAGGGA
55
340


 


57
76
524430
AACTGGAGCCACCAGCAGGG
45
341


 


58
77
146779
GAACTGGAGCCACCAGCAGG
57
83


 


59
78
524431
TGAACTGGAGCCACCAGCAG
85
342


 


60
79
524432
CTGAACTGGAGCCACCAGCA
90
343


 


61
80
505314
CCTGAACTGGAGCCACCAGC
93
85


 


62
81
524433
TCCTGAACTGGAGCCACCAG
79
344


 


63
82
524434
CTCCTGAACTGGAGCCACCA
82
345


 


65
84
524435
TGCTCCTGAACTGGAGCCAC
78
346


 


68
87
524436
TACTGCTCCTGAACTGGAGC
58
347


 


71
90
524437
GTTTACTGCTCCTGAACTGG
40
348


 


74
93
524438
AGGGTTTACTGCTCCTGAAC
45
349


 


77
96
524439
AACAGGGTTTACTGCTCCTG
69
350


 


80
99
524440
CGGAACAGGGTTTACTGCTC
67
351


 


83
102
524441
AGTCGGAACAGGGTTTACTG
47
352


 


86
105
524442
AGTAGTCGGAACAGGGTTTA
59
353


 


89
108
524443
GGCAGTAGTCGGAACAGGGT
47
354


 


92
111
524444
AGAGGCAGTAGTCGGAACAG
54
355


 


95
114
524445
GGGAGAGGCAGTAGTCGGAA
49
356


 


98
117
524446
TAAGGGAGAGGCAGTAGTCG
81
357


 


101
120
524447
CGATAAGGGAGAGGCAGTAG
86
358


 


104
123
524448
TGACGATAAGGGAGAGGCAG
79
359


 


107
126
524449
GATTGACGATAAGGGAGAGG
27
360


 


110
129
524450
GAAGATTGACGATAAGGGAG
53
361


 


113
132
524451
CGAGAAGATTGACGATAAGG
67
362


 


116
135
524452
CCTCGAGAAGATTGACGATA
84
363


 


119
138
524453
AATCCTCGAGAAGATTGACG
79
364


 


122
141
524454
CCCAATCCTCGAGAAGATTG
65
365


 


125
144
524455
GTCCCCAATCCTCGAGAAGA
66
366


 


128
147
524456
AGGGTCCCCAATCCTCGAGA
67
367


 


131
150
524457
CGCAGGGTCCCCAATCCTCG
76
368


 


134
153
524458
CAGCGCAGGGTCCCCAATCC
59
369


 


137
156
524459
GTTCAGCGCAGGGTCCCCAA
80
370


 


140
159
524460
CATGTTCAGCGCAGGGTCCC
90
371


 


143
162
524461
CTCCATGTTCAGCGCAGGGT
75
372


 


146
165
524462
GTTCTCCATGTTCAGCGCAG
54
373


 


149
168
524463
GATGTTCTCCATGTTCAGCG
27
374


 


152
171
524464
TGTGATGTTCTCCATGTTCA
72
375


 


158
177
524466
TCCTGATGTGATGTTCTCCA
91
376


 


161
180
524467
GAATCCTGATGTGATGTTCT
77
377


 


164
183
524468
TAGGAATCCTGATGTGATGT
77
378


 


167
186
524469
TCCTAGGAATCCTGATGTGA
94
379


 


170
189
524470
GGGTCCTAGGAATCCTGATG
56
380


 


188
207
524471
CGCCTGTAACACGAGAAGGG
65
381


 


191
210
524472
CCCCGCCTGTAACACGAGAA
71
382


 


194
213
524473
AAACCCCGCCTGTAACACGA
74
383


 


195
214
524474
AAAACCCCGCCTGTAACACG
72
384


 


196
215
505315
AAAAACCCCGCCTGTAACAC
52
87


 


197
216
524475
GAAAAACCCCGCCTGTAACA
38
385


 


198
217
524476
AGAAAAACCCCGCCTGTAAC
18
386


 


200
219
524477
CAAGAAAAACCCCGCCTGTA
86
387


 


203
222
524478
CAACAAGAAAAACCCCGCCT
84
388


 


204
223
524479
TCAACAAGAAAAACCCCGCC
80
389


 


205
224
505317
GTCAACAAGAAAAACCCCGC
84
89


 


206
225
524480
TGTCAACAAGAAAAACCCCG
79
390


 


207
226
524481
TTGTCAACAAGAAAAACCCC
76
391


 


209
228
524482
TCTTGTCAACAAGAAAAACC
86
392


 


212
231
524483
GATTCTTGTCAACAAGAAAA
57
393


 


215
234
524484
GAGGATTCTTGTCAACAAGA
51
394


 


218
237
524485
TGTGAGGATTCTTGTCAACA
83
395


 


221
240
524486
TATTGTGAGGATTCTTGTCA
61
396


 


224
243
524487
CGGTATTGTGAGGATTCTTG
74
397


 


227
246
524488
CTGCGGTATTGTGAGGATTC
49
398


 


230
249
524489
ACTCTGCGGTATTGTGAGGA
67
399


 


233
252
524490
TAGACTCTGCGGTATTGTGA
88
400


 


236
255
524491
GTCTAGACTCTGCGGTATTG
84
401


 


239
258
524492
CGAGTCTAGACTCTGCGGTA
82
402


 


242
261
524493
CCACGAGTCTAGACTCTGCG
94
403


 


243
262
524494
ACCACGAGTCTAGACTCTGC
87
404


 


244
263
146821
CACCACGAGTCTAGACTCTG
87
92


 


245
264
524495
CCACCACGAGTCTAGACTCT
80
405


 


246
265
524496
TCCACCACGAGTCTAGACTC
65
406


 


247
266
505318
GTCCACCACGAGTCTAGACT
65
96


 


248
267
524497
AGTCCACCACGAGTCTAGAC
46
407


 


249
268
524498
AAGTCCACCACGAGTCTAGA
54
408


 


250
269
509921
GAAGTCCACCACGAGTCTAG
35
97


 


251
270
509922
AGAAGTCCACCACGAGTCTA
51
99


 


252
271
509923
GAGAAGTCCACCACGAGTCT
49
101


 


253
272
505319
AGAGAAGTCCACCACGAGTC
60
103


 


254
273
509924
GAGAGAAGTCCACCACGAGT
46
105


 


255
274
509925
TGAGAGAAGTCCACCACGAG
79
108


 


256
275
505320
TTGAGAGAAGTCCACCACGA
84
111


 


257
276
524499
ATTGAGAGAAGTCCACCACG
83
409


 


260
279
524500
AAAATTGAGAGAAGTCCACC
71
410


 


263
282
524501
TAGAAAATTGAGAGAAGTCC
67
411


 


266
285
524502
CCCTAGAAAATTGAGAGAAG
88
412


 


269
288
524503
TCCCCCTAGAAAATTGAGAG
82
413


 


272
291
524504
AGTTCCCCCTAGAAAATTGA
66
414


 


275
294
524505
GGTAGTTCCCCCTAGAAAAT
0
415


 


278
297
524506
CACGGTAGTTCCCCCTAGAA
65
416


 


281
300
524507
ACACACGGTAGTTCCCCCTA
87
417


 


284
303
524508
AAGACACACGGTAGTTCCCC
76
418


 


287
306
524509
GCCAAGACACACGGTAGTTC
61
419


 


290
309
524510
TTGGCCAAGACACACGGTAG
87
420


 


291
310
524511
TTTGGCCAAGACACACGGTA
87
421


 


292
311
524512
TTTTGGCCAAGACACACGGT
93
422


 


293
312
505323
ATTTTGGCCAAGACACACGG
83
123


 


294
313
524513
AATTTTGGCCAAGACACACG
79
423


 


295
314
524514
GAATTTTGGCCAAGACACAC
74
424


 


298
317
524515
TGCGAATTTTGGCCAAGACA
78
425


 


300
319
524516
ACTGCGAATTTTGGCCAAGA
71
426


 


301
320
524517
GACTGCGAATTTTGGCCAAG
71
427


 


302
321
505325
GGACTGCGAATTTTGGCCAA
50
125


 


303
322
524518
GGGACTGCGAATTTTGGCCA
55
428


 


321
340
524519
GTGAGTGATTGGAGGTTGGG
68
429


 


324
343
524520
TTGGTGAGTGATTGGAGGTT
84
430


 


327
346
524521
AGGTTGGTGAGTGATTGGAG
64
431


 


330
349
524522
AGGAGGTTGGTGAGTGATTG
58
432


 


333
352
524523
GACAGGAGGTTGGTGAGTGA
62
433


 


336
355
524524
GAGGACAGGAGGTTGGTGAG
56
434


 


339
358
524525
TTGGAGGACAGGAGGTTGGT
81
435


 


342
361
524526
AAGTTGGAGGACAGGAGGTT
77
436


 


345
364
524527
GACAAGTTGGAGGACAGGAG
69
437


 


348
367
524528
CAGGACAAGTTGGAGGACAG
82
438


 


351
370
524529
AACCAGGACAAGTTGGAGGA
67
439


 


354
373
524530
GATAACCAGGACAAGTTGGA
53
440


 


357
376
524531
AGCGATAACCAGGACAAGTT
55
441


 


358
377
524532
CAGCGATAACCAGGACAAGT
84
442


 


359
378
524533
CCAGCGATAACCAGGACAAG
86
443


 


360
379
505326
TCCAGCGATAACCAGGACAA
79
126


 


361
380
524534
ATCCAGCGATAACCAGGACA
85
444


 


362
381
524535
CATCCAGCGATAACCAGGAC
90
445


 


364
383
524536
CACATCCAGCGATAACCAGG
82
446


 


365
384
524537
ACACATCCAGCGATAACCAG
72
447


 


366
385
505327
GACACATCCAGCGATAACCA
61
127


 


367
386
524538
AGACACATCCAGCGATAACC
79
448


 


368
387
524539
CAGACACATCCAGCGATAAC
73
449


 


370
389
524540
CGCAGACACATCCAGCGATA
94
450


 


373
392
524541
CGCCGCAGACACATCCAGCG
84
451


 


390
409
524542
AGAGGAAGATGATAAAACGC
45
452


 


393
412
524543
TGAAGAGGAAGATGATAAAA
62
453


 


396
415
524544
GGATGAAGAGGAAGATGATA
58
454


 


399
418
524545
GCAGGATGAAGAGGAAGATG
48
455


 


402
421
524546
GCAGCAGGATGAAGAGGAAG
60
456


 


405
424
524547
ATAGCAGCAGGATGAAGAGG
84
457


 


408
427
524548
GGCATAGCAGCAGGATGAAG
56
458


 


409
428
524549
AGGCATAGCAGCAGGATGAA
78
459


 


410
429
524550
GAGGCATAGCAGCAGGATGA
67
460


 


411
430
505329
TGAGGCATAGCAGCAGGATG
85
136


 


412
431
509926
ATGAGGCATAGCAGCAGGAT
84
139


 


413
432
509927
GATGAGGCATAGCAGCAGGA
68
142


 


414
433
505330
AGATGAGGCATAGCAGCAGG
82
20


 


415
434
509928
AAGATGAGGCATAGCAGCAG
83
22


 


416
435
509929
GAAGATGAGGCATAGCAGCA
80
24


 


417
436
509930
AGAAGATGAGGCATAGCAGC
78
26


 


418
437
146783
AAGAAGATGAGGCATAGCAG
80
28


 


419
438
524551
CAAGAAGATGAGGCATAGCA
55
461


 


422
441
524552
CAACAAGAAGATGAGGCATA
90
462


 


425
444
524553
AACCAACAAGAAGATGAGGC
82
463


 


428
447
524554
AAGAACCAACAAGAAGATGA
79
464


 


431
450
524555
CAGAAGAACCAACAAGAAGA
72
465


 


434
453
524556
GTCCAGAAGAACCAACAAGA
87
466


 


437
456
524557
ATAGTCCAGAAGAACCAACA
72
467


 


440
459
524558
TTGATAGTCCAGAAGAACCA
76
468


 


443
462
524559
ACCTTGATAGTCCAGAAGAA
78
469


 


446
465
524560
CATACCTTGATAGTCCAGAA
77
470


 


449
468
524561
CAACATACCTTGATAGTCCA
69
471


 


452
471
524562
GGGCAACATACCTTGATAGT
39
472


 


455
474
524563
AACGGGCAACATACCTTGAT
72
473


 


456
475
524564
AAACGGGCAACATACCTTGA
86
474


 


457
476
505332
CAAACGGGCAACATACCTTG
85
166


 


458
477
524565
ACAAACGGGCAACATACCTT
80
475


 


459
478
524566
GACAAACGGGCAACATACCT
42
476


 


461
480
524567
AGGACAAACGGGCAACATAC
47
477


 


464
483
524568
TAGAGGACAAACGGGCAACA
81
478


 


467
486
524569
AATTAGAGGACAAACGGGCA
72
479


 


470
489
524570
TGGAATTAGAGGACAAACGG
84
480


 


471
490
524571
CTGGAATTAGAGGACAAACG
86
481


 


472
491
505335
CCTGGAATTAGAGGACAAAC
89
174


 


473
492
524572
TCCTGGAATTAGAGGACAAA
92
482


 


474
493
524573
ATCCTGGAATTAGAGGACAA
86
483


 


476
495
524574
GGATCCTGGAATTAGAGGAC
76
484


 


479
498
524575
TGAGGATCCTGGAATTAGAG
77
485


 


482
501
524576
GGTTGAGGATCCTGGAATTA
62
486


 


485
504
524577
GGTGGTTGAGGATCCTGGAA
73
487


 


488
507
524578
GCTGGTGGTTGAGGATCCTG
84
488


 


491
510
524579
CGTGCTGGTGGTTGAGGATC
79
489


 


494
513
524580
TCCCGTGCTGGTGGTTGAGG
83
490


 


497
516
524581
TGGTCCCGTGCTGGTGGTTG
66
491


 


500
519
524582
GCATGGTCCCGTGCTGGTGG
77
492


 


503
522
524583
TCGGCATGGTCCCGTGCTGG
0
493


 


506
525
524584
GGTTCGGCATGGTCCCGTGC
56
494


 


509
528
524585
GCAGGTTCGGCATGGTCCCG
61
495


 


512
531
524586
CATGCAGGTTCGGCATGGTC
87
496


 


515
534
524587
AGTCATGCAGGTTCGGCATG
77
497


 


518
537
524588
AGTAGTCATGCAGGTTCGGC
64
498


 


521
540
524589
AGCAGTAGTCATGCAGGTTC
61
499


 


524
543
524590
TTGAGCAGTAGTCATGCAGG
86
500


 


527
546
524591
TCCTTGAGCAGTAGTCATGC
80
501


 


530
549
524592
GGTTCCTTGAGCAGTAGTCA
50
502


 


533
552
524593
AGAGGTTCCTTGAGCAGTAG
61
503


 


536
555
524594
CATAGAGGTTCCTTGAGCAG
89
504


 


539
558
524595
ATACATAGAGGTTCCTTGAG
87
505


 


542
561
524596
GGGATACATAGAGGTTCCTT
0
506


 


545
564
524597
GGAGGGATACATAGAGGTTC
38
507


 


548
567
524598
ACAGGAGGGATACATAGAGG
73
508


 


551
570
524599
GCAACAGGAGGGATACATAG
67
509


 


554
573
524600
ACAGCAACAGGAGGGATACA
72
510


 


557
576
524601
GGTACAGCAACAGGAGGGAT
59
511


 


560
579
524602
TTTGGTACAGCAACAGGAGG
81
512


 


563
582
524603
AGGTTTGGTACAGCAACAGG
74
513


 


566
585
524604
CGAAGGTTTGGTACAGCAAC
85
514


 


569
588
524605
GTCCGAAGGTTTGGTACAGC
76
515


 


572
591
524606
TCCGTCCGAAGGTTTGGTAC
80
516


 


575
594
524607
ATTTCCGTCCGAAGGTTTGG
88
517


 


578
597
524608
GCAATTTCCGTCCGAAGGTT
50
518


 


581
600
524609
GGTGCAATTTCCGTCCGAAG
55
519


 


584
603
524610
ACAGGTGCAATTTCCGTCCG
81
520


 


587
606
524611
AATACAGGTGCAATTTCCGT
88
521


 


590
609
524612
GGGAATACAGGTGCAATTTC
32
522


 


593
612
524613
GATGGGAATACAGGTGCAAT
49
523


 


608
627
524614
AGCCCAGGATGATGGGATGG
89
524


 


611
630
524615
GAAAGCCCAGGATGATGGGA
71
525


 


614
633
524616
TCCGAAAGCCCAGGATGATG
86
526


 


617
636
524617
TTTTCCGAAAGCCCAGGATG
97
527


 


620
639
524618
GAATTTTCCGAAAGCCCAGG
80
528


 


623
642
524619
TAGGAATTTTCCGAAAGCCC
95
529


 


626
645
524620
CCATAGGAATTTTCCGAAAG
88
530


 


629
648
524621
CTCCCATAGGAATTTTCCGA
83
531


 


632
651
524622
CCACTCCCATAGGAATTTTC
68
532


 


635
654
524623
GGCCCACTCCCATAGGAATT
60
533


 


638
657
524624
TGAGGCCCACTCCCATAGGA
57
534


 


641
660
524625
GGCTGAGGCCCACTCCCATA
62
535


 


644
663
524626
ACGGGCTGAGGCCCACTCCC
57
536


 


647
666
524627
GAAACGGGCTGAGGCCCACT
62
537


 


650
669
524628
GGAGAAACGGGCTGAGGCCC
31
538


 


653
672
524629
CCAGGAGAAACGGGCTGAGG
77
539


 


656
675
524630
GAGCCAGGAGAAACGGGCTG
48
540


 


659
678
524631
ACTGAGCCAGGAGAAACGGG
43
541


 


662
681
524632
TAAACTGAGCCAGGAGAAAC
67
542


 


665
684
524633
TAGTAAACTGAGCCAGGAGA
86
543


 


668
687
524634
CACTAGTAAACTGAGCCAGG
96
544


 


669
688
524635
GCACTAGTAAACTGAGCCAG
83
545


 


671
690
524636
TGGCACTAGTAAACTGAGCC
84
546


 


672
691
524637
ATGGCACTAGTAAACTGAGC
82
547


 


674
693
524638
AAATGGCACTAGTAAACTGA
74
548


 


677
696
524639
AACAAATGGCACTAGTAAAC
63
549


 


678
697
524640
GAACAAATGGCACTAGTAAA
67
550


 


679
698
505338
TGAACAAATGGCACTAGTAA
84
186


 


680
699
524641
CTGAACAAATGGCACTAGTA
95
551


 


681
700
524642
ACTGAACAAATGGCACTAGT
77
552


 


682
701
505339
CACTGAACAAATGGCACTAG
95
187


 


683
702
524643
CCACTGAACAAATGGCACTA
89
553


 


684
703
524644
ACCACTGAACAAATGGCACT
90
554


 


686
705
524646
GAACCACTGAACAAATGGCA
82
555


 


687
706
509931
CGAACCACTGAACAAATGGC
90
39


 


689
708
524647
TACGAACCACTGAACAAATG
79
556


 


690
709
146824
CTACGAACCACTGAACAAAT
72
557


 


692
711
524648
CCCTACGAACCACTGAACAA
73
558


 


693
712
524649
GCCCTACGAACCACTGAACA
83
559


 


695
714
524650
AAGCCCTACGAACCACTGAA
82
560


 


696
715
524651
AAAGCCCTACGAACCACTGA
81
561


 


697
716
505342
GAAAGCCCTACGAACCACTG
66
198


 


698
717
524652
GGAAAGCCCTACGAACCACT
59
562


 


699
718
524653
GGGAAAGCCCTACGAACCAC
46
563


 


718
737
524654
ACTGAAAGCCAAACAGTGGG
64
564


 


721
740
524655
ATAACTGAAAGCCAAACAGT
0
565


 


724
743
524656
CATATAACTGAAAGCCAAAC
70
566


 


727
746
524657
ATCCATATAACTGAAAGCCA
91
567


 


730
749
524658
ATCATCCATATAACTGAAAG
69
568


 


733
752
524659
CACATCATCCATATAACTGA
70
569


 


736
755
524660
TACCACATCATCCATATAAC
57
570


 


739
758
524661
CAATACCACATCATCCATAT
70
571


 


742
761
524662
CCCCAATACCACATCATCCA
85
572


 


745
764
524663
GGCCCCCAATACCACATCAT
70
573


 


748
767
524664
CTTGGCCCCCAATACCACAT
82
574


 


751
770
524665
AGACTTGGCCCCCAATACCA
77
575


 


754
773
524666
TACAGACTTGGCCCCCAATA
77
576


 


757
776
524667
CTGTACAGACTTGGCCCCCA
90
577


 


760
779
524668
ATGCTGTACAGACTTGGCCC
79
578


 


763
782
524669
AAGATGCTGTACAGACTTGG
79
579


 


766
785
524670
CTCAAGATGCTGTACAGACT
84
580


 


769
788
524671
GGACTCAAGATGCTGTACAG
24
581


 


772
791
524672
AAGGGACTCAAGATGCTGTA
57
582


 


775
794
524673
AAAAAGGGACTCAAGATGCT
66
583


 


778
797
524674
GGTAAAAAGGGACTCAAGAT
30
584


 


781
800
524675
AGCGGTAAAAAGGGACTCAA
68
585


 


784
803
524676
AACAGCGGTAAAAAGGGACT
67
586


 


787
806
524677
GGTAACAGCGGTAAAAAGGG
48
587


 


790
809
524678
ATTGGTAACAGCGGTAAAAA
81
588


 


793
812
524679
AAAATTGGTAACAGCGGTAA
89
589


 


796
815
524680
AAGAAAATTGGTAACAGCGG
84
590


 


799
818
524681
CAAAAGAAAATTGGTAACAG
41
591


 


802
821
524682
AGACAAAAGAAAATTGGTAA
51
592


 


805
824
524683
CAAAGACAAAAGAAAATTGG
66
593


 


808
827
524684
ACCCAAAGACAAAAGAAAAT
61
594


 


811
830
524685
TATACCCAAAGACAAAAGAA
79
595


 


814
833
524686
ATGTATACCCAAAGACAAAA
84
596


 


817
836
524687
TAAATGTATACCCAAAGACA
77
597


 


820
839
524688
GTTTAAATGTATACCCAAAG
80
598


 


821
840
524689
GGTTTAAATGTATACCCAAA
71
599


 


822
841
524690
GGGTTTAAATGTATACCCAA
85
600


 


823
842
505344
AGGGTTTAAATGTATACCCA
85
206


 


824
843
524691
TAGGGTTTAAATGTATACCC
90
601


 


825
844
524692
TTAGGGTTTAAATGTATACC
83
602


 


827
846
524693
TGTTAGGGTTTAAATGTATA
53
603


 


830
849
524694
TTTTGTTAGGGTTTAAATGT
67
604


 


845
864
524695
AACCCCATCTCTTTGTTTTG
81
605


 


848
867
524696
AGTAACCCCATCTCTTTGTT
71
606


 


851
870
524697
GAGAGTAACCCCATCTCTTT
65
607


 


854
873
524698
TCAGAGAGTAACCCCATCTC
96
608


 


857
876
524699
AATTCAGAGAGTAACCCCAT
94
609


 


860
879
524700
TAAAATTCAGAGAGTAACCC
71
610


 


863
882
524701
CCATAAAATTCAGAGAGTAA
90
611


 


866
885
524702
AACCCATAAAATTCAGAGAG
86
612


 


869
888
524703
CATAACCCATAAAATTCAGA
72
613


 


872
891
524704
TGACATAACCCATAAAATTC
81
614


 


875
894
524705
CAATGACATAACCCATAAAA
81
615


 


878
897
524706
TTCCAATGACATAACCCATA
95
616


 


881
900
524707
AACTTCCAATGACATAACCC
91
617


 


884
903
524708
CATAACTTCCAATGACATAA
83
618


 


887
906
524709
ACCCATAACTTCCAATGACA
95
619


 


890
909
524710
AGGACCCATAACTTCCAATG
66
620


 


893
912
524711
GCAAGGACCCATAACTTCCA
41
621


 


896
915
524712
GTGGCAAGGACCCATAACTT
53
622


 


899
918
524713
CTTGTGGCAAGGACCCATAA
91
623


 


902
921
524714
GTTCTTGTGGCAAGGACCCA
77
624


 


905
924
524715
TGTGTTCTTGTGGCAAGGAC
90
625


 


908
927
524716
TGATGTGTTCTTGTGGCAAG
90
626


 


911
930
524717
GTATGATGTGTTCTTGTGGC
82
627


 


914
933
524718
TTTGTATGATGTGTTCTTGT
95
628


 


930
949
524719
AAACATTCTTTGATTTTTTG
61
629


 


933
952
524720
CTAAAACATTCTTTGATTTT
43
630


 


936
955
524721
TTTCTAAAACATTCTTTGAT
90
631


 


939
958
524722
AGTTTTCTAAAACATTCTTT
75
632


 


942
961
524723
GGAAGTTTTCTAAAACATTC
52
633


 


945
964
524724
ATAGGAAGTTTTCTAAAACA
74
634


 


948
967
524725
TTAATAGGAAGTTTTCTAAA
40
635


 


951
970
524726
CTGTTAATAGGAAGTTTTCT
93
636


 


954
973
524727
GGCCTGTTAATAGGAAGTTT
87
637


 


957
976
524728
ATAGGCCTGTTAATAGGAAG
85
638


 


960
979
524729
TCAATAGGCCTGTTAATAGG
92
639


 


963
982
524730
CAATCAATAGGCCTGTTAAT
90
640


 


966
985
524731
TTCCAATCAATAGGCCTGTT
96
641


 


969
988
524732
ACTTTCCAATCAATAGGCCT
77
642


 


972
991
146826
CATACTTTCCAATCAATAGG
92
643


 


975
994
524733
TGACATACTTTCCAATCAAT
91
644


 


978
997
524734
CGTTGACATACTTTCCAATC
95
645


 


996
1015
524735
CCCAAAAGACCCACAATTCG
92
646


 


999
1018
524736
AAACCCAAAAGACCCACAAT
74
647


 


1002
1021
524737
GCAAAACCCAAAAGACCCAC
85
648


 


1005
1024
524738
GCAGCAAAACCCAAAAGACC
70
649


 


1025
1044
524739
AACCACATTGTGTAAATGGG
90
650


 


1028
1047
524740
GATAACCACATTGTGTAAAT
58
651


 


1031
1050
524741
CAGGATAACCACATTGTGTA
83
652


 


1034
1053
524742
ACGCAGGATAACCACATTGT
84
653


 


1037
1056
524743
TTAACGCAGGATAACCACAT
93
654


 


1040
1059
524744
GCATTAACGCAGGATAACCA
60
655


 


1043
1062
524745
AGGGCATTAACGCAGGATAA
58
656


 


1046
1065
524746
ACAAGGGCATTAACGCAGGA
75
657


 


1049
1068
524747
CATACAAGGGCATTAACGCA
89
658


 


1052
1071
524748
ATGCATACAAGGGCATTAAC
87
659


 


1055
1074
524749
TACATGCATACAAGGGCATT
86
660


 


1058
1077
524750
GAATACATGCATACAAGGGC
75
661


 


1061
1080
524751
ATTGAATACATGCATACAAG
81
662


 


1064
1083
524752
TAGATTGAATACATGCATAC
85
663


 


1067
1086
524753
GCTTAGATTGAATACATGCA
69
664


 


1070
1089
524754
CCTGCTTAGATTGAATACAT
90
665


 


1073
1092
524755
AAGCCTGCTTAGATTGAATA
76
666


 


1076
1095
524756
TGAAAGCCTGCTTAGATTGA
76
667


 


1079
1098
524757
AAGTGAAAGCCTGCTTAGAT
68
668


 


1082
1101
524758
AGAAAGTGAAAGCCTGCTTA
81
669


 


1085
1104
524759
GCGAGAAAGTGAAAGCCTGC
61
670


 


1088
1107
524760
TTGGCGAGAAAGTGAAAGCC
89
671


 


1091
1110
524761
AAGTTGGCGAGAAAGTGAAA
74
672


 


1094
1113
524762
TGTAAGTTGGCGAGAAAGTG
85
673


 


1097
1116
524763
CCTTGTAAGTTGGCGAGAAA
90
674


 


1100
1119
524764
AGGCCTTGTAAGTTGGCGAG
93
675


 


1103
1122
524765
GAAAGGCCTTGTAAGTTGGC
78
676


 


1106
1125
524766
ACAGAAAGGCCTTGTAAGTT
76
677


 


1109
1128
524767
TACACAGAAAGGCCTTGTAA
94
678


 


1112
1131
524768
GTTTACACAGAAAGGCCTTG
80
679


 


1115
1134
524769
ATTGTTTACACAGAAAGGCC
83
680


 


1118
1137
524770
GGTATTGTTTACACAGAAAG
63
681


 


1121
1140
524771
TCAGGTATTGTTTACACAGA
93
682


 


1124
1143
524772
GGTTCAGGTATTGTTTACAC
68
683


 


1127
1146
524773
AAAGGTTCAGGTATTGTTTA
82
684


 


1130
1149
524774
GGTAAAGGTTCAGGTATTGT
68
685


 


1150
1169
524775
TGGCCGTTGCCGGGCAACGG
74
686


 


1153
1172
524776
ACCTGGCCGTTGCCGGGCAA
77
687


 


1156
1175
524777
CAGACCTGGCCGTTGCCGGG
88
688


 


1159
1178
524778
GCACAGACCTGGCCGTTGCC
80
689


 


1162
1181
524779
TTGGCACAGACCTGGCCGTT
85
690


 


1165
1184
524780
CACTTGGCACAGACCTGGCC
93
691


 


1168
1187
524781
AAACACTTGGCACAGACCTG
90
692


 


1169
1188
524782
CAAACACTTGGCACAGACCT
75
693


 


1170
1189
505345
GCAAACACTTGGCACAGACC
78
207


 


1171
1190
524783
AGCAAACACTTGGCACAGAC
84
694


 


1172
1191
524784
CAGCAAACACTTGGCACAGA
90
695


 


1174
1193
524785
GTCAGCAAACACTTGGCACA
79
696


 


1200
1219
524786
ACCAAGCCCCAGCCAGTGGG
57
697


 


1203
1222
524787
ATGACCAAGCCCCAGCCAGT
74
698


 


1206
1225
524788
CCCATGACCAAGCCCCAGCC
90
699


 


1209
1228
524789
TGGCCCATGACCAAGCCCCA
96
700


 


1212
1231
524790
TGATGGCCCATGACCAAGCC
79
701


 


1215
1234
524791
CGCTGATGGCCCATGACCAA
97
702


 


1218
1237
524792
ACGCGCTGATGGCCCATGAC
98
703


 


1221
1240
524793
CGCACGCGCTGATGGCCCAT
98
704


 


1224
1243
524794
CCACGCACGCGCTGATGGCC
98
705


 


1227
1246
524795
GTTCCACGCACGCGCTGATG
98
706


 


1230
1249
524796
AAGGTTCCACGCACGCGCTG
99
707


 


1233
1252
524797
GAAAAGGTTCCACGCACGCG
97
708


 


1236
1255
524798
GCCGAAAAGGTTCCACGCAC
98
709


 


1239
1258
524799
GGAGCCGAAAAGGTTCCACG
75
710


 


1242
1261
524800
AGAGGAGCCGAAAAGGTTCC
79
711


 


1245
1264
524801
GGCAGAGGAGCCGAAAAGGT
98
712


 


1248
1267
524802
ATCGGCAGAGGAGCCGAAAA
73
713


 


1251
1270
524803
TGGATCGGCAGAGGAGCCGA
91
714


 


1254
1273
524804
GTATGGATCGGCAGAGGAGC
98
715


 


1257
1276
524805
GCAGTATGGATCGGCAGAGG
98
716


 


1258
1277
524806
CGCAGTATGGATCGGCAGAG
98
717


 


1259
1278
505346
CCGCAGTATGGATCGGCAGA
98
210


 


1260
1279
146785
TCCGCAGTATGGATCGGCAG
98
718


 


1261
1280
524807
TTCCGCAGTATGGATCGGCA
98
719


 


1262
1281
505347
GTTCCGCAGTATGGATCGGC
98
212


 


1263
1282
524808
AGTTCCGCAGTATGGATCGG
96
720


 


1264
1283
524809
GAGTTCCGCAGTATGGATCG
97
721


 


1266
1285
524810
AGGAGTTCCGCAGTATGGAT
96
722


 


1269
1288
524811
GCTAGGAGTTCCGCAGTATG
96
723


 


1272
1291
524812
GCGGCTAGGAGTTCCGCAGT
75
724


 


1275
1294
524813
CAAGCGGCTAGGAGTTCCGC
86
725


 


1278
1297
524814
AAACAAGCGGCTAGGAGTTC
73
726


 


1281
1300
524815
GCAAAACAAGCGGCTAGGAG
71
727


 


1282
1301
524816
AGCAAAACAAGCGGCTAGGA
89
728


 


1283
1302
505352
GAGCAAAACAAGCGGCTAGG
76
217


 


1284
1303
524817
CGAGCAAAACAAGCGGCTAG
78
729


 


1285
1304
524818
GCGAGCAAAACAAGCGGCTA
71
730


 


1286
1305
505353
TGCGAGCAAAACAAGCGGCT
82
218


 


1287
1306
524819
CTGCGAGCAAAACAAGCGGC
82
731


 


1288
1307
524820
GCTGCGAGCAAAACAAGCGG
67
732


 


1290
1309
524821
CTGCTGCGAGCAAAACAAGC
79
733


 


1293
1312
524822
GACCTGCTGCGAGCAAAACA
87
734


 


1296
1315
524823
CCAGACCTGCTGCGAGCAAA
94
735


 


1299
1318
524824
GCTCCAGACCTGCTGCGAGC
80
736


 


1302
1321
524825
TTTGCTCCAGACCTGCTGCG
70
737


 


1305
1324
524826
ATGTTTGCTCCAGACCTGCT
75
738


 


1308
1327
524827
ATAATGTTTGCTCCAGACCT
55
739


 


1311
1330
524828
CCGATAATGTTTGCTCCAGA
87
740


 


1314
1333
524829
GTCCCGATAATGTTTGCTCC
80
741


 


1317
1336
524830
TCAGTCCCGATAATGTTTGC
76
742


 


1320
1339
524831
TTATCAGTCCCGATAATGTT
53
743


 


1577
1596
146786
GTGAAGCGAAGTGCACACGG
96
224









Example 11
Antisense Inhibition of HBV Viral mRNA in HepG2.2.15 Cells by MOE Gapmers
Additional antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. Several of the antisense oligonucleotides from the studies described above were also included in the assay. Cultured HepG2.2.15 cells at a density of 28,000 cells per well were transfected using LipofectAMINE 2000® reagent with 70 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 was used to measure mRNA levels. The mRNA levels of some of the gapmers were also measured using RTS3372. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in Tables 24 and 25 were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has an MOE sugar modification. Each nucleoside in the central gap segment has a deoxy sugar modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
“Viral Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. Each gapmer listed in Table 24 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1). Each gapmer listed in Table 25 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1286 (a permuted version of GENBANK Accession No. U95551.1). ‘n/a.’ indicates that the inhibition data for that particular gapmer was not measured with that particular primer probe set.







TABLE 24







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides targeted


to SEQ ID NO: 1 (RTS3370 and RTS3372)

















RTS3370
RTS3372
SEQ


Start
Stop


%
%
ID


Site
Site
ISIS No
Sequence
inhibition
inhibition
NO
















1323
1342
524832
GAGTTATCAGTCCCGATAAT
63
n/a
744


 


1326
1345
524833
ACAGAGTTATCAGTCCCGAT
82
n/a
745


 


1329
1348
524834
ACAACAGAGTTATCAGTCCC
52
n/a
746


 


1332
1351
524835
AGGACAACAGAGTTATCAGT
57
n/a
747


 


1335
1354
524836
GAGAGGACAACAGAGTTATC
49
n/a
748


 


1338
1357
524837
CGGGAGAGGACAACAGAGTT
0
n/a
749


 


1341
1360
524838
TTGCGGGAGAGGACAACAGA
17
n/a
750


 


1344
1363
524839
TATTTGCGGGAGAGGACAAC
30
n/a
751


 


1347
1366
524840
GTATATTTGCGGGAGAGGAC
22
n/a
752


 


1350
1369
524841
GATGTATATTTGCGGGAGAG
32
n/a
753


 


1353
1372
524842
TACGATGTATATTTGCGGGA
76
n/a
754


 


1356
1375
524843
GGATACGATGTATATTTGCG
76
n/a
755


 


1359
1378
524844
CATGGATACGATGTATATTT
87
n/a
756


 


1362
1381
524845
AGCCATGGATACGATGTATA
70
n/a
757


 


1365
1384
524846
AGCAGCCATGGATACGATGT
22
n/a
758


 


1368
1387
524847
CCTAGCAGCCATGGATACGA
67
n/a
759


 


1371
1390
524848
CAGCCTAGCAGCCATGGATA
56
n/a
760


 


1374
1393
524849
GCACAGCCTAGCAGCCATGG
38
n/a
761


 


1377
1396
524850
GCAGCACAGCCTAGCAGCCA
11
n/a
762


 


1380
1399
524851
TTGGCAGCACAGCCTAGCAG
34
n/a
763


 


1383
1402
524852
CAGTTGGCAGCACAGCCTAG
47
n/a
764


 


1386
1405
524853
ATCCAGTTGGCAGCACAGCC
45
n/a
765


 


1389
1408
524854
AGGATCCAGTTGGCAGCACA
36
n/a
766


 


1392
1411
524855
CGCAGGATCCAGTTGGCAGC
41
n/a
767


 


1395
1414
524856
CCGCGCAGGATCCAGTTGGC
72
n/a
768


 


1398
1417
524857
GTCCCGCGCAGGATCCAGTT
55
n/a
769


 


1457
1476
524858
AGCGACCCCGAGAAGGGTCG
17
n/a
770


 


1460
1479
524859
CCAAGCGACCCCGAGAAGGG
45
n/a
771


 


1463
1482
524860
GTCCCAAGCGACCCCGAGAA
8
n/a
772


 


1466
1485
524861
AGAGTCCCAAGCGACCCCGA
51
n/a
773


 


1469
1488
524862
GAGAGAGTCCCAAGCGACCC
28
n/a
774


 


1472
1491
524863
GACGAGAGAGTCCCAAGCGA
37
n/a
775


 


1492
1511
524864
GAACGGCAGACGGAGAAGGG
27
n/a
776


 


1498
1517
524866
CGGTCGGAACGGCAGACGGA
78
n/a
777


 


1501
1520
524867
GGTCGGTCGGAACGGCAGAC
78
n/a
778


 


1504
1523
524868
CGTGGTCGGTCGGAACGGCA
79
n/a
779


 


1507
1526
524869
CCCCGTGGTCGGTCGGAACG
70
n/a
780


 


1510
1529
524870
GCGCCCCGTGGTCGGTCGGA
78
n/a
781


 


1513
1532
524871
GGTGCGCCCCGTGGTCGGTC
74
n/a
782


 


1514
1533
524872
AGGTGCGCCCCGTGGTCGGT
63
n/a
783


 


1515
1534
505354
GAGGTGCGCCCCGTGGTCGG
70
n/a
220


 


1516
1535
524873
AGAGGTGCGCCCCGTGGTCG
72
n/a
784


 


1517
1536
524874
GAGAGGTGCGCCCCGTGGTC
49
n/a
785


 


1518
1537
505355
AGAGAGGTGCGCCCCGTGGT
64
n/a
221


 


1519
1538
524875
AAGAGAGGTGCGCCCCGTGG
57
n/a
786


 


1520
1539
524876
AAAGAGAGGTGCGCCCCGTG
63
n/a
787


 


1521
1540
505356
TAAAGAGAGGTGCGCCCCGT
68
n/a
222


 


1522
1541
524877
GTAAAGAGAGGTGCGCCCCG
50
n/a
788


 


1523
1542
524878
CGTAAAGAGAGGTGCGCCCC
64
n/a
789


 


1550
1569
524879
GATGAGAAGGCACAGACGGG
70
n/a
790


 


1553
1572
524880
GCAGATGAGAAGGCACAGAC
81
n/a
791


 


1556
1575
524881
CCGGCAGATGAGAAGGCACA
80
n/a
792


 


1559
1578
524882
GGTCCGGCAGATGAGAAGGC
84
n/a
793


 


1562
1581
524883
CACGGTCCGGCAGATGAGAA
79
n/a
794


 


1565
1584
524884
GCACACGGTCCGGCAGATGA
83
n/a
795


 


1568
1587
524885
AGTGCACACGGTCCGGCAGA
77
n/a
796


 


1571
1590
524886
CGAAGTGCACACGGTCCGGC
89
n/a
797


 


1574
1593
524887
AAGCGAAGTGCACACGGTCC
85
n/a
798


 


1575
1594
524888
GAAGCGAAGTGCACACGGTC
83
n/a
799


 


1576
1595
524889
TGAAGCGAAGTGCACACGGT
83
n/a
800


 


1577
1596
146786
GTGAAGCGAAGTGCACACGG
88
85
224


 


1578
1597
524890
GGTGAAGCGAAGTGCACACG
83
n/a
801


 


1579
1598
524891
AGGTGAAGCGAAGTGCACAC
82
n/a
802


 


1580
1599
505357
GAGGTGAAGCGAAGTGCACA
79
n/a
803


 


1581
1600
524892
AGAGGTGAAGCGAAGTGCAC
73
n/a
804


 


1582
1601
524893
CAGAGGTGAAGCGAAGTGCA
80
n/a
805


 


1583
1602
505358
GCAGAGGTGAAGCGAAGTGC
84
n/a
226


 


1584
1603
524894
TGCAGAGGTGAAGCGAAGTG
74
n/a
806


 


1585
1604
524895
GTGCAGAGGTGAAGCGAAGT
72
n/a
807


 


1586
1605
505359
CGTGCAGAGGTGAAGCGAAG
78
n/a
227


 


1604
1623
524896
ACGGTGGTCTCCATGCGACG
79
n/a
808


 


1607
1626
524897
TTCACGGTGGTCTCCATGCG
75
n/a
809


 


1630
1649
524898
CCTTGGGCAACATTCGGTGG
77
n/a
810


 


1633
1652
524899
AGACCTTGGGCAACATTCGG
76
n/a
811


 


1636
1655
524900
GTAAGACCTTGGGCAACATT
73
n/a
812


 


1639
1658
524901
TATGTAAGACCTTGGGCAAC
60
n/a
813


 


1642
1661
524902
TCTTATGTAAGACCTTGGGC
72
n/a
814


 


1645
1664
524903
TCCTCTTATGTAAGACCTTG
75
n/a
815


 


1648
1667
524904
GAGTCCTCTTATGTAAGACC
65
n/a
816


 


1651
1670
524905
CAAGAGTCCTCTTATGTAAG
76
n/a
817


 


1654
1673
524906
GTCCAAGAGTCCTCTTATGT
78
n/a
818


 


1657
1676
524907
AGAGTCCAAGAGTCCTCTTA
82
n/a
819


 


1660
1679
524908
CAGAGAGTCCAAGAGTCCTC
82
n/a
820


 


1663
1682
524909
TTGCAGAGAGTCCAAGAGTC
76
n/a
821


 


1666
1685
524910
ACATTGCAGAGAGTCCAAGA
76
n/a
822


 


1669
1688
524911
TTGACATTGCAGAGAGTCCA
74
n/a
823


 


1689
1708
524912
GTATGCCTCAAGGTCGGTCG
76
n/a
824


 


1692
1711
524913
GAAGTATGCCTCAAGGTCGG
73
n/a
825


 


1695
1714
524914
TTTGAAGTATGCCTCAAGGT
76
n/a
826


 


1698
1717
524915
GTCTTTGAAGTATGCCTCAA
75
n/a
827


 


1701
1720
524916
ACAGTCTTTGAAGTATGCCT
77
n/a
828


 


1704
1723
524917
CAAACAGTCTTTGAAGTATG
55
n/a
829


 


1707
1726
524918
AAACAAACAGTCTTTGAAGT
59
n/a
830


 


1710
1729
524919
TTTAAACAAACAGTCTTTGA
53
n/a
831


 


1713
1732
524920
GTCTTTAAACAAACAGTCTT
3
n/a
832


 


1716
1735
524921
CCAGTCTTTAAACAAACAGT
75
n/a
833


 


1719
1738
524922
CTCCCAGTCTTTAAACAAAC
70
n/a
834


 


1722
1741
524923
CTCCTCCCAGTCTTTAAACA
68
n/a
835


 


1725
1744
524924
CAACTCCTCCCAGTCTTTAA
62
n/a
836


 


1728
1747
524925
CCCCAACTCCTCCCAGTCTT
63
n/a
837


 


1731
1750
524926
CTCCCCCAACTCCTCCCAGT
62
n/a
838


 


1734
1753
524927
CTCCTCCCCCAACTCCTCCC
55
n/a
839


 


1737
1756
524928
AATCTCCTCCCCCAACTCCT
61
n/a
840


 


1740
1759
524929
TCTAATCTCCTCCCCCAACT
61
n/a
841


 


1743
1762
524930
TAATCTAATCTCCTCCCCCA
70
n/a
842


 


1746
1765
524931
CTTTAATCTAATCTCCTCCC
74
n/a
843


 


1749
1768
524932
GACCTTTAATCTAATCTCCT
74
n/a
844


 


1752
1771
524933
AAAGACCTTTAATCTAATCT
60
n/a
845


 


1755
1774
524934
TACAAAGACCTTTAATCTAA
55
n/a
846


 


1758
1777
524935
TAGTACAAAGACCTTTAATC
54
n/a
847


 


1761
1780
524936
TCCTAGTACAAAGACCTTTA
69
n/a
848


 


1764
1783
524937
GCCTCCTAGTACAAAGACCT
72
n/a
849


 


1767
1786
524938
ACAGCCTCCTAGTACAAAGA
60
n/a
850


 


1770
1789
524939
CCTACAGCCTCCTAGTACAA
66
n/a
851


 


1773
1792
524940
ATGCCTACAGCCTCCTAGTA
70
n/a
852


 


1776
1795
524941
TTTATGCCTACAGCCTCCTA
63
n/a
853


 


1777
1796
524942
ATTTATGCCTACAGCCTCCT
70
n/a
854


 


1778
1797
509932
AATTTATGCCTACAGCCTCC
68
n/a
46


 


1779
1798
509933
CAATTTATGCCTACAGCCTC
68
n/a
48


 


1780
1799
509934
CCAATTTATGCCTACAGCCT
65
n/a
50


 


1781
1800
509935
ACCAATTTATGCCTACAGCC
64
n/a
52


 


1782
1801
524943
GACCAATTTATGCCTACAGC
57
n/a
855


 


1783
1802
524944
AGACCAATTTATGCCTACAG
60
n/a
856


 


1785
1804
524945
GCAGACCAATTTATGCCTAC
54
n/a
857


 


1788
1807
524946
TGCGCAGACCAATTTATGCC
68
n/a
858


 


1791
1810
524947
TGGTGCGCAGACCAATTTAT
64
n/a
859


 


1794
1813
524948
TGCTGGTGCGCAGACCAATT
75
n/a
860


 


1797
1816
524949
TGGTGCTGGTGCGCAGACCA
68
n/a
861


 


1800
1819
524950
GCATGGTGCTGGTGCGCAGA
69
n/a
862


 


1803
1822
524951
GTTGCATGGTGCTGGTGCGC
59
n/a
863


 


1807
1826
524952
AAAAGTTGCATGGTGCTGGT
61
n/a
864


 


1810
1829
524953
TGAAAAAGTTGCATGGTGCT
60
n/a
865


 


1813
1832
524954
AGGTGAAAAAGTTGCATGGT
61
n/a
866


 


1816
1835
524955
CAGAGGTGAAAAAGTTGCAT
63
n/a
867


 


1819
1838
524956
AGGCAGAGGTGAAAAAGTTG
57
n/a
868


 


1822
1841
524957
ATTAGGCAGAGGTGAAAAAG
50
n/a
869


 


1823
1842
524958
GATTAGGCAGAGGTGAAAAA
57
n/a
870


 


1825
1844
524959
ATGATTAGGCAGAGGTGAAA
54
n/a
871


 


1828
1847
524960
GAGATGATTAGGCAGAGGTG
59
n/a
872


 


1831
1850
524961
CAAGAGATGATTAGGCAGAG
61
n/a
873


 


1834
1853
524962
GAACAAGAGATGATTAGGCA
56
n/a
874


 


1837
1856
524963
CATGAACAAGAGATGATTAG
24
n/a
875


 


1840
1859
524964
GGACATGAACAAGAGATGAT
54
n/a
876


 


1843
1862
524965
GTAGGACATGAACAAGAGAT
52
n/a
877


 


1846
1865
524966
ACAGTAGGACATGAACAAGA
47
n/a
878


 


1849
1868
524967
TGAACAGTAGGACATGAACA
33
n/a
879


 


1852
1871
524968
GCTTGAACAGTAGGACATGA
44
n/a
880


 


1855
1874
524969
GAGGCTTGAACAGTAGGACA
43
n/a
881


 


1858
1877
524970
TTGGAGGCTTGAACAGTAGG
28
n/a
882


 


1862
1881
524971
CAGCTTGGAGGCTTGAACAG
30
n/a
883


 


1871
1890
524972
CCCAAGGCACAGCTTGGAGG
38
n/a
884


 


1874
1893
524973
CCACCCAAGGCACAGCTTGG
47
n/a
885


 


1877
1896
524974
AAGCCACCCAAGGCACAGCT
49
n/a
886


 


1880
1899
524975
CCAAAGCCACCCAAGGCACA
32
n/a
887


 


1883
1902
524976
GCCCCAAAGCCACCCAAGGC
56
n/a
888


 


1886
1905
524977
CATGCCCCAAAGCCACCCAA
63
n/a
889


 


1889
1908
524978
GTCCATGCCCCAAAGCCACC
64
n/a
890


 


1892
1911
524979
GATGTCCATGCCCCAAAGCC
65
n/a
891


 


1895
1914
524980
GTCGATGTCCATGCCCCAAA
80
n/a
892


 


1898
1917
524981
AGGGTCGATGTCCATGCCCC
79
n/a
893


 


1901
1920
524982
ATAAGGGTCGATGTCCATGC
79
n/a
894


 


1904
1923
524983
TTTATAAGGGTCGATGTCCA
71
n/a
895


 


1907
1926
524984
TTCTTTATAAGGGTCGATGT
77
n/a
896


 


1910
1929
524985
AAATTCTTTATAAGGGTCGA
79
n/a
897


 


1913
1932
524986
TCCAAATTCTTTATAAGGGT
80
n/a
898


 


1916
1935
524987
AGCTCCAAATTCTTTATAAG
80
n/a
899


 


1919
1938
524988
AGTAGCTCCAAATTCTTTAT
76
n/a
900


 


1922
1941
524989
CACAGTAGCTCCAAATTCTT
59
n/a
901


 


1925
1944
524990
CTCCACAGTAGCTCCAAATT
46
n/a
902


 


1928
1947
524991
TAACTCCACAGTAGCTCCAA
63
n/a
903


 


1931
1950
524992
GAGTAACTCCACAGTAGCTC
65
n/a
904


 


1934
1953
524993
CGAGAGTAACTCCACAGTAG
69
n/a
905


 


1937
1956
524994
AAACGAGAGTAACTCCACAG
61
n/a
906


 


1940
1959
524995
CAAAAACGAGAGTAACTCCA
46
n/a
907


 


1943
1962
524996
AGGCAAAAACGAGAGTAACT
39
n/a
908


 


1946
1965
524997
AGAAGGCAAAAACGAGAGTA
53
n/a
909


 


1949
1968
524998
GTCAGAAGGCAAAAACGAGA
56
n/a
910


 


1952
1971
524999
GAAGTCAGAAGGCAAAAACG
49
n/a
911


 


1955
1974
525000
AAAGAAGTCAGAAGGCAAAA
29
n/a
912


 


1958
1977
525001
AGGAAAGAAGTCAGAAGGCA
41
n/a
913


 


1961
1980
525002
TGAAGGAAAGAAGTCAGAAG
34
n/a
914


 


1964
1983
525003
TACTGAAGGAAAGAAGTCAG
26
n/a
915


 


1984
2003
525004
GCGGTATCTAGAAGATCTCG
24
n/a
916


 


1987
2006
525005
GAGGCGGTATCTAGAAGATC
29
n/a
917


 


1990
2009
525006
GCTGAGGCGGTATCTAGAAG
29
n/a
918


 


1993
2012
525007
AGAGCTGAGGCGGTATCTAG
13
n/a
919


 


1996
2015
525008
TACAGAGCTGAGGCGGTATC
6
n/a
920


 


1999
2018
525009
CGATACAGAGCTGAGGCGGT
3
n/a
921


 


2002
2021
525010
TCCCGATACAGAGCTGAGGC
27
n/a
922


 


2005
2024
525011
GCTTCCCGATACAGAGCTGA
43
n/a
923


 


2008
2027
525012
AAGGCTTCCCGATACAGAGC
33
n/a
924


 


2011
2030
525013
TCTAAGGCTTCCCGATACAG
34
n/a
925


 


2014
2033
525014
GACTCTAAGGCTTCCCGATA
38
n/a
926


 


2017
2036
525015
GGAGACTCTAAGGCTTCCCG
16
n/a
927


 


2020
2039
525016
TCAGGAGACTCTAAGGCTTC
16
n/a
928


 


2023
2042
525017
TGCTCAGGAGACTCTAAGGC
14
n/a
929


 


2026
2045
525018
CAATGCTCAGGAGACTCTAA
34
n/a
930


 


2029
2048
525019
GAACAATGCTCAGGAGACTC
32
n/a
931


 


2032
2051
525020
GGTGAACAATGCTCAGGAGA
9
n/a
932


 


2035
2054
525021
TGAGGTGAACAATGCTCAGG
50
n/a
933


 


2038
2057
525022
TGGTGAGGTGAACAATGCTC
54
n/a
934


 


2041
2060
525023
GTATGGTGAGGTGAACAATG
47
n/a
935


 


2044
2063
525024
GCAGTATGGTGAGGTGAACA
40
n/a
936


 


2047
2066
525025
AGTGCAGTATGGTGAGGTGA
35
n/a
937


 


2050
2069
525026
CTGAGTGCAGTATGGTGAGG
43
n/a
938


 


2053
2072
525027
TGCCTGAGTGCAGTATGGTG
45
n/a
939


 


2056
2075
525028
GCTTGCCTGAGTGCAGTATG
42
n/a
940


 


2059
2078
525029
ATTGCTTGCCTGAGTGCAGT
39
n/a
941


 


2062
2081
525030
AGAATTGCTTGCCTGAGTGC
27
n/a
942


 


2065
2084
525031
CAAAGAATTGCTTGCCTGAG
42
n/a
943


 


2068
2087
525032
CAGCAAAGAATTGCTTGCCT
49
n/a
944


 


2071
2090
525033
CCCCAGCAAAGAATTGCTTG
41
n/a
945


 


2074
2093
525034
TCCCCCCAGCAAAGAATTGC
39
n/a
946


 


2077
2096
525035
AGTTCCCCCCAGCAAAGAAT
39
n/a
947


 


2080
2099
525036
ATTAGTTCCCCCCAGCAAAG
43
n/a
948


 


2083
2102
525037
GTCATTAGTTCCCCCCAGCA
64
n/a
949


 


2086
2105
525038
AGAGTCATTAGTTCCCCCCA
45
n/a
950


 


2089
2108
525039
GCTAGAGTCATTAGTTCCCC
58
n/a
951


 


2092
2111
525040
GTAGCTAGAGTCATTAGTTC
45
n/a
952


 


2095
2114
525041
CAGGTAGCTAGAGTCATTAG
44
n/a
953


 


2098
2117
525042
ACCCAGGTAGCTAGAGTCAT
39
n/a
954


 


2101
2120
525043
CCCACCCAGGTAGCTAGAGT
51
n/a
955


 


2104
2123
525044
ACACCCACCCAGGTAGCTAG
27
n/a
956


 


2107
2126
525045
TTAACACCCACCCAGGTAGC
41
n/a
957


 


2110
2129
525046
AAATTAACACCCACCCAGGT
44
n/a
958


 


2113
2132
525047
TCCAAATTAACACCCACCCA
29
n/a
959


 


2116
2135
525048
TCTTCCAAATTAACACCCAC
31
n/a
960


 


2119
2138
525049
GGATCTTCCAAATTAACACC
42
n/a
961


 


2122
2141
525050
GCTGGATCTTCCAAATTAAC
53
n/a
962


 


2125
2144
525051
GATGCTGGATCTTCCAAATT
41
n/a
963


 


2128
2147
525052
CTAGATGCTGGATCTTCCAA
62
n/a
964


 


2131
2150
525053
TCTCTAGATGCTGGATCTTC
41
83
965


 


2134
2153
525054
AGGTCTCTAGATGCTGGATC
26
73
966


 


2137
2156
525055
ACTAGGTCTCTAGATGCTGG
36
74
967


 


2140
2159
525056
ACTACTAGGTCTCTAGATGC
22
63
968


 


2143
2162
525057
CTGACTACTAGGTCTCTAGA
28
80
969


 


2146
2165
525058
TAACTGACTACTAGGTCTCT
47
83
970


 


2149
2168
525059
ACATAACTGACTACTAGGTC
31
77
971


 


2152
2171
525060
TTGACATAACTGACTACTAG
34
75
972


 


2155
2174
525061
GTGTTGACATAACTGACTAC
42
75
973


 


2158
2177
525062
TTAGTGTTGACATAACTGAC
48
81
974


 


2161
2180
525063
ATATTAGTGTTGACATAACT
33
73
975


 


2164
2183
525064
CCCATATTAGTGTTGACATA
41
82
976


 


2167
2186
525065
AGGCCCATATTAGTGTTGAC
39
77
977


 


2170
2189
525066
TTTAGGCCCATATTAGTGTT
46
83
978


 


2173
2192
525067
AACTTTAGGCCCATATTAGT
38
69
979


 


2176
2195
525068
CTGAACTTTAGGCCCATATT
41
85
980


 


2179
2198
525069
TGCCTGAACTTTAGGCCCAT
38
81
981


 


2182
2201
525070
AGTTGCCTGAACTTTAGGCC
17
67
982


 


2185
2204
525071
AAGAGTTGCCTGAACTTTAG
27
62
983


 


2188
2207
525072
CACAAGAGTTGCCTGAACTT
27
64
984


 


2191
2210
525073
AACCACAAGAGTTGCCTGAA
41
80
985


 


2194
2213
525074
TGAAACCACAAGAGTTGCCT
32
75
986


 


2197
2216
525075
ATGTGAAACCACAAGAGTTG
43
67
987


 


2200
2219
525076
GAAATGTGAAACCACAAGAG
34
74
988


 


2203
2222
525077
CAAGAAATGTGAAACCACAA
22
65
989


 


2206
2225
525078
AGACAAGAAATGTGAAACCA
39
70
990


 


2209
2228
525079
GTGAGACAAGAAATGTGAAA
32
74
991


 


2212
2231
525080
AAAGTGAGACAAGAAATGTG
30
63
992


 


2215
2234
525081
CCAAAAGTGAGACAAGAAAT
25
58
993


 


2218
2237
525082
CTTCCAAAAGTGAGACAAGA
36
74
994


 


2221
2240
525083
TCTCTTCCAAAAGTGAGACA
42
84
995


 


2224
2243
525084
GTTTCTCTTCCAAAAGTGAG
33
75
996


 


2227
2246
525085
ACGGTTTCTCTTCCAAAAGT
32
68
997


 


2230
2249
525086
ATAACGGTTTCTCTTCCAAA
51
80
998


 


2233
2252
525087
TCTATAACGGTTTCTCTTCC
36
77
999


 


2236
2255
525088
TACTCTATAACGGTTTCTCT
23
69
1000


 


2239
2258
525089
AAATACTCTATAACGGTTTC
45
77
1001


 


2242
2261
525090
ACCAAATACTCTATAACGGT
57
82
1002


 


2245
2264
525091
GACACCAAATACTCTATAAC
36
77
1003


 


2248
2267
525092
AAAGACACCAAATACTCTAT
42
80
1004


 


2251
2270
525093
CCGAAAGACACCAAATACTC
41
89
1005


 


2254
2273
525094
ACTCCGAAAGACACCAAATA
29
73
1006


 


2257
2276
525095
CACACTCCGAAAGACACCAA
33
92
1007


 


2260
2279
525096
ATCCACACTCCGAAAGACAC
18
74
1008


 


2263
2282
525097
CGAATCCACACTCCGAAAGA
30
57
1009


 


2266
2285
525098
GTGCGAATCCACACTCCGAA
28
67
1010


 


2269
2288
146789
GGAGTGCGAATCCACACTCC
37
72
1011


 


2272
2291
525099
GGAGGAGTGCGAATCCACAC
36
64
1012


 


2275
2294
525100
GCTGGAGGAGTGCGAATCCA
52
90
1013


 


2278
2297
525101
TAAGCTGGAGGAGTGCGAAT
49
96
1014


 


2281
2300
525102
CTATAAGCTGGAGGAGTGCG
37
96
1015


 


2284
2303
525103
GGTCTATAAGCTGGAGGAGT
30
97
1016


 


2287
2306
525104
GGTGGTCTATAAGCTGGAGG
22
77
1017


 


2290
2309
525105
TTTGGTGGTCTATAAGCTGG
41
76
1018


 


2293
2312
525106
GCATTTGGTGGTCTATAAGC
39
76
1019


 


2313
2332
525107
GAAGTGTTGATAGGATAGGG
27
97
1020


 


2316
2335
525108
CCGGAAGTGTTGATAGGATA
42
97
1021


 


2319
2338
525109
TTTCCGGAAGTGTTGATAGG
48
99
1022


 


2322
2341
525110
TAGTTTCCGGAAGTGTTGAT
18
98
1023


 


2325
2344
525111
CAGTAGTTTCCGGAAGTGTT
19
98
1024


 


2328
2347
525112
CAACAGTAGTTTCCGGAAGT
29
96
1025


 


2331
2350
525113
TAACAACAGTAGTTTCCGGA
39
95
1026


 


2334
2353
525114
GTCTAACAACAGTAGTTTCC
40
99
1027


 


2369
2388
525115
CGAGGGAGTTCTTCTTCTAG
42
98
1028


 


2372
2391
525116
AGGCGAGGGAGTTCTTCTTC
31
97
1029


 


2375
2394
525117
GCGAGGCGAGGGAGTTCTTC
22
98
1030


 


2379
2398
525118
GTCTGCGAGGCGAGGGAGTT
20
99
1031


 


2398
2417
525119
CGCGGCGATTGAGACCTTCG
26
97
1032


 


2401
2420
525120
CGACGCGGCGATTGAGACCT
23
97
1033


 


2404
2423
525121
CTGCGACGCGGCGATTGAGA
47
92
1034


 


2407
2426
525122
CTTCTGCGACGCGGCGATTG
27
74
1035


 


2410
2429
525123
GATCTTCTGCGACGCGGCGA
36
87
1036


 


2413
2432
146790
TGAGATCTTCTGCGACGCGG
25
85
1037


 


2416
2435
525124
GATTGAGATCTTCTGCGACG
17
84
1038


 


2419
2438
525125
CGAGATTGAGATCTTCTGCG
24
82
1039


 


2422
2441
525126
TCCCGAGATTGAGATCTTCT
29
74
1040


 


2425
2444
525127
GGTTCCCGAGATTGAGATCT
14
79
1041


 


2428
2447
525128
TGAGGTTCCCGAGATTGAGA
41
76
1042


 


2431
2450
525129
CATTGAGGTTCCCGAGATTG
39
72
1043


 


2434
2453
525130
TAACATTGAGGTTCCCGAGA
37
71
1044


 


2437
2456
525131
TACTAACATTGAGGTTCCCG
42
76
1045


 


2440
2459
525132
GAATACTAACATTGAGGTTC
21
75
1046


 


2443
2462
525133
AAGGAATACTAACATTGAGG
36
75
1047


 


2446
2465
525134
TCCAAGGAATACTAACATTG
29
77
1048


 


2449
2468
525135
GAGTCCAAGGAATACTAACA
32
76
1049


 


2452
2471
525136
TATGAGTCCAAGGAATACTA
23
62
1050


 


2455
2474
525137
CCTTATGAGTCCAAGGAATA
27
57
1051


 


2458
2477
525138
CCACCTTATGAGTCCAAGGA
52
82
1052


 


2461
2480
525139
TCCCCACCTTATGAGTCCAA
46
80
1053


 


2464
2483
525140
AGTTCCCCACCTTATGAGTC
14
59
1054


 


2467
2486
525141
TAAAGTTCCCCACCTTATGA
20
45
1055


 


2470
2489
525142
CAGTAAAGTTCCCCACCTTA
14
72
1056


 


2473
2492
525143
GACCAGTAAAGTTCCCCACC
30
77
1057


 


2476
2495
525144
AAAGACCAGTAAAGTTCCCC
19
72
1058


 


2479
2498
525145
AATAAAGACCAGTAAAGTTC
18
55
1059


 


2482
2501
525146
AAGAATAAAGACCAGTAAAG
16
51
1060


 


2485
2504
525147
TAGAAGAATAAAGACCAGTA
22
68
1061


 


2488
2507
525148
CAGTAGAAGAATAAAGACCA
13
59
1062


 


2491
2510
525149
GTACAGTAGAAGAATAAAGA
0
45
1063


 


2494
2513
525150
CAGGTACAGTAGAAGAATAA
31
62
1064


 


2497
2516
525151
AGACAGGTACAGTAGAAGAA
8
62
1065


 


2500
2519
525152
TAAAGACAGGTACAGTAGAA
29
61
1066


 


2503
2522
525153
GATTAAAGACAGGTACAGTA
28
67
1067


 


2506
2525
525154
GAGGATTAAAGACAGGTACA
38
76
1068


 


2509
2528
525155
AATGAGGATTAAAGACAGGT
30
72
1069


 


2512
2531
525156
TCCAATGAGGATTAAAGACA
24
67
1070


 


2515
2534
525157
TTTTCCAATGAGGATTAAAG
0
44
1071


 


2518
2537
525158
GTGTTTTCCAATGAGGATTA
20
74
1072


 


2521
2540
525159
ATGGTGTTTTCCAATGAGGA
30
71
1073


 


2524
2543
525160
AAGATGGTGTTTTCCAATGA
22
68
1074


 


2527
2546
525161
GAAAAGATGGTGTTTTCCAA
19
61
1075


 


2530
2549
525162
TAGGAAAAGATGGTGTTTTC
14
52
1076


 


2533
2552
525163
TATTAGGAAAAGATGGTGTT
1
47
1077


 


2536
2555
525164
GTATATTAGGAAAAGATGGT
0
60
1078


 


2539
2558
525165
AATGTATATTAGGAAAAGAT
0
30
1079


 


2542
2561
525166
GTAAATGTATATTAGGAAAA
1
18
1080


 


2545
2564
525167
GGTGTAAATGTATATTAGGA
23
72
1081


 


2548
2567
525168
CTTGGTGTAAATGTATATTA
32
75
1082


 


2551
2570
525169
TGTCTTGGTGTAAATGTATA
12
65
1083


 


2554
2573
525170
TAATGTCTTGGTGTAAATGT
3
51
1084


 


2557
2576
525171
TGATAATGTCTTGGTGTAAA
24
62
1085


 


2560
2579
525172
TTTTGATAATGTCTTGGTGT
18
66
1086


 


2563
2582
525173
ATTTTTTGATAATGTCTTGG
11
63
1087


 


2566
2585
525174
CACATTTTTTGATAATGTCT
20
68
1088


 


2569
2588
525175
GTTCACATTTTTTGATAATG
38
68
1089


 


2572
2591
525176
ACTGTTCACATTTTTTGATA
12
61
1090


 


2575
2594
525177
CAAACTGTTCACATTTTTTG
25
56
1091


 


2578
2597
525178
CTACAAACTGTTCACATTTT
21
47
1092


 


2581
2600
525179
GGCCTACAAACTGTTCACAT
28
83
1093


 


2584
2603
525180
GTGGGCCTACAAACTGTTCA
7
72
1094


 


2587
2606
525181
TAAGTGGGCCTACAAACTGT
26
75
1095


 


2590
2609
525182
CTGTAAGTGGGCCTACAAAC
35
78
1096


 


2593
2612
525183
TAACTGTAAGTGGGCCTACA
29
69
1097


 


2596
2615
525184
CATTAACTGTAAGTGGGCCT
22
73
1098


 


2599
2618
525185
TCTCATTAACTGTAAGTGGG
31
81
1099


 


2602
2621
525186
TTTTCTCATTAACTGTAAGT
15
58
1100


 


2605
2624
525187
TTCTTTTCTCATTAACTGTA
14
71
1101


 


2608
2627
525188
ATCTTCTTTTCTCATTAACT
19
71
1102


 


2611
2630
525189
GCAATCTTCTTTTCTCATTA
36
79
1103


 


2614
2633
525190
ATTGCAATCTTCTTTTCTCA
38
82
1104


 


2617
2636
525191
TCAATTGCAATCTTCTTTTC
23
61
1105


 


2620
2639
525192
TAATCAATTGCAATCTTCTT
10
67
1106


 


2623
2642
525193
GCATAATCAATTGCAATCTT
27
71
1107


 


2626
2645
525194
CAGGCATAATCAATTGCAAT
23
71
1108


 


2629
2648
525195
TAGCAGGCATAATCAATTGC
30
77
1109


 


2632
2651
525196
ACCTAGCAGGCATAATCAAT
7
70
1110


 


2635
2654
525197
AAAACCTAGCAGGCATAATC
47
70
1111


 


2638
2657
525198
GATAAAACCTAGCAGGCATA
41
81
1112


 


2641
2660
525199
TTGGATAAAACCTAGCAGGC
30
78
1113


 


2644
2663
525200
CCTTTGGATAAAACCTAGCA
31
76
1114


 


2647
2666
525201
TAACCTTTGGATAAAACCTA
25
63
1115


 


2650
2669
525202
TGGTAACCTTTGGATAAAAC
24
76
1116


 


2653
2672
525203
ATTTGGTAACCTTTGGATAA
20
64
1117


 


2656
2675
525204
AATATTTGGTAACCTTTGGA
16
77
1118


 


2659
2678
525205
GTAAATATTTGGTAACCTTT
39
80
1119


 


2662
2681
525206
ATGGTAAATATTTGGTAACC
40
75
1120


 


2665
2684
525207
CCAATGGTAAATATTTGGTA
38
75
1121


 


2668
2687
525208
TATCCAATGGTAAATATTTG
0
0
1122


 


2671
2690
525209
CCTTATCCAATGGTAAATAT
28
57
1123


 


2674
2693
525210
TACCCTTATCCAATGGTAAA
18
71
1124


 


2677
2696
525211
TAATACCCTTATCCAATGGT
35
76
1125


 


2680
2699
525212
GTTTAATACCCTTATCCAAT
41
77
1126


 


2683
2702
525213
AAGGTTTAATACCCTTATCC
11
79
1127


 


2686
2705
525214
AATAAGGTTTAATACCCTTA
35
75
1128


 


2689
2708
525215
GATAATAAGGTTTAATACCC
22
54
1129


 


2692
2711
525216
CTGGATAATAAGGTTTAATA
19
35
1130


 


2695
2714
525217
GTTCTGGATAATAAGGTTTA
24
58
1131


 


2698
2717
525218
GATGTTCTGGATAATAAGGT
20
73
1132


 


2701
2720
525219
CTAGATGTTCTGGATAATAA
26
66
1133


 


2704
2723
525220
TAACTAGATGTTCTGGATAA
21
66
1134


 


2707
2726
525221
GATTAACTAGATGTTCTGGA
30
78
1135


 


2710
2729
525222
AATGATTAACTAGATGTTCT
30
61
1136


 


2713
2732
525223
AGTAATGATTAACTAGATGT
9
57
1137


 


2716
2735
525224
GGAAGTAATGATTAACTAGA
18
72
1138


 


2719
2738
525225
TTTGGAAGTAATGATTAACT
7
67
1139


 


2722
2741
525226
TAGTTTGGAAGTAATGATTA
2
30
1140


 


2725
2744
525227
GTCTAGTTTGGAAGTAATGA
27
78
1141


 


2728
2747
525228
AGTGTCTAGTTTGGAAGTAA
27
75
1142


 


2731
2750
525229
AATAGTGTCTAGTTTGGAAG
34
73
1143


 


2734
2753
525230
GTAAATAGTGTCTAGTTTGG
28
68
1144


 


2737
2756
525231
TGTGTAAATAGTGTCTAGTT
27
79
1145


 


2740
2759
525232
GAGTGTGTAAATAGTGTCTA
27
71
1146


 


2743
2762
525233
ATAGAGTGTGTAAATAGTGT
17
75
1147


 


2746
2765
525234
TCCATAGAGTGTGTAAATAG
18
75
1148


 


2749
2768
525235
CCTTCCATAGAGTGTGTAAA
23
80
1149


 


2752
2771
525236
CCGCCTTCCATAGAGTGTGT
26
82
1150


 


2755
2774
525237
TACCCGCCTTCCATAGAGTG
19
80
1151


 


2758
2777
525238
ATATACCCGCCTTCCATAGA
0
67
1152


 


2761
2780
525239
ATAATATACCCGCCTTCCAT
19
70
1153


 


2764
2783
525240
TATATAATATACCCGCCTTC
9
73
1154


 


2767
2786
525241
TCTTATATAATATACCCGCC
20
80
1155


 


2770
2789
525242
CTCTCTTATATAATATACCC
29
76
1156


 


2773
2792
525243
TTTCTCTCTTATATAATATA
16
58
1157


 


2776
2795
525244
TTGTTTCTCTCTTATATAAT
26
57
1158


 


2779
2798
525245
GTGTTGTTTCTCTCTTATAT
35
85
1159


 


2782
2801
525246
TATGTGTTGTTTCTCTCTTA
34
82
1160


 


2785
2804
525247
CGCTATGTGTTGTTTCTCTC
34
86
1161


 


2802
2821
525248
TGACCCACAAAATGAGGCGC
17
71
1162


 


2805
2824
525249
TGGTGACCCACAAAATGAGG
31
67
1163


 


2808
2827
525250
ATATGGTGACCCACAAAATG
38
69
1164


 


2811
2830
525251
AGAATATGGTGACCCACAAA
37
77
1165


 


2814
2833
525252
CCAAGAATATGGTGACCCAC
35
79
1166


 


2817
2836
146831
TTCCCAAGAATATGGTGACC
27
75
1167


 


2820
2839
525253
TTGTTCCCAAGAATATGGTG
33
69
1168


 


2823
2842
525254
ATCTTGTTCCCAAGAATATG
27
65
1169


 


2826
2845
525255
TAGATCTTGTTCCCAAGAAT
31
70
1170


 


2829
2848
525256
CTGTAGATCTTGTTCCCAAG
42
81
1171


 


2832
2851
525257
ATGCTGTAGATCTTGTTCCC
34
80
1172


 


2835
2854
525258
CCCATGCTGTAGATCTTGTT
38
80
1173


 


2838
2857
525259
TGCCCCATGCTGTAGATCTT
36
80
1174


 


2841
2860
525260
TTCTGCCCCATGCTGTAGAT
32
74
1175


 


2844
2863
525261
AGATTCTGCCCCATGCTGTA
27
75
1176


 


2847
2866
525262
GAAAGATTCTGCCCCATGCT
34
70
1177


 


2850
2869
525263
GTGGAAAGATTCTGCCCCAT
22
76
1178


 


2853
2872
525264
CTGGTGGAAAGATTCTGCCC
36
72
1179


 


2856
2875
525265
TTGCTGGTGGAAAGATTCTG
32
71
1180


 


2859
2878
525266
GGATTGCTGGTGGAAAGATT
20
74
1181


 


2862
2881
525267
AGAGGATTGCTGGTGGAAAG
25
73
1182


 


2865
2884
525268
CCCAGAGGATTGCTGGTGGA
40
82
1183


 


2868
2887
525269
AATCCCAGAGGATTGCTGGT
32
79
1184


 


2871
2890
525270
AAGAATCCCAGAGGATTGCT
23
69
1185


 


2874
2893
525271
GGAAAGAATCCCAGAGGATT
10
66
1186


 


2877
2896
525272
TCGGGAAAGAATCCCAGAGG
29
73
1187


 


2880
2899
525273
TGGTCGGGAAAGAATCCCAG
31
77
1188


 


2883
2902
525274
TGGTGGTCGGGAAAGAATCC
38
71
1189


 


2886
2905
525275
AACTGGTGGTCGGGAAAGAA
33
78
1190


 


2889
2908
525276
TCCAACTGGTGGTCGGGAAA
29
76
1191


 


2892
2911
525277
GGATCCAACTGGTGGTCGGG
19
81
1192


 


2895
2914
525278
GCTGGATCCAACTGGTGGTC
24
74
1193


 


2898
2917
525279
AAGGCTGGATCCAACTGGTG
33
83
1194


 


2901
2920
525280
CTGAAGGCTGGATCCAACTG
18
81
1195


 


2904
2923
525286
GCTCTGAAGGCTGGATCCAA
40
79
1196


 


2907
2926
525287
TTTGCTCTGAAGGCTGGATC
34
69
1197


 


2910
2929
525288
GTGTTTGCTCTGAAGGCTGG
38
72
1198


 


2913
2932
525289
GCTGTGTTTGCTCTGAAGGC
40
82
1199


 


2916
2935
525290
TTTGCTGTGTTTGCTCTGAA
44
78
1200


 


2919
2938
525291
GGATTTGCTGTGTTTGCTCT
38
76
1201


 


2922
2941
525292
TCTGGATTTGCTGTGTTTGC
28
79
1202


 


2925
2944
525293
CAATCTGGATTTGCTGTGTT
26
61
1203


 


2928
2947
525294
TCCCAATCTGGATTTGCTGT
32
68
1204


 


2931
2950
525295
AAGTCCCAATCTGGATTTGC
33
59
1205


 


2934
2953
146832
TTGAAGTCCCAATCTGGATT
17
35
1206


 


2937
2956
525296
GGATTGAAGTCCCAATCTGG
35
62
1207


 


2940
2959
525297
TTGGGATTGAAGTCCCAATC
10
36
1208


 


2943
2962
525298
TTGTTGGGATTGAAGTCCCA
24
49
1209


 


2946
2965
525299
TCCTTGTTGGGATTGAAGTC
16
52
1210


 


2949
2968
525300
GTGTCCTTGTTGGGATTGAA
18
71
1211


 


2952
2971
525301
CAGGTGTCCTTGTTGGGATT
25
73
1212


 


2955
2974
525302
GGCCAGGTGTCCTTGTTGGG
31
70
1213


 


2958
2977
525303
TCTGGCCAGGTGTCCTTGTT
29
75
1214


 


2978
2997
525304
CAGCTCCTACCTTGTTGGCG
29
71
1215


 


2981
3000
525305
CTCCAGCTCCTACCTTGTTG
19
63
1216


 


2984
3003
525306
ATGCTCCAGCTCCTACCTTG
35
75
1217


 


2987
3006
525307
CGAATGCTCCAGCTCCTACC
13
77
1218


 


2990
3009
525308
GCCCGAATGCTCCAGCTCCT
28
72
1219


 


2993
3012
525309
CCAGCCCGAATGCTCCAGCT
32
77
1220


 


2996
3015
525310
AACCCAGCCCGAATGCTCCA
34
72
1221


 


2999
3018
525311
TGAAACCCAGCCCGAATGCT
28
69
1222


 


3002
3021
525312
GGGTGAAACCCAGCCCGAAT
18
68
1223


 


3020
3039
525313
AAAGGCCTCCGTGCGGTGGG
36
77
1224


 


3023
3042
525314
CCAAAAGGCCTCCGTGCGGT
34
83
1225


 


3026
3045
525315
ACCCCAAAAGGCCTCCGTGC
28
70
1226


 


3029
3048
525316
TCCACCCCAAAAGGCCTCCG
26
65
1227


 


3032
3051
525317
GGCTCCACCCCAAAAGGCCT
19
36
1228


 


3035
3054
525318
GAGGGCTCCACCCCAAAAGG
14
36
1229


 


3038
3057
525319
CCTGAGGGCTCCACCCCAAA
32
71
1230


 


3041
3060
525320
GAGCCTGAGGGCTCCACCCC
37
61
1231


 


3044
3063
525321
CCTGAGCCTGAGGGCTCCAC
42
70
1232


 


3047
3066
525322
TGCCCTGAGCCTGAGGGCTC
24
56
1233


 


3050
3069
525323
GTATGCCCTGAGCCTGAGGG
14
75
1234


 


3053
3072
525324
GTAGTATGCCCTGAGCCTGA
29
83
1235


 


3056
3075
525325
TTTGTAGTATGCCCTGAGCC
32
61
1236


 


3059
3078
525326
AAGTTTGTAGTATGCCCTGA
35
70
1237


 


3062
3081
525327
GCAAAGTTTGTAGTATGCCC
37
61
1238


 


3065
3084
525328
CTGGCAAAGTTTGTAGTATG
26
63
1239


 


3068
3087
525329
TTGCTGGCAAAGTTTGTAGT
37
74
1240


 


3071
3090
525330
GATTTGCTGGCAAAGTTTGT
20
56
1241


 


3074
3093
525331
GCGGATTTGCTGGCAAAGTT
28
80
1242


 


3077
3096
525332
GAGGCGGATTTGCTGGCAAA
38
74
1243


 


3080
3099
525333
CAGGAGGCGGATTTGCTGGC
41
66
1244


 


3083
3102
525334
AGGCAGGAGGCGGATTTGCT
27
55
1245


 


3086
3105
525335
TGGAGGCAGGAGGCGGATTT
13
17
1246


 


3089
3108
525336
TGGTGGAGGCAGGAGGCGGA
7
21
1247


 


3092
3111
525337
GATTGGTGGAGGCAGGAGGC
21
44
1248


 


3095
3114
525338
GGCGATTGGTGGAGGCAGGA
31
65
1249


 


3098
3117
525339
TCTGGCGATTGGTGGAGGCA
15
76
1250


 


3101
3120
525340
CTGTCTGGCGATTGGTGGAG
35
73
1251


 


3104
3123
525341
TTCCTGTCTGGCGATTGGTG
32
72
1252


 


3107
3126
525342
GCCTTCCTGTCTGGCGATTG
28
64
1253


 


3110
3129
525343
GCTGCCTTCCTGTCTGGCGA
25
69
1254


 


3113
3132
525344
TAGGCTGCCTTCCTGTCTGG
32
79
1255


 


3116
3135
525345
GGGTAGGCTGCCTTCCTGTC
35
80
1256


 


3134
3153
525346
TCAAAGGTGGAGACAGCGGG
4
57
1257


 


3137
3156
525347
TTCTCAAAGGTGGAGACAGC
32
72
1258


 


3140
3159
525348
TGTTTCTCAAAGGTGGAGAC
32
66
1259


 


3143
3162
525349
GAGTGTTTCTCAAAGGTGGA
34
63
1260


 


3146
3165
525350
GATGAGTGTTTCTCAAAGGT
35
68
1261


 


3149
3168
525351
GAGGATGAGTGTTTCTCAAA
36
84
1262


 


3152
3171
525352
CCTGAGGATGAGTGTTTCTC
44
77
1263


 


3155
3174
525353
TGGCCTGAGGATGAGTGTTT
32
72
1264


 


3158
3177
525354
GCATGGCCTGAGGATGAGTG
27
73
1265


 


3162
3181
525355
CACTGCATGGCCTGAGGATG
40
69
1266
















TABLE 25







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides targeted


to SEQ ID NO: 1286 (RTS3370 and RTS3372)













Viral
Viral


RTS3370
RTS3372



Start
Stop


%
%
SEQ ID


Site
Site
ISIS No
Sequence
inhibition
inhibition
NO
















85
104
525356
TTCCACTGCATGGCCTGAGG
53
78
1267


 


88
107
525357
GAATTCCACTGCATGGCCTG
44
68
1268


 


91
110
525358
GTGGAATTCCACTGCATGGC
42
80
1269


 


94
113
525359
GTTGTGGAATTCCACTGCAT
45
77
1270


 


97
116
525360
AAGGTTGTGGAATTCCACTG
65
67
1271


 


100
119
525361
TGAAAGGTTGTGGAATTCCA
56
61
1272









Example 12
Dose-Dependent Inhibition of Viral HBV RNA in HepG2.2.15 Cells by MOE Gapmers
Certain gapmers from the study described in Examples 11 and 12 were tested at various doses in human HepG2.2.15 cells. Cells were plated at a density of 28,000 cells per well and transfected using LipofectAMINE 2000® reagent with 5.56 nM, 16.67 nM, 50.0 nM, and 150.0 nM concentrations of antisense oligonucleotide, as specified in Table 26. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.
The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented in Table 26. As illustrated in Table 26, HBV mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.







TABLE 26







Dose-dependent antisense inhibition of HBV


RNA in HepG2.2.15 cells using RTS3370












ISIS No
5.5556 nM
16.6667 nM
50.0 nM
150.0 nM
IC50 (nM)















146785
0
0
14
66
120.8


146786
40
64
78
88
8.5


505314
23
35
58
84
28.8


505339
28
42
62
84
23.2


505347
9
21
45
75
53.5


514469
11
22
69
79
35.4


524493
13
39
56
81
32.8


524540
15
38
54
80
34.0


524617
14
32
78
83
27.1


524619
33
42
60
84
21.3


524634
20
45
63
80
26.3


524641
39
49
62
86
14.9


524698
34
34
49
64
47.4


524699
25
31
44
63
66.1


524706
29
20
36
58
128.8


524709
32
26
48
56
89.1


524718
46
41
61
79
15.8


524731
49
53
68
83
8.1


524734
42
31
35
64
87.2


524767
19
38
62
84
27.8


524768
35
38
62
75
23.5


524769
16
26
61
75
38.1


524806
0
0
0
35
>150.0


524807
3
22
39
74
60.2


524907
22
35
63
80
29.1


524908
25
45
67
78
22.9


524976
7
3
0
16
>150.0


524978
6
0
0
27
>150.0


524979
3
0
11
34
>150.0


524980
18
51
48
59
51.5


524981
16
27
49
61
65.8


524982
21
19
29
54
>150.0


524983
23
40
50
60
53.2


524984
19
25
45
74
50.0


524985
13
19
40
56
107.2


524986
29
48
46
64
39.3


524987
17
0
43
61
102.8


524988
22
39
52
63
47.6


524991
0
7
19
20
>150.0


524997
17
0
1
9
>150.0


524998
1
5
8
34
>150.0


525095
5
0
0
18
>150.0


525100
14
5
14
26
>150.0


525101
0
0
15
19
>150.0


525102
0
0
18
23
>150.0


525103
0
0
3
15
>150.0


525179
18
7
9
18
>150.0


525245
0
0
8
8
>150.0


525247
12
15
16
23
>150.0


525289
1
1
15
30
>150.0


525314
17
0
18
25
>150.0


525324
0
6
13
16
>150.0


525351
28
13
22
30
>150.0









Some of the ISIS-oligonucleotides were also tested using primer probe set RTS3372. The results are presented in Table 27.







TABLE 27







Dose-dependent antisense inhibition of HBV


RNA in HepG2.2.15 cells using RTS3372












ISIS No
5.5556 nM
16.6667 nM
50.0 nM
150.0 nM
IC50 (nM)















146785
0
0
0
51
>150.0


146786
41
68
81
91
7.9


505347
0
13
44
75
59.7


524103
0
0
1
9
>150.0


524245
0
0
6
10
>150.0


524767
18
46
60
85
25.8


524768
34
41
66
79
20.5


524769
12
38
60
77
34.5


524806
0
0
0
0
>150.0


524807
0
9
34
70
78.6


524907
20
41
62
84
26.4


524908
27
45
66
82
21.3


524976
0
0
0
16
>150.0


524978
3
0
0
22
>150.0


524979
0
0
0
33
>150.0


524980
28
51
52
67
30.1


524981
7
29
51
66
55.8


524982
22
29
37
63
83.5


524983
20
51
43
62
50.9


524984
20
30
38
75
51.7


524985
30
33
40
60
83.6


524986
25
51
51
66
33.8


524987
19
0
24
65
157.6


524988
12
41
45
62
59.2


524991
0
0
4
8
>150.0


524997
19
0
0
15
>150.0


524998
0
0
1
42
>150.0


525095
0
0
0
17
>150.0


525100
10
0
4
19
>150.0


525101
10
0
21
25
>150.0


525102
0
0
10
15
>150.0


525247
11
12
15
28
>150.0


525289
0
9
11
33
>150.0


525314
1
0
18
24
>150.0


525324
9
8
15
10
>150.0









Example 13
Dose-Dependent Inhibition of Viral HBV RNA in HepG2.2.15 Cells by MOE Gapmers
Certain gapmers from the studies described above were tested at various doses in human HepG2.2.15 cells. Cells were plated at a density of 30,000 cells per well and transfected using LipofectAMINE 2000® reagent with 7.8125 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125.0 nM and 250.0 nM concentrations of antisense oligonucleotide, as specified in Table 28. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.
The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented in Table 28. As illustrated in Table 28, HBV mRNA levels were reduced in a dose-dependent manner in several antisense oligonucleotide treated cells.







TABLE 28







Dose-dependent antisense inhibition of 


HBV RNA in HepG2.2.15 cells using RTS3370














ISIS
7.8125 
15.625
31.25
62.5
125.0
250.0
IC50


No
nM
nM
nM
nM
nM
nM
(nM)

















146786
0
0
14
49
33
50
161.2


510100
0
17
30
28
44
53
177.8


510106
0
4
0
0
29
0
>250.0


509934
0
0
0
7
16
0
>250.0


510116
0
0
8
21
27
25
>250.0


505347
31
3
30
63
80
81
48.7









Example 14
Antisense Inhibition of HBV Viral mRNA in HepG2 Cells by MOE Gapmers
Antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables. ISIS 146786, 509934, ISIS 509959, and ISIS 510100, from the studies described above, were also included. Cultured HepG2 cells at a density of 28,000 cells per well were transfected using LipofectAMINE2000® with 70 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 (forward sequence CTTGGTCATGGGCCATCAG, designated herein as SEQ ID NO: 2; reverse sequence CGGCTAGGAGTTCCGCAGTA, designated herein as SEQ ID NO: 3; probe sequence TGCGTGGAACCTTTTCGGCTCC, designated herein as SEQ ID NO: 4) was used to measure mRNA levels. Levels were also measured using primer probe set RTS3371 (forward sequence CCAAACCTTCGGACGGAAA, designated herein as SEQ ID NO: 311; reverse sequence TGAGGCCCACTCCCATAGG, designated herein as SEQ ID NO: 312; probe sequence CCCATCATCCTGGGCTTTCGGAAAAT, designated herein as SEQ ID NO: 313). HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells. In some of the assays shown in Tables 32, 35, 42, 45, and 46, the potency of ISIS 146786 was measured in two wells in a single plate. In those cases, the values of inhibition levels in both wells have been presented.
The newly designed chimeric antisense oligonucleotides in Tables below were designed as either 2-9-5 MOE gapmers, 2-9-6 MOE gapmers, 2-10-8 MOE gapmers, 3-9-4 MOE gapmers, 3-9-5 MOE gapmers, 3-10-3 MOE gapmers, 3-10-4 MOE gapmers, 3-10-7 MOE gapmers, 4-9-3 MOE gapmers, 4-9-4 MOE gapmers, 4-10-6 MOE gapmers, 5-9-2 MOE gapmers, 5-9-3 MOE gapmers, 5-10-5 MOE gapmers, 6-9-2 MOE gapmers, 6-10-4 MOE gapmers, 7-10-3 MOE gapmers, or 8-10-2 MOE gapmers. The 2-9-5 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising two and five nucleosides respectively. The 2-9-6 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising two and six nucleosides respectively. The 2-10-8 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising two and eight nucleosides respectively. The 3-9-4 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three and four nucleosides respectively. The 3-9-5 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three and five nucleosides respectively. The 3-10-3 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three nucleosides each. The 3-10-4 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three and four nucleosides respectively. The 3-10-7 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three and seven nucleosides respectively. The 4-9-3 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising four and three nucleosides respectively. The 4-9-4 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising four nucleosides each. The 4-10-6 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising four and six nucleosides respectively. The 5-9-2 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five and two nucleosides respectively. The 5-9-3 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five and three nucleosides respectively. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleosides each. The 6-9-2 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising six and two nucleosides respectively. The 6-10-4 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising six and four nucleosides respectively. The 7-10-3 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising seven and three nucleosides respectively. The 8-10-2 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising eight and two nucleosides respectively. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has an MOE sugar modification. The ‘Motif’ column indicates the motifs with the number of nucleosides in the wings and the gap segment of each of the oligonucleotides. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
“Viral Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. The ‘Motif’ column indicates the gap and wing structure of each gapmer. Each gapmer listed in the Tables is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1). The potency of the newly designed oligonucleotides was compared with ISIS 146786, 509934, ISIS 509959, and ISIS 510100, the information of which have been placed at the top of each table.







TABLE 29







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
5-10-5
50
224


 


411
427
GGCATAGCAGCAGGATG
510100
3-10-4
62
17


 


58
74
CTGGAGCCACCAGCAGG
552276
5-9-3
42
1288


 


59
75
ACTGGAGCCACCAGCAG
552277
5-9-3
46
1289


 


60
76
AACTGGAGCCACCAGCA
552278
5-9-3
31
1290


 


61
77
GAACTGGAGCCACCAGC
552279
5-9-3
41
1291


 


253
269
GAAGTCCACCACGAGTC
552280
5-9-3
5
9


 


254
270
AGAAGTCCACCACGAGT
552281
5-9-3
11
10


 


255
271
GAGAAGTCCACCACGAG
552282
5-9-3
20
11


 


256
272
AGAGAAGTCCACCACGA
552283
5-9-3
28
12


 


411
427
GGCATAGCAGCAGGATG
552230
4-9-4
57
17


 


411
427
GGCATAGCAGCAGGATG
552284
5-9-3
0
17


 


412
428
AGGCATAGCAGCAGGAT
552231
4-9-4
29
18


 


412
428
AGGCATAGCAGCAGGAT
552285
5-9-3
61
18


 


413
429
GAGGCATAGCAGCAGGA
552232
4-9-4
35
19


 


413
429
GAGGCATAGCAGCAGGA
552286
5-9-3
47
19


 


414
430
TGAGGCATAGCAGCAGG
552233
4-9-4
38
21


 


414
430
TGAGGCATAGCAGCAGG
552287
5-9-3
45
21


 


415
431
ATGAGGCATAGCAGCAG
552234
4-9-4
0
23


 


415
431
ATGAGGCATAGCAGCAG
552288
5-9-3
50
23


 


416
432
GATGAGGCATAGCAGCA
552235
4-9-4
0
25


 


416
432
GATGAGGCATAGCAGCA
552289
5-9-3
46
25


 


417
433
AGATGAGGCATAGCAGC
552236
4-9-4
45
27


 


417
433
AGATGAGGCATAGCAGC
552290
5-9-3
41
27


 


418
434
AAGATGAGGCATAGCAG
552237
4-9-4
44
29


 


418
434
AAGATGAGGCATAGCAG
552291
5-9-3
26
29


 


670
686
ACTAGTAAACTGAGCCA
552239
4-9-4
62
1292


 


670
686
ACTAGTAAACTGAGCCA
552293
5-9-3
67
1292


 


671
687
CACTAGTAAACTGAGCC
552240
4-9-4
61
1293


 


671
687
CACTAGTAAACTGAGCC
552294
5-9-3
71
1293


 


672
688
GCACTAGTAAACTGAGC
552241
4-9-4
55
1294


 


672
688
GCACTAGTAAACTGAGC
552295
5-9-3
58
1294


 


687
703
ACCACTGAACAAATGGC
552242
4-9-4
60
40


 


687
703
ACCACTGAACAAATGGC
552296
5-9-3
59
40


 


688
704
AACCACTGAACAAATGG
552243
4-9-4
57
41


 


688
704
AACCACTGAACAAATGG
552297
5-9-3
55
41


 


689
705
GAACCACTGAACAAATG
552244
4-9-4
33
42


 


689
705
GAACCACTGAACAAATG
552298
5-9-3
48
42


 


690
706
CGAACCACTGAACAAAT
552245
4-9-4
48
43


 


690
706
CGAACCACTGAACAAAT
552299
5-9-3
34
43


 


1261
1277
CGCAGTATGGATCGGCA
552246
4-9-4
81
1295


 


1261
1277
CGCAGTATGGATCGGCA
552300
5-9-3
56
1295


 


1262
1278
CCGCAGTATGGATCGGC
552247
4-9-4
87
1296


 


1262
1278
CCGCAGTATGGATCGGC
552301
5-9-3
86
1296


 


1263
1279
TCCGCAGTATGGATCGG
552248
4-9-4
72
1297


 


1263
1279
TCCGCAGTATGGATCGG
552302
5-9-3
77
1297


 


1264
1280
TTCCGCAGTATGGATCG
552249
4-9-4
56
1298


 


1264
1280
TTCCGCAGTATGGATCG
552303
5-9-3
65
1298


 


1265
1281
GTTCCGCAGTATGGATC
552250
4-9-4
52
1299


 


1265
1281
GTTCCGCAGTATGGATC
552304
5-9-3
57
1299


 


1266
1282
AGTTCCGCAGTATGGAT
552251
4-9-4
43
1300


 


1266
1282
AGTTCCGCAGTATGGAT
552305
5-9-3
56
1300


 


1267
1283
GAGTTCCGCAGTATGGA
552252
4-9-4
62
1301


 


1267
1283
GAGTTCCGCAGTATGGA
552306
5-9-3
75
1301


 


1268
1284
GGAGTTCCGCAGTATGG
552253
4-9-4
82
1302


 


1268
1284
GGAGTTCCGCAGTATGG
552307
5-9-3
90
1302


 


1269
1285
AGGAGTTCCGCAGTATG
552254
4-9-4
74
1303


 


1577
1593
AAGCGAAGTGCACACGG
552255
4-9-4
78
1304


 


1578
1594
GAAGCGAAGTGCACACG
552256
4-9-4
65
1305


 


1579
1595
TGAAGCGAAGTGCACAC
552257
4-9-4
62
1306


 


1580
1596
GTGAAGCGAAGTGCACA
552258
4-9-4
72
1307


 


1581
1597
GGTGAAGCGAAGTGCAC
552259
4-9-4
63
1308


 


1582
1598
AGGTGAAGCGAAGTGCA
552260
4-9-4
58
1309


 


1583
1599
GAGGTGAAGCGAAGTGC
552261
4-9-4
63
1310


 


1584
1600
AGAGGTGAAGCGAAGTG
552262
4-9-4
50
1311


 


1585
1601
CAGAGGTGAAGCGAAGT
552263
4-9-4
60
1312


 


1586
1602
GCAGAGGTGAAGCGAAG
552264
4-9-4
52
1313


 


1587
1603
TGCAGAGGTGAAGCGAA
552265
4-9-4
68
1314


 


1588
1604
GTGCAGAGGTGAAGCGA
552266
4-9-4
62
1315


 


1589
1605
CGTGCAGAGGTGAAGCG
552267
4-9-4
58
1316


 


1590
1606
ACGTGCAGAGGTGAAGC
552268
4-9-4
62
1317


 


1778
1794
TTATGCCTACAGCCTCC
552269
4-9-4
52
47


 


1779
1795
TTTATGCCTACAGCCTC
552270
4-9-4
54
49


 


1780
1796
ATTTATGCCTACAGCCT
552271
4-9-4
58
51


 


1781
1797
AATTTATGCCTACAGCC
552272
4-9-4
40
53


 


1782
1798
CAATTTATGCCTACAGC
552273
4-9-4
34
54


 


1783
1799
CCAATTTATGCCTACAG
552274
4-9-4
34
55


 


1784
1800
ACCAATTTATGCCTACA
552275
4-9-4
39
56
















TABLE 30







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
5-10-5
49
224


 


414
429
GAGGCATAGCAGCAGG
509959
3-10-3
43
145


 


411
427
GGCATAGCAGCAGGATG
510100
3-10-4
54
17


 


58
73
TGGAGCCACCAGCAGG
552384
2-9-5
29
1318


 


58
73
TGGAGCCACCAGCAGG
552440
3-9-4
58
1318


 


59
74
CTGGAGCCACCAGCAG
552385
2-9-5
57
1319


 


59
74
CTGGAGCCACCAGCAG
552441
3-9-4
42
1319


 


60
75
ACTGGAGCCACCAGCA
552386
2-9-5
53
1320


 


60
75
ACTGGAGCCACCAGCA
552442
3-9-4
53
1320


 


61
76
AACTGGAGCCACCAGC
552387
2-9-5
48
1321


 


61
76
AACTGGAGCCACCAGC
552443
3-9-4
59
1321


 


62
77
GAACTGGAGCCACCAG
552388
2-9-5
40
86


 


62
77
GAACTGGAGCCACCAG
552444
3-9-4
51
86


 


411
426
GCATAGCAGCAGGATG
552389
2-9-5
39
137


 


411
426
GCATAGCAGCAGGATG
552445
3-9-4
60
137


 


412
427
GGCATAGCAGCAGGAT
552390
2-9-5
52
140


 


412
427
GGCATAGCAGCAGGAT
552446
3-9-4
54
140


 


413
428
AGGCATAGCAGCAGGA
552391
2-9-5
57
143


 


413
428
AGGCATAGCAGCAGGA
552447
3-9-4
54
143


 


414
429
GAGGCATAGCAGCAGG
552392
2-9-5
0
145


 


414
429
GAGGCATAGCAGCAGG
552448
3-9-4
58
145


 


415
430
TGAGGCATAGCAGCAG
552393
2-9-5
59
147


 


415
430
TGAGGCATAGCAGCAG
552449
3-9-4
60
147


 


416
431
ATGAGGCATAGCAGCA
552394
2-9-5
53
149


 


416
431
ATGAGGCATAGCAGCA
552450
3-9-4
53
149


 


417
432
GATGAGGCATAGCAGC
552395
2-9-5
57
151


 


417
432
GATGAGGCATAGCAGC
552451
3-9-4
39
151


 


418
433
AGATGAGGCATAGCAG
552396
2-9-5
62
153


 


418
433
AGATGAGGCATAGCAG
552452
3-9-4
57
153


 


457
473
ACGGGCAACATACCTTG
552238
4-9-4
38
33


 


457
473
ACGGGCAACATACCTTG
552292
5-9-3
48
33


 


457
473
ACGGGCAACATACCTTG
552346
6-9-2
0
33


 


457
472
CGGGCAACATACCTTG
552397
2-9-5
63
167


 


457
472
CGGGCAACATACCTTG
552453
3-9-4
56
167


 


458
473
ACGGGCAACATACCTT
552398
2-9-5
61
168


 


458
473
ACGGGCAACATACCTT
552454
3-9-4
48
168


 


670
685
CTAGTAAACTGAGCCA
552399
2-9-5
52
181


 


671
686
ACTAGTAAACTGAGCC
552400
2-9-5
57
1322


 


672
687
CACTAGTAAACTGAGC
552401
2-9-5
52
1323


 


673
688
GCACTAGTAAACTGAG
552402
2-9-5
54
1324


 


687
702
CCACTGAACAAATGGC
552403
2-9-5
74
188


 


688
703
ACCACTGAACAAATGG
552404
2-9-5
43
190


 


689
704
AACCACTGAACAAATG
552405
2-9-5
15
191


 


690
705
GAACCACTGAACAAAT
552406
2-9-5
37
192


 


691
706
CGAACCACTGAACAAA
552407
2-9-5
37
194


 


1261
1276
GCAGTATGGATCGGCA
552408
2-9-5
76
211


 


1262
1277
CGCAGTATGGATCGGC
552409
2-9-5
76
1325


 


1263
1278
CCGCAGTATGGATCGG
552410
2-9-5
63
1326


 


1264
1279
TCCGCAGTATGGATCG
552411
2-9-5
70
1327


 


1265
1280
TTCCGCAGTATGGATC
552412
2-9-5
62
1328


 


1266
1281
GTTCCGCAGTATGGAT
552413
2-9-5
56
1329


 


1267
1282
AGTTCCGCAGTATGGA
552414
2-9-5
63
1330


 


1268
1283
GAGTTCCGCAGTATGG
552415
2-9-5
52
1331


 


1269
1284
GGAGTTCCGCAGTATG
552416
2-9-5
67
1332


 


1270
1285
AGGAGTTCCGCAGTAT
552417
2-9-5
50
1333


 


1577
1592
AGCGAAGTGCACACGG
552418
2-9-5
79
1334


 


1578
1593
AAGCGAAGTGCACACG
552419
2-9-5
70
1335


 


1579
1594
GAAGCGAAGTGCACAC
552420
2-9-5
71
1336


 


1580
1595
TGAAGCGAAGTGCACA
552421
2-9-5
69
1337


 


1581
1596
GTGAAGCGAAGTGCAC
552422
2-9-5
68
1338


 


1582
1597
GGTGAAGCGAAGTGCA
552423
2-9-5
65
1339


 


1583
1598
AGGTGAAGCGAAGTGC
552424
2-9-5
70
1340


 


1584
1599
GAGGTGAAGCGAAGTG
552425
2-9-5
51
1341


 


1585
1600
AGAGGTGAAGCGAAGT
552426
2-9-5
40
1342


 


1586
1601
CAGAGGTGAAGCGAAG
552427
2-9-5
35
1343


 


1587
1602
GCAGAGGTGAAGCGAA
552428
2-9-5
58
1344


 


1588
1603
TGCAGAGGTGAAGCGA
552429
2-9-5
46
1345


 


1589
1604
GTGCAGAGGTGAAGCG
552430
2-9-5
53
1346


 


1590
1605
CGTGCAGAGGTGAAGC
552431
2-9-5
51
1347


 


1591
1606
ACGTGCAGAGGTGAAG
552432
2-9-5
57
1348


 


1778
1793
TATGCCTACAGCCTCC
552433
2-9-5
54
230


 


1779
1794
TTATGCCTACAGCCTC
552434
2-9-5
44
231


 


1780
1795
TTTATGCCTACAGCCT
552435
2-9-5
46
232


 


1781
1796
ATTTATGCCTACAGCC
552436
2-9-5
36
233


 


1782
1797
AATTTATGCCTACAGC
552437
2-9-5
27
234


 


1783
1798
CAATTTATGCCTACAG
552438
2-9-5
27
235


 


1784
1799
CCAATTTATGCCTACA
552439
2-9-5
13
236
















TABLE 31







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
5-10-5
35
224


 


414
429
GAGGCATAGCAGCAGG
509959
3-10-3
52
145


 


58
73
TGGAGCCACCAGCAGG
552496
4-9-3
47
1318


 


59
74
CTGGAGCCACCAGCAG
552497
4-9-3
57
1319


 


60
75
ACTGGAGCCACCAGCA
552498
4-9-3
45
1320


 


61
76
AACTGGAGCCACCAGC
552499
4-9-3
52
1321


 


62
77
GAACTGGAGCCACCAG
552500
4-9-3
46
86


 


411
426
GCATAGCAGCAGGATG
552501
4-9-3
44
137


 


412
427
GGCATAGCAGCAGGAT
552502
4-9-3
57
140


 


413
428
AGGCATAGCAGCAGGA
552503
4-9-3
52
143


 


414
429
GAGGCATAGCAGCAGG
552504
4-9-3
45
145


 


415
430
TGAGGCATAGCAGCAG
552505
4-9-3
56
147


 


416
431
ATGAGGCATAGCAGCA
552506
4-9-3
54
149


 


417
432
GATGAGGCATAGCAGC
552507
4-9-3
34
151


 


418
433
AGATGAGGCATAGCAG
552508
4-9-3
34
153


 


457
472
CGGGCAACATACCTTG
552509
4-9-3
48
167


 


458
473
ACGGGCAACATACCTT
552510
4-9-3
50
168


 


670
685
CTAGTAAACTGAGCCA
552455
3-9-4
66
181


 


670
685
CTAGTAAACTGAGCCA
552511
4-9-3
66
181


 


671
686
ACTAGTAAACTGAGCC
552456
3-9-4
64
1322


 


671
686
ACTAGTAAACTGAGCC
552512
4-9-3
62
1322


 


672
687
CACTAGTAAACTGAGC
552457
3-9-4
14
1323


 


672
687
CACTAGTAAACTGAGC
552513
4-9-3
56
1323


 


673
688
GCACTAGTAAACTGAG
552458
3-9-4
59
1324


 


673
688
GCACTAGTAAACTGAG
552514
4-9-3
52
1324


 


687
702
CCACTGAACAAATGGC
552459
3-9-4
69
188


 


687
702
CCACTGAACAAATGGC
552515
4-9-3
57
188


 


688
703
ACCACTGAACAAATGG
552460
3-9-4
0
190


 


688
703
ACCACTGAACAAATGG
552516
4-9-3
54
190


 


689
704
AACCACTGAACAAATG
552461
3-9-4
20
191


 


689
704
AACCACTGAACAAATG
552517
4-9-3
52
191


 


690
705
GAACCACTGAACAAAT
552462
3-9-4
46
192


 


690
705
GAACCACTGAACAAAT
552518
4-9-3
34
192


 


691
706
CGAACCACTGAACAAA
552463
3-9-4
48
194


 


691
706
CGAACCACTGAACAAA
552519
4-9-3
44
194


 


1261
1276
GCAGTATGGATCGGCA
552464
3-9-4
81
211


 


1261
1276
GCAGTATGGATCGGCA
552520
4-9-3
69
211


 


1262
1277
CGCAGTATGGATCGGC
552465
3-9-4
84
1325


 


1262
1277
CGCAGTATGGATCGGC
552521
4-9-3
80
1325


 


1263
1278
CCGCAGTATGGATCGG
552466
3-9-4
75
1326


 


1263
1278
CCGCAGTATGGATCGG
552522
4-9-3
76
1326


 


1264
1279
TCCGCAGTATGGATCG
552467
3-9-4
65
1327


 


1264
1279
TCCGCAGTATGGATCG
552523
4-9-3
71
1327


 


1265
1280
TTCCGCAGTATGGATC
552468
3-9-4
53
1328


 


1265
1280
TTCCGCAGTATGGATC
552524
4-9-3
43
1328


 


1266
1281
GTTCCGCAGTATGGAT
552469
3-9-4
51
1329


 


1266
1281
GTTCCGCAGTATGGAT
552525
4-9-3
57
1329


 


1267
1282
AGTTCCGCAGTATGGA
552470
3-9-4
46
1330


 


1267
1282
AGTTCCGCAGTATGGA
552526
4-9-3
60
1330


 


1268
1283
GAGTTCCGCAGTATGG
552471
3-9-4
54
1331


 


1268
1283
GAGTTCCGCAGTATGG
552527
4-9-3
72
1331


 


1269
1284
GGAGTTCCGCAGTATG
552472
3-9-4
78
1332


 


1269
1284
GGAGTTCCGCAGTATG
552528
4-9-3
78
1332


 


1270
1285
AGGAGTTCCGCAGTAT
552473
3-9-4
67
1333


 


1270
1285
AGGAGTTCCGCAGTAT
552529
4-9-3
77
1333


 


1577
1592
AGCGAAGTGCACACGG
552474
3-9-4
79
1334


 


1577
1592
AGCGAAGTGCACACGG
552530
4-9-3
78
1334


 


1578
1593
AAGCGAAGTGCACACG
552475
3-9-4
74
1335


 


1578
1593
AAGCGAAGTGCACACG
552531
4-9-3
68
1335


 


1579
1594
GAAGCGAAGTGCACAC
552476
3-9-4
52
1336


 


1580
1595
TGAAGCGAAGTGCACA
552477
3-9-4
76
1337


 


1581
1596
GTGAAGCGAAGTGCAC
552478
3-9-4
70
1338


 


1582
1597
GGTGAAGCGAAGTGCA
552479
3-9-4
67
1339


 


1583
1598
AGGTGAAGCGAAGTGC
552480
3-9-4
68
1340


 


1584
1599
GAGGTGAAGCGAAGTG
552481
3-9-4
57
1341


 


1585
1600
AGAGGTGAAGCGAAGT
552482
3-9-4
51
1342


 


1586
1601
CAGAGGTGAAGCGAAG
552483
3-9-4
48
1343


 


1587
1602
GCAGAGGTGAAGCGAA
552484
3-9-4
58
1344


 


1588
1603
TGCAGAGGTGAAGCGA
552485
3-9-4
51
1345


 


1589
1604
GTGCAGAGGTGAAGCG
552486
3-9-4
55
1346


 


1590
1605
CGTGCAGAGGTGAAGC
552487
3-9-4
62
1347


 


1591
1606
ACGTGCAGAGGTGAAG
552488
3-9-4
51
1348


 


1778
1793
TATGCCTACAGCCTCC
552489
3-9-4
49
230


 


1779
1794
TTATGCCTACAGCCTC
552490
3-9-4
51
231


 


1780
1795
TTTATGCCTACAGCCT
552491
3-9-4
51
232


 


1781
1796
ATTTATGCCTACAGCC
552492
3-9-4
38
233


 


1782
1797
AATTTATGCCTACAGC
552493
3-9-4
52
234


 


1783
1798
CAATTTATGCCTACAG
552494
3-9-4
17
235


 


1784
1799
CCAATTTATGCCTACA
552495
3-9-4
49
236
















TABLE 32







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
5-10-5
43
224







52



 


414
429
GAGGCATAGCAGCAGG
509959
3-10-3
38
145


 


58
73
TGGAGCCACCAGCAGG
552552
5-9-2
33
1318


 


59
74
CTGGAGCCACCAGCAG
552553
5-9-2
46
1319


 


60
75
ACTGGAGCCACCAGCA
552554
5-9-2
54
1320


 


61
76
AACTGGAGCCACCAGC
552555
5-9-2
50
1321


 


62
77
GAACTGGAGCCACCAG
552556
5-9-2
46
86


 


411
426
GCATAGCAGCAGGATG
552557
5-9-2
57
137


 


412
427
GGCATAGCAGCAGGAT
552558
5-9-2
55
140


 


413
428
AGGCATAGCAGCAGGA
552559
5-9-2
66
143


 


414
429
GAGGCATAGCAGCAGG
552560
5-9-2
44
145


 


415
430
TGAGGCATAGCAGCAG
552561
5-9-2
48
147


 


416
431
ATGAGGCATAGCAGCA
552562
5-9-2
52
149


 


417
432
GATGAGGCATAGCAGC
552563
5-9-2
45
151


 


418
433
AGATGAGGCATAGCAG
552564
5-9-2
41
153


 


457
472
CGGGCAACATACCTTG
552565
5-9-2
54
167


 


458
473
ACGGGCAACATACCTT
552566
5-9-2
56
168


 


670
685
CTAGTAAACTGAGCCA
552567
5-9-2
71
181


 


671
686
ACTAGTAAACTGAGCC
552568
5-9-2
64
1322


 


672
687
CACTAGTAAACTGAGC
552569
5-9-2
59
1323


 


673
688
GCACTAGTAAACTGAG
552570
5-9-2
60
1324


 


687
702
CCACTGAACAAATGGC
552571
5-9-2
55
188


 


688
703
ACCACTGAACAAATGG
552572
5-9-2
60
190


 


689
704
AACCACTGAACAAATG
552573
5-9-2
24
191


 


690
705
GAACCACTGAACAAAT
552574
5-9-2
34
192


 


691
706
CGAACCACTGAACAAA
552575
5-9-2
36
194


 


1261
1276
GCAGTATGGATCGGCA
552576
5-9-2
67
211


 


1262
1277
CGCAGTATGGATCGGC
552577
5-9-2
64
1325


 


1263
1278
CCGCAGTATGGATCGG
552578
5-9-2
75
1326


 


1264
1279
TCCGCAGTATGGATCG
552579
5-9-2
75
1327


 


1265
1280
TTCCGCAGTATGGATC
552580
5-9-2
59
1328


 


1266
1281
GTTCCGCAGTATGGAT
552581
5-9-2
54
1329


 


1267
1282
AGTTCCGCAGTATGGA
552582
5-9-2
61
1330


 


1268
1283
GAGTTCCGCAGTATGG
552583
5-9-2
69
1331


 


1269
1284
GGAGTTCCGCAGTATG
552584
5-9-2
74
1332


 


1270
1285
AGGAGTTCCGCAGTAT
552585
5-9-2
62
1333


 


1577
1592
AGCGAAGTGCACACGG
552586
5-9-2
79
1334


 


1578
1593
AAGCGAAGTGCACACG
552587
5-9-2
71
1335


 


1579
1594
GAAGCGAAGTGCACAC
552532
4-9-3
48
1336


 


1579
1594
GAAGCGAAGTGCACAC
552588
5-9-2
70
1336


 


1580
1595
TGAAGCGAAGTGCACA
552533
4-9-3
43
1337


 


1580
1595
TGAAGCGAAGTGCACA
552589
5-9-2
59
1337


 


1581
1596
GTGAAGCGAAGTGCAC
552534
4-9-3
62
1338


 


1581
1596
GTGAAGCGAAGTGCAC
552590
5-9-2
70
1338


 


1582
1597
GGTGAAGCGAAGTGCA
552535
4-9-3
55
1339


 


1582
1597
GGTGAAGCGAAGTGCA
552591
5-9-2
51
1339


 


1583
1598
AGGTGAAGCGAAGTGC
552536
4-9-3
3
1340


 


1583
1598
AGGTGAAGCGAAGTGC
552592
5-9-2
50
1340


 


1584
1599
GAGGTGAAGCGAAGTG
552537
4-9-3
14
1341


 


1584
1599
GAGGTGAAGCGAAGTG
552593
5-9-2
46
1341


 


1585
1600
AGAGGTGAAGCGAAGT
552538
4-9-3
52
1342


 


1585
1600
AGAGGTGAAGCGAAGT
552594
5-9-2
55
1342


 


1586
1601
CAGAGGTGAAGCGAAG
552539
4-9-3
47
1343


 


1586
1601
CAGAGGTGAAGCGAAG
552595
5-9-2
60
1343


 


1587
1602
GCAGAGGTGAAGCGAA
552540
4-9-3
60
1344


 


1587
1602
GCAGAGGTGAAGCGAA
552596
5-9-2
63
1344


 


1588
1603
TGCAGAGGTGAAGCGA
552541
4-9-3
60
1345


 


1588
1603
TGCAGAGGTGAAGCGA
552597
5-9-2
61
1345


 


1589
1604
GTGCAGAGGTGAAGCG
552542
4-9-3
64
1346


 


1589
1604
GTGCAGAGGTGAAGCG
552598
5-9-2
57
1346


 


1590
1605
CGTGCAGAGGTGAAGC
552543
4-9-3
46
1347


 


1590
1605
CGTGCAGAGGTGAAGC
552600
5-9-2
59
1347


 


1591
1606
ACGTGCAGAGGTGAAG
552544
4-9-3
53
1348


 


1591
1606
ACGTGCAGAGGTGAAG
552602
5-9-2
6
1348


 


1778
1793
TATGCCTACAGCCTCC
552545
4-9-3
33
230


 


1778
1793
TATGCCTACAGCCTCC
552604
5-9-2
47
230


 


1779
1794
TTATGCCTACAGCCTC
552546
4-9-3
42
231


 


1779
1794
TTATGCCTACAGCCTC
552606
5-9-2
53
231


 


1780
1795
TTTATGCCTACAGCCT
552547
4-9-3
51
232


 


1780
1795
TTTATGCCTACAGCCT
552608
5-9-2
53
232


 


1781
1796
ATTTATGCCTACAGCC
552548
4-9-3
52
233


 


1781
1796
ATTTATGCCTACAGCC
552610
5-9-2
47
233


 


1782
1797
AATTTATGCCTACAGC
552549
4-9-3
38
234


 


1782
1797
AATTTATGCCTACAGC
552612
5-9-2
39
234


 


1783
1798
CAATTTATGCCTACAG
552550
4-9-3
19
235


 


1783
1798
CAATTTATGCCTACAG
552614
5-9-2
24
235


 


1784
1799
CCAATTTATGCCTACA
552551
4-9-3
24
236


 


1784
1799
CCAATTTATGCCTACA
552616
5-9-2
15
236
















TABLE 33







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
5-10-5
51
224


 


1780
1799
CCAATTTATGCCTACAGCCT
509934
5-10-5
76
50


 


58
77
GAACTGGAGCCACCAGCAGG
552007
6-10-4
61
83


 


58
77
GAACTGGAGCCACCAGCAGG
552039
7-10-3
84
83


 


253
272
AGAGAAGTCCACCACGAGTC
552008
6-10-4
48
103


 


253
272
AGAGAAGTCCACCACGAGTC
552040
7-10-3
48
103


 


411
430
TGAGGCATAGCAGCAGGATG
552009
6-10-4
77
136


 


411
430
TGAGGCATAGCAGCAGGATG
552041
7-10-3
73
136


 


412
431
ATGAGGCATAGCAGCAGGAT
552010
6-10-4
63
139


 


412
431
ATGAGGCATAGCAGCAGGAT
552042
7-10-3
66
139


 


413
432
GATGAGGCATAGCAGCAGGA
552011
6-10-4
52
142


 


413
432
GATGAGGCATAGCAGCAGGA
552043
7-10-3
54
142


 


414
433
AGATGAGGCATAGCAGCAGG
552012
6-10-4
73
20


 


414
433
AGATGAGGCATAGCAGCAGG
552044
7-10-3
86
20


 


415
434
AAGATGAGGCATAGCAGCAG
552013
6-10-4
73
22


 


415
434
AAGATGAGGCATAGCAGCAG
552045
7-10-3
65
22


 


416
435
GAAGATGAGGCATAGCAGCA
552014
6-10-4
76
24


 


416
435
GAAGATGAGGCATAGCAGCA
552046
7-10-3
93
24


 


417
436
AGAAGATGAGGCATAGCAGC
552015
6-10-4
70
26


 


417
436
AGAAGATGAGGCATAGCAGC
552047
7-10-3
77
26


 


418
437
AAGAAGATGAGGCATAGCAG
552016
6-10-4
61
28


 


418
437
AAGAAGATGAGGCATAGCAG
552048
7-10-3
66
28


 


687
706
CGAACCACTGAACAAATGGC
552017
6-10-4
73
39


 


687
706
CGAACCACTGAACAAATGGC
552049
7-10-3
73
39


 


1261
1280
TTCCGCAGTATGGATCGGCA
552018
6-10-4
98
719


 


1261
1280
TTCCGCAGTATGGATCGGCA
552050
7-10-3
98
719


 


1262
1281
GTTCCGCAGTATGGATCGGC
552019
6-10-4
98
212


 


1262
1281
GTTCCGCAGTATGGATCGGC
552051
7-10-3
99
212


 


1263
1282
AGTTCCGCAGTATGGATCGG
551986
4-10-6
92
720


 


1263
1282
AGTTCCGCAGTATGGATCGG
552020
6-10-4
97
720


 


1263
1282
AGTTCCGCAGTATGGATCGG
552052
7-10-3
98
720


 


1264
1283
GAGTTCCGCAGTATGGATCG
551987
4-10-6
95
721


 


1264
1283
GAGTTCCGCAGTATGGATCG
552021
6-10-4
97
721


 


1264
1283
GAGTTCCGCAGTATGGATCG
552053
7-10-3
98
721


 


1265
1284
GGAGTTCCGCAGTATGGATC
551988
4-10-6
50
1349


 


1265
1284
GGAGTTCCGCAGTATGGATC
552005
5-10-5
99
1349


 


1265
1284
GGAGTTCCGCAGTATGGATC
552022
6-10-4
99
1349


 


1265
1284
GGAGTTCCGCAGTATGGATC
552054
7-10-3
99
1349


 


1266
1285
AGGAGTTCCGCAGTATGGAT
551989
4-10-6
96
722


 


1266
1285
AGGAGTTCCGCAGTATGGAT
552023
6-10-4
99
722


 


1266
1285
AGGAGTTCCGCAGTATGGAT
552055
7-10-3
98
722


 


1577
1596
GTGAAGCGAAGTGCACACGG
551990
4-10-6
86
224


 


1577
1596
GTGAAGCGAAGTGCACACGG
552024
6-10-4
89
224


 


1577
1596
GTGAAGCGAAGTGCACACGG
552056
7-10-3
88
224


 


1578
1597
GGTGAAGCGAAGTGCACACG
551991
4-10-6
0
801


 


1578
1597
GGTGAAGCGAAGTGCACACG
552025
6-10-4
90
801


 


1578
1597
GGTGAAGCGAAGTGCACACG
552057
7-10-3
92
801


 


1579
1598
AGGTGAAGCGAAGTGCACAC
551992
4-10-6
72
802


 


1579
1598
AGGTGAAGCGAAGTGCACAC
552026
6-10-4
88
802


 


1579
1598
AGGTGAAGCGAAGTGCACAC
552058
7-10-3
86
802


 


1580
1599
GAGGTGAAGCGAAGTGCACA
551993
4-10-6
82
225


 


1580
1599
GAGGTGAAGCGAAGTGCACA
552027
6-10-4
87
225


 


1580
1599
GAGGTGAAGCGAAGTGCACA
552059
7-10-3
88
225


 


1581
1600
AGAGGTGAAGCGAAGTGCAC
551994
4-10-6
85
804


 


1581
1600
AGAGGTGAAGCGAAGTGCAC
552028
6-10-4
83
804


 


1581
1600
AGAGGTGAAGCGAAGTGCAC
552060
7-10-3
82
804


 


1582
1601
CAGAGGTGAAGCGAAGTGCA
551995
4-10-6
84
805


 


1582
1601
CAGAGGTGAAGCGAAGTGCA
552029
6-10-4
88
805


 


1582
1601
CAGAGGTGAAGCGAAGTGCA
552061
7-10-3
85
805


 


1583
1602
GCAGAGGTGAAGCGAAGTGC
551996
4-10-6
87
226


 


1583
1602
GCAGAGGTGAAGCGAAGTGC
552030
6-10-4
88
226


 


1583
1602
GCAGAGGTGAAGCGAAGTGC
552062
7-10-3
85
226


 


1584
1603
TGCAGAGGTGAAGCGAAGTG
551997
4-10-6
83
806


 


1584
1603
TGCAGAGGTGAAGCGAAGTG
552031
6-10-4
82
806


 


1585
1604
GTGCAGAGGTGAAGCGAAGT
551998
4-10-6
85
807


 


1585
1604
GTGCAGAGGTGAAGCGAAGT
552032
6-10-4
87
807


 


1586
1605
CGTGCAGAGGTGAAGCGAAG
551999
4-10-6
82
227


 


1586
1605
CGTGCAGAGGTGAAGCGAAG
552033
6-10-4
87
227


 


1587
1606
ACGTGCAGAGGTGAAGCGAA
552000
4-10-6
83
1350


 


1587
1606
ACGTGCAGAGGTGAAGCGAA
552006
5-10-5
88
1350


 


1587
1606
ACGTGCAGAGGTGAAGCGAA
552034
6-10-4
89
1350


 


1778
1797
AATTTATGCCTACAGCCTCC
552001
4-10-6
65
46


 


1778
1797
AATTTATGCCTACAGCCTCC
552035
6-10-4
60
46


 


1779
1798
CAATTTATGCCTACAGCCTC
552002
4-10-6
63
48


 


1779
1798
CAATTTATGCCTACAGCCTC
552036
6-10-4
65
48


 


1780
1799
CCAATTTATGCCTACAGCCT
552003
4-10-6
65
50


 


1780
1799
CCAATTTATGCCTACAGCCT
552037
6-10-4
58
50


 


1781
1800
ACCAATTTATGCCTACAGCC
552004
4-10-6
58
52


 


1781
1800
ACCAATTTATGCCTACAGCC
552038
6-10-4
70
52
















TABLE 34







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
5-10-5
64
224


 


411
427
GGCATAGCAGCAGGATG
510100
3-10-4
62
17


 


58
74
CTGGAGCCACCAGCAGG
552168
3-9-5
79
1288


 


58
74
CTGGAGCCACCAGCAGG
552222
4-9-4
79
1288


 


59
75
ACTGGAGCCACCAGCAG
552169
3-9-5
67
1289


 


59
75
ACTGGAGCCACCAGCAG
552223
4-9-4
40
1289


 


60
76
AACTGGAGCCACCAGCA
552170
3-9-5
69
1290


 


60
76
AACTGGAGCCACCAGCA
552224
4-9-4
64
1290


 


61
77
GAACTGGAGCCACCAGC
552171
3-9-5
65
1291


 


61
77
GAACTGGAGCCACCAGC
552225
4-9-4
69
1291


 


253
269
GAAGTCCACCACGAGTC
552172
3-9-5
33
9


 


253
269
GAAGTCCACCACGAGTC
552226
4-9-4
48
9


 


254
270
AGAAGTCCACCACGAGT
552173
3-9-5
41
10


 


254
270
AGAAGTCCACCACGAGT
552227
4-9-4
32
10


 


255
271
GAGAAGTCCACCACGAG
552174
3-9-5
31
11


 


255
271
GAGAAGTCCACCACGAG
552228
4-9-4
42
11


 


256
272
AGAGAAGTCCACCACGA
552175
3-9-5
59
12


 


411
427
GGCATAGCAGCAGGATG
552176
3-9-5
68
17


 


412
428
AGGCATAGCAGCAGGAT
552177
3-9-5
55
18


 


413
429
GAGGCATAGCAGCAGGA
552178
3-9-5
66
19


 


414
430
TGAGGCATAGCAGCAGG
552179
3-9-5
70
21


 


415
431
ATGAGGCATAGCAGCAG
552180
3-9-5
66
23


 


416
432
GATGAGGCATAGCAGCA
552181
3-9-5
51
25


 


417
433
AGATGAGGCATAGCAGC
552182
3-9-5
69
27


 


418
434
AAGATGAGGCATAGCAG
552183
3-9-5
69
29


 


457
473
ACGGGCAACATACCTTG
552184
3-9-5
43
33


 


670
686
ACTAGTAAACTGAGCCA
552185
3-9-5
66
1292


 


671
687
CACTAGTAAACTGAGCC
552186
3-9-5
54
1293


 


672
688
GCACTAGTAAACTGAGC
552187
3-9-5
74
1294


 


687
703
ACCACTGAACAAATGGC
552188
3-9-5
78
40


 


688
704
AACCACTGAACAAATGG
552189
3-9-5
57
41


 


689
705
GAACCACTGAACAAATG
552190
3-9-5
39
42


 


690
706
CGAACCACTGAACAAAT
552191
3-9-5
60
43


 


1261
1277
CGCAGTATGGATCGGCA
552192
3-9-5
85
1295


 


1262
1278
CCGCAGTATGGATCGGC
552193
3-9-5
86
1296


 


1263
1279
TCCGCAGTATGGATCGG
552194
3-9-5
68
1297


 


1264
1280
TTCCGCAGTATGGATCG
552195
3-9-5
73
1298


 


1265
1281
GTTCCGCAGTATGGATC
552196
3-9-5
60
1299


 


1266
1282
AGTTCCGCAGTATGGAT
552197
3-9-5
60
1300


 


1267
1283
GAGTTCCGCAGTATGGA
552198
3-9-5
61
1301


 


1268
1284
GGAGTTCCGCAGTATGG
552199
3-9-5
89
1302


 


1269
1285
AGGAGTTCCGCAGTATG
552200
3-9-5
85
1303


 


1577
1593
AAGCGAAGTGCACACGG
552201
3-9-5
81
1304


 


1578
1594
GAAGCGAAGTGCACACG
552202
3-9-5
76
1305


 


1579
1595
TGAAGCGAAGTGCACAC
552203
3-9-5
74
1306


 


1580
1596
GTGAAGCGAAGTGCACA
552204
3-9-5
71
1307


 


1581
1597
GGTGAAGCGAAGTGCAC
552151
2-9-6
77
1308


 


1581
1597
GGTGAAGCGAAGTGCAC
552205
3-9-5
78
1308


 


1582
1598
AGGTGAAGCGAAGTGCA
552152
2-9-6
72
1309


 


1582
1598
AGGTGAAGCGAAGTGCA
552206
3-9-5
77
1309


 


1583
1599
GAGGTGAAGCGAAGTGC
552153
2-9-6
67
1310


 


1583
1599
GAGGTGAAGCGAAGTGC
552207
3-9-5
81
1310


 


1584
1600
AGAGGTGAAGCGAAGTG
552154
2-9-6
56
1311


 


1584
1600
AGAGGTGAAGCGAAGTG
552208
3-9-5
70
1311


 


1585
1601
CAGAGGTGAAGCGAAGT
552155
2-9-6
61
1312


 


1585
1601
CAGAGGTGAAGCGAAGT
552209
3-9-5
63
1312


 


1586
1602
GCAGAGGTGAAGCGAAG
552156
2-9-6
20
1313


 


1586
1602
GCAGAGGTGAAGCGAAG
552210
3-9-5
75
1313


 


1587
1603
TGCAGAGGTGAAGCGAA
552157
2-9-6
39
1314


 


1587
1603
TGCAGAGGTGAAGCGAA
552211
3-9-5
75
1314


 


1588
1604
GTGCAGAGGTGAAGCGA
552158
2-9-6
70
1315


 


1588
1604
GTGCAGAGGTGAAGCGA
552212
3-9-5
67
1315


 


1589
1605
CGTGCAGAGGTGAAGCG
552159
2-9-6
74
1316


 


1589
1605
CGTGCAGAGGTGAAGCG
552213
3-9-5
70
1316


 


1590
1606
ACGTGCAGAGGTGAAGC
552160
2-9-6
78
1317


 


1590
1606
ACGTGCAGAGGTGAAGC
552214
3-9-5
79
1317


 


1778
1794
TTATGCCTACAGCCTCC
552161
2-9-6
56
47


 


1778
1794
TTATGCCTACAGCCTCC
552215
3-9-5
61
47


 


1779
1795
TTTATGCCTACAGCCTC
552162
2-9-6
64
49


 


1779
1795
TTTATGCCTACAGCCTC
552216
3-9-5
62
49


 


1780
1796
ATTTATGCCTACAGCCT
552163
2-9-6
71
51


 


1780
1796
ATTTATGCCTACAGCCT
552217
3-9-5
58
51


 


1781
1797
AATTTATGCCTACAGCC
552164
2-9-6
52
53


 


1781
1797
AATTTATGCCTACAGCC
552218
3-9-5
56
53


 


1782
1798
CAATTTATGCCTACAGC
552165
2-9-6
53
54


 


1782
1798
CAATTTATGCCTACAGC
552219
3-9-5
33
54


 


1783
1799
CCAATTTATGCCTACAG
552166
2-9-6
41
55


 


1783
1799
CCAATTTATGCCTACAG
552220
3-9-5
53
55


 


1784
1800
ACCAATTTATGCCTACA
552167
2-9-6
54
56


 


1784
1800
ACCAATTTATGCCTACA
552221
3-9-5
31
56
















TABLE 35







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
5-10-5
60
224







85



 


1780
1799
CCAATTTATGCCTACAGCCT
509934
5-10-5
76
50


 


411
427
GGCATAGCAGCAGGATG
510100
3-10-4
73
17


 


58
77
GAACTGGAGCCACCAGCAGG
552071
8-10-2
79
83


 


58
74
CTGGAGCCACCAGCAGG
552114
2-9-6
66
1288


 


59
75
ACTGGAGCCACCAGCAG
552115
2-9-6
70
1289


 


60
76
AACTGGAGCCACCAGCA
552116
2-9-6
68
1290


 


61
77
GAACTGGAGCCACCAGC
552117
2-9-6
70
1291


 


253
272
AGAGAAGTCCACCACGAGTC
552072
8-10-2
50
103


 


253
269
GAAGTCCACCACGAGTC
552118
2-9-6
66
9


 


254
270
AGAAGTCCACCACGAGT
552119
2-9-6
62
10


 


255
271
GAGAAGTCCACCACGAG
552120
2-9-6
35
11


 


256
272
AGAGAAGTCCACCACGA
552121
2-9-6
39
12


 


411
430
TGAGGCATAGCAGCAGGATG
552073
8-10-2
80
136


 


411
427
GGCATAGCAGCAGGATG
552122
2-9-6
55
17


 


412
431
ATGAGGCATAGCAGCAGGAT
552074
8-10-2
73
139


 


412
428
AGGCATAGCAGCAGGAT
552123
2-9-6
75
18


 


413
432
GATGAGGCATAGCAGCAGGA
552075
8-10-2
78
142


 


413
429
GAGGCATAGCAGCAGGA
552124
2-9-6
64
19


 


414
433
AGATGAGGCATAGCAGCAGG
552076
8-10-2
70
20


 


414
430
TGAGGCATAGCAGCAGG
552125
2-9-6
73
21


 


415
434
AAGATGAGGCATAGCAGCAG
552077
8-10-2
83
22


 


415
431
ATGAGGCATAGCAGCAG
552126
2-9-6
64
23


 


416
435
GAAGATGAGGCATAGCAGCA
552078
8-10-2
80
24


 


416
432
GATGAGGCATAGCAGCA
552127
2-9-6
72
25


 


417
436
AGAAGATGAGGCATAGCAGC
552079
8-10-2
86
26


 


417
433
AGATGAGGCATAGCAGC
552128
2-9-6
76
27


 


418
437
AAGAAGATGAGGCATAGCAG
552080
8-10-2
83
28


 


418
434
AAGATGAGGCATAGCAG
552129
2-9-6
72
29


 


670
686
ACTAGTAAACTGAGCCA
552131
2-9-6
61
1292


 


671
687
CACTAGTAAACTGAGCC
552132
2-9-6
73
1293


 


672
688
GCACTAGTAAACTGAGC
552133
2-9-6
75
1294


 


687
706
CGAACCACTGAACAAATGGC
552081
8-10-2
76
39


 


687
703
ACCACTGAACAAATGGC
552134
2-9-6
58
40


 


688
704
AACCACTGAACAAATGG
552135
2-9-6
67
41


 


689
705
GAACCACTGAACAAATG
552136
2-9-6
65
42


 


690
706
CGAACCACTGAACAAAT
552137
2-9-6
55
43


 


1261
1280
TTCCGCAGTATGGATCGGCA
552082
8-10-2
98
719


 


1261
1277
CGCAGTATGGATCGGCA
552138
2-9-6
82
1295


 


1262
1281
GTTCCGCAGTATGGATCGGC
552083
8-10-2
99
212


 


1262
1278
CCGCAGTATGGATCGGC
552139
2-9-6
86
1296


 


1263
1282
AGTTCCGCAGTATGGATCGG
552084
8-10-2
99
720


 


1263
1279
TCCGCAGTATGGATCGG
552140
2-9-6
74
1297


 


1264
1283
GAGTTCCGCAGTATGGATCG
552085
8-10-2
100
721


 


1264
1280
TTCCGCAGTATGGATCG
552141
2-9-6
67
1298


 


1265
1284
GGAGTTCCGCAGTATGGATC
552086
8-10-2
100
1349


 


1265
1281
GTTCCGCAGTATGGATC
552142
2-9-6
45
1299


 


1266
1285
AGGAGTTCCGCAGTATGGAT
552087
8-10-2
100
722


 


1266
1282
AGTTCCGCAGTATGGAT
552143
2-9-6
68
1300


 


1267
1283
GAGTTCCGCAGTATGGA
552144
2-9-6
78
1301


 


1268
1284
GGAGTTCCGCAGTATGG
552145
2-9-6
88
1302


 


1269
1285
AGGAGTTCCGCAGTATG
552146
2-9-6
81
1303


 


1577
1596
GTGAAGCGAAGTGCACACGG
552088
8-10-2
95
224


 


1577
1593
AAGCGAAGTGCACACGG
552147
2-9-6
88
1304


 


1578
1597
GGTGAAGCGAAGTGCACACG
552089
8-10-2
93
801


 


1578
1594
GAAGCGAAGTGCACACG
552148
2-9-6
79
1305


 


1579
1598
AGGTGAAGCGAAGTGCACAC
552090
8-10-2
87
802


 


1579
1595
TGAAGCGAAGTGCACAC
552149
2-9-6
81
1306


 


1580
1599
GAGGTGAAGCGAAGTGCACA
552091
8-10-2
88
225


 


1581
1600
AGAGGTGAAGCGAAGTGCAC
552092
8-10-2
90
804


 


1582
1601
CAGAGGTGAAGCGAAGTGCA
552093
8-10-2
91
805


 


1583
1602
GCAGAGGTGAAGCGAAGTGC
552094
8-10-2
88
226


 


1584
1603
TGCAGAGGTGAAGCGAAGTG
552063
7-10-3
81
806


 


1584
1603
TGCAGAGGTGAAGCGAAGTG
552095
8-10-2
89
806


 


1585
1604
GTGCAGAGGTGAAGCGAAGT
552064
7-10-3
85
807


 


1585
1604
GTGCAGAGGTGAAGCGAAGT
552096
8-10-2
92
807


 


1586
1605
CGTGCAGAGGTGAAGCGAAG
552065
7-10-3
86
227


 


1586
1605
CGTGCAGAGGTGAAGCGAAG
552097
8-10-2
93
227


 


1587
1606
ACGTGCAGAGGTGAAGCGAA
552066
7-10-3
33
1350


 


1587
1606
ACGTGCAGAGGTGAAGCGAA
552098
8-10-2
88
1350


 


1778
1797
AATTTATGCCTACAGCCTCC
552067
7-10-3
50
46


 


1778
1797
AATTTATGCCTACAGCCTCC
552099
8-10-2
70
46


 


1779
1798
CAATTTATGCCTACAGCCTC
552068
7-10-3
73
48


 


1779
1798
CAATTTATGCCTACAGCCTC
552100
8-10-2
70
48


 


1780
1799
CCAATTTATGCCTACAGCCT
552069
7-10-3
73
50


 


1780
1799
CCAATTTATGCCTACAGCCT
552101
8-10-2
76
50


 


1781
1800
ACCAATTTATGCCTACAGCC
552070
7-10-3
71
52


 


1781
1800
ACCAATTTATGCCTACAGCC
552102
8-10-2
64
52
















TABLE 36







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
5-10-5
84
224


 


411
427
GGCATAGCAGCAGGATG
510100
3-10-4
76
17


 


58
74
CTGGAGCCACCAGCAGG
552330
6-9-2
54
1288


 


59
75
ACTGGAGCCACCAGCAG
552331
6-9-2
66
1289


 


60
76
AACTGGAGCCACCAGCA
552332
6-9-2
70
1290


 


61
77
GAACTGGAGCCACCAGC
552333
6-9-2
55
1291


 


253
269
GAAGTCCACCACGAGTC
552334
6-9-2
42
9


 


254
270
AGAAGTCCACCACGAGT
552335
6-9-2
39
10


 


255
271
GAGAAGTCCACCACGAG
552336
6-9-2
27
11


 


256
272
AGAGAAGTCCACCACGA
552337
6-9-2
74
12


 


411
427
GGCATAGCAGCAGGATG
552338
6-9-2
68
17


 


412
428
AGGCATAGCAGCAGGAT
552339
6-9-2
71
18


 


413
429
GAGGCATAGCAGCAGGA
552340
6-9-2
61
19


 


414
430
TGAGGCATAGCAGCAGG
552341
6-9-2
58
21


 


415
431
ATGAGGCATAGCAGCAG
552342
6-9-2
55
23


 


416
432
GATGAGGCATAGCAGCA
552343
6-9-2
63
25


 


417
433
AGATGAGGCATAGCAGC
552344
6-9-2
51
27


 


418
434
AAGATGAGGCATAGCAG
552345
6-9-2
65
29


 


457
473
ACGGGCAACATACCTTG
552346
6-9-2
0
33


 


670
686
ACTAGTAAACTGAGCCA
552347
6-9-2
84
1292


 


671
687
CACTAGTAAACTGAGCC
552348
6-9-2
87
1293


 


672
688
GCACTAGTAAACTGAGC
552349
6-9-2
74
1294


 


687
703
ACCACTGAACAAATGGC
552350
6-9-2
59
40


 


688
704
AACCACTGAACAAATGG
552351
6-9-2
60
41


 


689
705
GAACCACTGAACAAATG
552352
6-9-2
53
42


 


690
706
CGAACCACTGAACAAAT
552353
6-9-2
0
43


 


1261
1277
CGCAGTATGGATCGGCA
552354
6-9-2
83
1295


 


1262
1278
CCGCAGTATGGATCGGC
552355
6-9-2
90
1296


 


1263
1279
TCCGCAGTATGGATCGG
552356
6-9-2
0
1297


 


1264
1280
TTCCGCAGTATGGATCG
552357
6-9-2
45
1298


 


1265
1281
GTTCCGCAGTATGGATC
552358
6-9-2
74
1299


 


1266
1282
AGTTCCGCAGTATGGAT
552359
6-9-2
72
1300


 


1267
1283
GAGTTCCGCAGTATGGA
552360
6-9-2
87
1301


 


1268
1284
GGAGTTCCGCAGTATGG
552361
6-9-2
96
1302


 


1269
1285
AGGAGTTCCGCAGTATG
552308
5-9-3
81
1303


 


1269
1285
AGGAGTTCCGCAGTATG
552362
6-9-2
92
1303


 


1577
1593
AAGCGAAGTGCACACGG
552309
5-9-3
77
1304


 


1577
1593
AAGCGAAGTGCACACGG
552363
6-9-2
92
1304


 


1578
1594
GAAGCGAAGTGCACACG
552310
5-9-3
80
1305


 


1578
1594
GAAGCGAAGTGCACACG
552364
6-9-2
87
1305


 


1579
1595
TGAAGCGAAGTGCACAC
552311
5-9-3
13
1306


 


1579
1595
TGAAGCGAAGTGCACAC
552365
6-9-2
84
1306


 


1580
1596
GTGAAGCGAAGTGCACA
552150
2-9-6
73
1307


 


1580
1596
GTGAAGCGAAGTGCACA
552312
5-9-3
77
1307


 


1580
1596
GTGAAGCGAAGTGCACA
552366
6-9-2
87
1307


 


1581
1597
GGTGAAGCGAAGTGCAC
552313
5-9-3
64
1308


 


1581
1597
GGTGAAGCGAAGTGCAC
552367
6-9-2
85
1308


 


1582
1598
AGGTGAAGCGAAGTGCA
552314
5-9-3
73
1309


 


1582
1598
AGGTGAAGCGAAGTGCA
552368
6-9-2
77
1309


 


1583
1599
GAGGTGAAGCGAAGTGC
552315
5-9-3
75
1310


 


1583
1599
GAGGTGAAGCGAAGTGC
552369
6-9-2
75
1310


 


1584
1600
AGAGGTGAAGCGAAGTG
552316
5-9-3
64
1311


 


1584
1600
AGAGGTGAAGCGAAGTG
552370
6-9-2
63
1311


 


1585
1601
CAGAGGTGAAGCGAAGT
552317
5-9-3
99
1312


 


1585
1601
CAGAGGTGAAGCGAAGT
552371
6-9-2
81
1312


 


1586
1602
GCAGAGGTGAAGCGAAG
552318
5-9-3
76
1313


 


1586
1602
GCAGAGGTGAAGCGAAG
552372
6-9-2
65
1313


 


1587
1603
TGCAGAGGTGAAGCGAA
552319
5-9-3
55
1314


 


1587
1603
TGCAGAGGTGAAGCGAA
552373
6-9-2
74
1314


 


1588
1604
GTGCAGAGGTGAAGCGA
552320
5-9-3
68
1315


 


1588
1604
GTGCAGAGGTGAAGCGA
552374
6-9-2
78
1315


 


1589
1605
CGTGCAGAGGTGAAGCG
552321
5-9-3
74
1316


 


1589
1605
CGTGCAGAGGTGAAGCG
552375
6-9-2
81
1316


 


1590
1606
ACGTGCAGAGGTGAAGC
552322
5-9-3
73
1317


 


1590
1606
ACGTGCAGAGGTGAAGC
552376
6-9-2
78
1317


 


1778
1794
TTATGCCTACAGCCTCC
552323
5-9-3
75
47


 


1778
1794
TTATGCCTACAGCCTCC
552377
6-9-2
70
47


 


1779
1795
TTTATGCCTACAGCCTC
552324
5-9-3
0
49


 


1779
1795
TTTATGCCTACAGCCTC
552378
6-9-2
72
49


 


1780
1796
ATTTATGCCTACAGCCT
552325
5-9-3
70
51


 


1780
1796
ATTTATGCCTACAGCCT
552379
6-9-2
74
51


 


1781
1797
AATTTATGCCTACAGCC
552326
5-9-3
63
53


 


1781
1797
AATTTATGCCTACAGCC
552380
6-9-2
53
53


 


1782
1798
CAATTTATGCCTACAGC
552327
5-9-3
30
54


 


1782
1798
CAATTTATGCCTACAGC
552381
6-9-2
26
54


 


1783
1799
CCAATTTATGCCTACAG
552328
5-9-3
25
55


 


1783
1799
CCAATTTATGCCTACAG
552382
6-9-2
13
55


 


1784
1800
ACCAATTTATGCCTACA
552329
5-9-3
33
56


 


1784
1800
ACCAATTTATGCCTACA
552383
6-9-2
5
56
















TABLE 37







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1780
1799
CCAATTTATGCCTACAGCCT
509934
5-10-5
30
50


 


58
77
GAACTGGAGCCACCAGCAGG
551909
2-10-8
62
83


 


58
77
GAACTGGAGCCACCAGCAGG
551941
3-10-7
74
83


 


58
77
GAACTGGAGCCACCAGCAGG
551973
4-10-6
64
83


 


253
272
AGAGAAGTCCACCACGAGTC
551910
2-10-8
52
103


 


253
272
AGAGAAGTCCACCACGAGTC
551942
3-10-7
54
103


 


253
272
AGAGAAGTCCACCACGAGTC
551974
4-10-6
51
103


 


411
430
TGAGGCATAGCAGCAGGATG
551911
2-10-8
58
136


 


411
430
TGAGGCATAGCAGCAGGATG
551943
3-10-7
64
136


 


411
430
TGAGGCATAGCAGCAGGATG
551975
4-10-6
57
136


 


412
431
ATGAGGCATAGCAGCAGGAT
551912
2-10-8
59
139


 


412
431
ATGAGGCATAGCAGCAGGAT
551944
3-10-7
66
139


 


412
431
ATGAGGCATAGCAGCAGGAT
551976
4-10-6
57
139


 


413
432
GATGAGGCATAGCAGCAGGA
551913
2-10-8
58
142


 


413
432
GATGAGGCATAGCAGCAGGA
551945
3-10-7
56
142


 


413
432
GATGAGGCATAGCAGCAGGA
551977
4-10-6
56
142


 


414
433
AGATGAGGCATAGCAGCAGG
551914
2-10-8
0
20


 


414
433
AGATGAGGCATAGCAGCAGG
551946
3-10-7
48
20


 


414
433
AGATGAGGCATAGCAGCAGG
551978
4-10-6
53
20


 


415
434
AAGATGAGGCATAGCAGCAG
551915
2-10-8
44
22


 


415
434
AAGATGAGGCATAGCAGCAG
551947
3-10-7
53
22


 


415
434
AAGATGAGGCATAGCAGCAG
551979
4-10-6
64
22


 


416
435
GAAGATGAGGCATAGCAGCA
551916
2-10-8
57
24


 


416
435
GAAGATGAGGCATAGCAGCA
551948
3-10-7
68
24


 


416
435
GAAGATGAGGCATAGCAGCA
551980
4-10-6
56
24


 


417
436
AGAAGATGAGGCATAGCAGC
551917
2-10-8
58
26


 


417
436
AGAAGATGAGGCATAGCAGC
551949
3-10-7
64
26


 


417
436
AGAAGATGAGGCATAGCAGC
551981
4-10-6
63
26


 


418
437
AAGAAGATGAGGCATAGCAG
551918
2-10-8
59
28


 


418
437
AAGAAGATGAGGCATAGCAG
551950
3-10-7
71
28


 


418
437
AAGAAGATGAGGCATAGCAG
551982
4-10-6
63
28


 


687
706
CGAACCACTGAACAAATGGC
551919
2-10-8
76
39


 


687
706
CGAACCACTGAACAAATGGC
551951
3-10-7
71
39


 


687
706
CGAACCACTGAACAAATGGC
551983
4-10-6
73
39


 


1261
1280
TTCCGCAGTATGGATCGGCA
551920
2-10-8
68
719


 


1261
1280
TTCCGCAGTATGGATCGGCA
551952
3-10-7
76
719


 


1261
1280
TTCCGCAGTATGGATCGGCA
551984
4-10-6
81
719


 


1262
1281
GTTCCGCAGTATGGATCGGC
551921
2-10-8
83
212


 


1262
1281
GTTCCGCAGTATGGATCGGC
551953
3-10-7
82
212


 


1262
1281
GTTCCGCAGTATGGATCGGC
551985
4-10-6
76
212


 


1263
1282
AGTTCCGCAGTATGGATCGG
551922
2-10-8
73
720


 


1263
1282
AGTTCCGCAGTATGGATCGG
551954
3-10-7
68
720


 


1264
1283
GAGTTCCGCAGTATGGATCG
551923
2-10-8
59
721


 


1264
1283
GAGTTCCGCAGTATGGATCG
551955
3-10-7
71
721


 


1265
1284
GGAGTTCCGCAGTATGGATC
551924
2-10-8
80
1349


 


1265
1284
GGAGTTCCGCAGTATGGATC
551956
3-10-7
80
1349


 


1266
1285
AGGAGTTCCGCAGTATGGAT
551925
2-10-8
82
722


 


1266
1285
AGGAGTTCCGCAGTATGGAT
551957
3-10-7
88
722


 


1577
1596
GTGAAGCGAAGTGCACACGG
551926
2-10-8
71
224


 


1577
1596
GTGAAGCGAAGTGCACACGG
551958
3-10-7
74
224


 


1578
1597
GGTGAAGCGAAGTGCACACG
551927
2-10-8
68
801


 


1578
1597
GGTGAAGCGAAGTGCACACG
551959
3-10-7
69
801


 


1579
1598
AGGTGAAGCGAAGTGCACAC
551928
2-10-8
69
802


 


1579
1598
AGGTGAAGCGAAGTGCACAC
551960
3-10-7
62
802


 


1580
1599
GAGGTGAAGCGAAGTGCACA
551929
2-10-8
54
225


 


1580
1599
GAGGTGAAGCGAAGTGCACA
551961
3-10-7
20
225


 


1581
1600
AGAGGTGAAGCGAAGTGCAC
551930
2-10-8
53
804


 


1581
1600
AGAGGTGAAGCGAAGTGCAC
551962
3-10-7
60
804


 


1582
1601
CAGAGGTGAAGCGAAGTGCA
551931
2-10-8
47
805


 


1582
1601
CAGAGGTGAAGCGAAGTGCA
551963
3-10-7
63
805


 


1583
1602
GCAGAGGTGAAGCGAAGTGC
551932
2-10-8
68
226


 


1583
1602
GCAGAGGTGAAGCGAAGTGC
551964
3-10-7
56
226


 


1584
1603
TGCAGAGGTGAAGCGAAGTG
551933
2-10-8
72
806


 


1584
1603
TGCAGAGGTGAAGCGAAGTG
551965
3-10-7
67
806


 


1585
1604
GTGCAGAGGTGAAGCGAAGT
551934
2-10-8
64
807


 


1585
1604
GTGCAGAGGTGAAGCGAAGT
551966
3-10-7
73
807


 


1586
1605
CGTGCAGAGGTGAAGCGAAG
551935
2-10-8
68
227


 


1586
1605
CGTGCAGAGGTGAAGCGAAG
551967
3-10-7
60
227


 


1587
1606
ACGTGCAGAGGTGAAGCGAA
551936
2-10-8
67
1350


 


1587
1606
ACGTGCAGAGGTGAAGCGAA
551968
3-10-7
63
1350


 


1778
1797
AATTTATGCCTACAGCCTCC
551937
2-10-8
47
46


 


1778
1797
AATTTATGCCTACAGCCTCC
551969
3-10-7
36
46


 


1779
1798
CAATTTATGCCTACAGCCTC
551938
2-10-8
41
48


 


1779
1798
CAATTTATGCCTACAGCCTC
551970
3-10-7
43
48


 


1780
1799
CCAATTTATGCCTACAGCCT
551939
2-10-8
53
50


 


1780
1799
CCAATTTATGCCTACAGCCT
551971
3-10-7
55
50


 


1781
1800
ACCAATTTATGCCTACAGCC
551940
2-10-8
50
52


 


1781
1800
ACCAATTTATGCCTACAGCC
551972
3-10-7
58
52
















TABLE 38







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1780
1799
CCAATTTATGCCTACAGCCT
509934
5-10-5
21
50


 


58
77
GAACTGGAGCCACCAGCAGG
551909
2-10-8
52
83


 


58
77
GAACTGGAGCCACCAGCAGG
551941
3-10-7
62
83


 


58
77
GAACTGGAGCCACCAGCAGG
551973
4-10-6
58
83


 


253
272
AGAGAAGTCCACCACGAGTC
551910
2-10-8
48
103


 


253
272
AGAGAAGTCCACCACGAGTC
551942
3-10-7
36
103


 


253
272
AGAGAAGTCCACCACGAGTC
551974
4-10-6
45
103


 


411
430
TGAGGCATAGCAGCAGGATG
551911
2-10-8
61
136


 


411
430
TGAGGCATAGCAGCAGGATG
551943
3-10-7
56
136


 


411
430
TGAGGCATAGCAGCAGGATG
551975
4-10-6
60
136


 


412
431
ATGAGGCATAGCAGCAGGAT
551912
2-10-8
53
139


 


412
431
ATGAGGCATAGCAGCAGGAT
551944
3-10-7
48
139


 


412
431
ATGAGGCATAGCAGCAGGAT
551976
4-10-6
48
139


 


413
432
GATGAGGCATAGCAGCAGGA
551913
2-10-8
53
142


 


413
432
GATGAGGCATAGCAGCAGGA
551945
3-10-7
54
142


 


413
432
GATGAGGCATAGCAGCAGGA
551977
4-10-6
48
142


 


414
433
AGATGAGGCATAGCAGCAGG
551914
2-10-8
0
20


 


414
433
AGATGAGGCATAGCAGCAGG
551946
3-10-7
56
20


 


414
433
AGATGAGGCATAGCAGCAGG
551978
4-10-6
36
20


 


415
434
AAGATGAGGCATAGCAGCAG
551915
2-10-8
47
22


 


415
434
AAGATGAGGCATAGCAGCAG
551947
3-10-7
45
22


 


415
434
AAGATGAGGCATAGCAGCAG
551979
4-10-6
54
22


 


416
435
GAAGATGAGGCATAGCAGCA
551916
2-10-8
44
24


 


416
435
GAAGATGAGGCATAGCAGCA
551948
3-10-7
59
24


 


416
435
GAAGATGAGGCATAGCAGCA
551980
4-10-6
49
24


 


417
436
AGAAGATGAGGCATAGCAGC
551917
2-10-8
48
26


 


417
436
AGAAGATGAGGCATAGCAGC
551949
3-10-7
60
26


 


417
436
AGAAGATGAGGCATAGCAGC
551981
4-10-6
57
26


 


418
437
AAGAAGATGAGGCATAGCAG
551918
2-10-8
53
28


 


418
437
AAGAAGATGAGGCATAGCAG
551950
3-10-7
57
28


 


418
437
AAGAAGATGAGGCATAGCAG
551982
4-10-6
57
28


 


687
706
CGAACCACTGAACAAATGGC
551919
2-10-8
65
39


 


687
706
CGAACCACTGAACAAATGGC
551951
3-10-7
57
39


 


687
706
CGAACCACTGAACAAATGGC
551983
4-10-6
53
39


 


1261
1280
TTCCGCAGTATGGATCGGCA
551920
2-10-8
57
719


 


1261
1280
TTCCGCAGTATGGATCGGCA
551952
3-10-7
67
719


 


1261
1280
TTCCGCAGTATGGATCGGCA
551984
4-10-6
62
719


 


1262
1281
GTTCCGCAGTATGGATCGGC
551921
2-10-8
60
212


 


1262
1281
GTTCCGCAGTATGGATCGGC
551953
3-10-7
57
212


 


1262
1281
GTTCCGCAGTATGGATCGGC
551985
4-10-6
58
212


 


1263
1282
AGTTCCGCAGTATGGATCGG
551922
2-10-8
63
720


 


1263
1282
AGTTCCGCAGTATGGATCGG
551954
3-10-7
61
720


 


1264
1283
GAGTTCCGCAGTATGGATCG
551923
2-10-8
50
721


 


1264
1283
GAGTTCCGCAGTATGGATCG
551955
3-10-7
44
721


 


1265
1284
GGAGTTCCGCAGTATGGATC
551924
2-10-8
52
1349


 


1265
1284
GGAGTTCCGCAGTATGGATC
551956
3-10-7
46
1349


 


1266
1285
AGGAGTTCCGCAGTATGGAT
551925
2-10-8
54
722


 


1266
1285
AGGAGTTCCGCAGTATGGAT
551957
3-10-7
51
722


 


1577
1596
GTGAAGCGAAGTGCACACGG
551926
2-10-8
70
224


 


1577
1596
GTGAAGCGAAGTGCACACGG
551958
3-10-7
72
224


 


1578
1597
GGTGAAGCGAAGTGCACACG
551927
2-10-8
60
801


 


1578
1597
GGTGAAGCGAAGTGCACACG
551959
3-10-7
61
801


 


1579
1598
AGGTGAAGCGAAGTGCACAC
551928
2-10-8
57
802


 


1579
1598
AGGTGAAGCGAAGTGCACAC
551960
3-10-7
58
802


 


1580
1599
GAGGTGAAGCGAAGTGCACA
551929
2-10-8
49
225


 


1580
1599
GAGGTGAAGCGAAGTGCACA
551961
3-10-7
26
225


 


1581
1600
AGAGGTGAAGCGAAGTGCAC
551930
2-10-8
54
804


 


1581
1600
AGAGGTGAAGCGAAGTGCAC
551962
3-10-7
57
804


 


1582
1601
CAGAGGTGAAGCGAAGTGCA
551931
2-10-8
46
805


 


1582
1601
CAGAGGTGAAGCGAAGTGCA
551963
3-10-7
56
805


 


1583
1602
GCAGAGGTGAAGCGAAGTGC
551932
2-10-8
57
226


 


1583
1602
GCAGAGGTGAAGCGAAGTGC
551964
3-10-7
53
226


 


1584
1603
TGCAGAGGTGAAGCGAAGTG
551933
2-10-8
65
806


 


1584
1603
TGCAGAGGTGAAGCGAAGTG
551965
3-10-7
54
806


 


1585
1604
GTGCAGAGGTGAAGCGAAGT
551934
2-10-8
58
807


 


1585
1604
GTGCAGAGGTGAAGCGAAGT
551966
3-10-7
69
807


 


1586
1605
CGTGCAGAGGTGAAGCGAAG
551935
2-10-8
63
227


 


1586
1605
CGTGCAGAGGTGAAGCGAAG
551967
3-10-7
53
227


 


1587
1606
ACGTGCAGAGGTGAAGCGAA
551936
2-10-8
67
1350


 


1587
1606
ACGTGCAGAGGTGAAGCGAA
551968
3-10-7
60
1350


 


1778
1797
AATTTATGCCTACAGCCTCC
551937
2-10-8
51
46


 


1778
1797
AATTTATGCCTACAGCCTCC
551969
3-10-7
42
46


 


1779
1798
CAATTTATGCCTACAGCCTC
551938
2-10-8
40
48


 


1779
1798
CAATTTATGCCTACAGCCTC
551970
3-10-7
38
48


 


1780
1799
CCAATTTATGCCTACAGCCT
551939
2-10-8
32
50


 


1780
1799
CCAATTTATGCCTACAGCCT
551971
3-10-7
46
50


 


1781
1800
ACCAATTTATGCCTACAGCC
551940
2-10-8
39
52


 


1781
1800
ACCAATTTATGCCTACAGCC
551972
3-10-7
51
52
















TABLE 39







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
5-10-5
40
224


 


411
427
GGCATAGCAGCAGGATG
510100
3-10-4
60
17


 


58
74
CTGGAGCCACCAGCAGG
552276
5-9-3
44
1288


 


59
75
ACTGGAGCCACCAGCAG
552277
5-9-3
39
1289


 


60
76
AACTGGAGCCACCAGCA
552278
5-9-3
37
1290


 


61
77
GAACTGGAGCCACCAGC
552279
5-9-3
50
1291


 


253
269
GAAGTCCACCACGAGTC
552280
5-9-3
2
9


 


254
270
AGAAGTCCACCACGAGT
552281
5-9-3
0
10


 


255
271
GAGAAGTCCACCACGAG
552282
5-9-3
13
11


 


256
272
AGAGAAGTCCACCACGA
552229
4-9-4
17
12


 


256
272
AGAGAAGTCCACCACGA
552283
5-9-3
27
12


 


411
427
GGCATAGCAGCAGGATG
552230
4-9-4
53
17


 


411
427
GGCATAGCAGCAGGATG
552284
5-9-3
0
17


 


412
428
AGGCATAGCAGCAGGAT
552231
4-9-4
31
18


 


412
428
AGGCATAGCAGCAGGAT
552285
5-9-3
56
18


 


413
429
GAGGCATAGCAGCAGGA
552232
4-9-4
35
19


 


413
429
GAGGCATAGCAGCAGGA
552286
5-9-3
43
19


 


414
430
TGAGGCATAGCAGCAGG
552233
4-9-4
40
21


 


414
430
TGAGGCATAGCAGCAGG
552287
5-9-3
44
21


 


415
431
ATGAGGCATAGCAGCAG
552234
4-9-4
0
23


 


415
431
ATGAGGCATAGCAGCAG
552288
5-9-3
44
23


 


416
432
GATGAGGCATAGCAGCA
552235
4-9-4
13
25


 


416
432
GATGAGGCATAGCAGCA
552289
5-9-3
21
25


 


417
433
AGATGAGGCATAGCAGC
552236
4-9-4
40
27


 


417
433
AGATGAGGCATAGCAGC
552290
5-9-3
34
27


 


418
434
AAGATGAGGCATAGCAG
552237
4-9-4
37
29


 


418
434
AAGATGAGGCATAGCAG
552291
5-9-3
34
29


 


670
686
ACTAGTAAACTGAGCCA
552239
4-9-4
58
1292


 


670
686
ACTAGTAAACTGAGCCA
552293
5-9-3
61
1292


 


671
687
CACTAGTAAACTGAGCC
552240
4-9-4
54
1293


 


671
687
CACTAGTAAACTGAGCC
552294
5-9-3
62
1293


 


672
688
GCACTAGTAAACTGAGC
552241
4-9-4
47
1294


 


672
688
GCACTAGTAAACTGAGC
552295
5-9-3
63
1294


 


687
703
ACCACTGAACAAATGGC
552242
4-9-4
61
40


 


687
703
ACCACTGAACAAATGGC
552296
5-9-3
61
40


 


688
704
AACCACTGAACAAATGG
552243
4-9-4
55
41


 


688
704
AACCACTGAACAAATGG
552297
5-9-3
52
41


 


689
705
GAACCACTGAACAAATG
552244
4-9-4
45
42


 


689
705
GAACCACTGAACAAATG
552298
5-9-3
27
42


 


690
706
CGAACCACTGAACAAAT
552245
4-9-4
41
43


 


690
706
CGAACCACTGAACAAAT
552299
5-9-3
32
43


 


1261
1277
CGCAGTATGGATCGGCA
552246
4-9-4
67
1295


 


1261
1277
CGCAGTATGGATCGGCA
552300
5-9-3
57
1295


 


1262
1278
CCGCAGTATGGATCGGC
552247
4-9-4
74
1296


 


1262
1278
CCGCAGTATGGATCGGC
552301
5-9-3
76
1296


 


1263
1279
TCCGCAGTATGGATCGG
552248
4-9-4
65
1297


 


1263
1279
TCCGCAGTATGGATCGG
552302
5-9-3
68
1297


 


1264
1280
TTCCGCAGTATGGATCG
552249
4-9-4
38
1298


 


1264
1280
TTCCGCAGTATGGATCG
552303
5-9-3
59
1298


 


1265
1281
GTTCCGCAGTATGGATC
552250
4-9-4
43
1299


 


1265
1281
GTTCCGCAGTATGGATC
552304
5-9-3
30
1299


 


1266
1282
AGTTCCGCAGTATGGAT
552251
4-9-4
52
1300


 


1266
1282
AGTTCCGCAGTATGGAT
552305
5-9-3
49
1300


 


1267
1283
GAGTTCCGCAGTATGGA
552252
4-9-4
51
1301


 


1267
1283
GAGTTCCGCAGTATGGA
552306
5-9-3
56
1301


 


1268
1284
GGAGTTCCGCAGTATGG
552253
4-9-4
47
1302


 


1268
1284
GGAGTTCCGCAGTATGG
552307
5-9-3
49
1302


 


1269
1285
AGGAGTTCCGCAGTATG
552254
4-9-4
50
1303


 


1577
1593
AAGCGAAGTGCACACGG
552255
4-9-4
64
1304


 


1578
1594
GAAGCGAAGTGCACACG
552256
4-9-4
57
1305


 


1579
1595
TGAAGCGAAGTGCACAC
552257
4-9-4
51
1306


 


1580
1596
GTGAAGCGAAGTGCACA
552258
4-9-4
62
1307


 


1581
1597
GGTGAAGCGAAGTGCAC
552259
4-9-4
59
1308


 


1582
1598
AGGTGAAGCGAAGTGCA
552260
4-9-4
56
1309


 


1583
1599
GAGGTGAAGCGAAGTGC
552261
4-9-4
54
1310


 


1584
1600
AGAGGTGAAGCGAAGTG
552262
4-9-4
47
1311


 


1585
1601
CAGAGGTGAAGCGAAGT
552263
4-9-4
45
1312


 


1586
1602
GCAGAGGTGAAGCGAAG
552264
4-9-4
52
1313


 


1587
1603
TGCAGAGGTGAAGCGAA
552265
4-9-4
58
1314


 


1588
1604
GTGCAGAGGTGAAGCGA
552266
4-9-4
54
1315


 


1589
1605
CGTGCAGAGGTGAAGCG
552267
4-9-4
43
1316


 


1590
1606
ACGTGCAGAGGTGAAGC
552268
4-9-4
57
1317


 


1778
1794
TTATGCCTACAGCCTCC
552269
4-9-4
34
47


 


1779
1795
TTTATGCCTACAGCCTC
552270
4-9-4
37
49


 


1780
1796
ATTTATGCCTACAGCCT
552271
4-9-4
42
51


 


1781
1797
AATTTATGCCTACAGCC
552272
4-9-4
36
53


 


1782
1798
CAATTTATGCCTACAGC
552273
4-9-4
25
54


 


1783
1799
CCAATTTATGCCTACAG
552274
4-9-4
11
55


 


1784
1800
ACCAATTTATGCCTACA
552275
4-9-4
38
56
















TABLE 40







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
5-10-5
38
1354


 


414
429
GAGGCATAGCAGCAGG
509959
3-10-3
49
145


 


411
427
GGCATAGCAGCAGGATG
510100
3-10-4
55
17


 


58
73
TGGAGCCACCAGCAGG
552384
2-9-5
41
1318


 


58
73
TGGAGCCACCAGCAGG
552440
3-9-4
57
1318


 


59
74
CTGGAGCCACCAGCAG
552385
2-9-5
53
1319


 


59
74
CTGGAGCCACCAGCAG
552441
3-9-4
38
1319


 


60
75
ACTGGAGCCACCAGCA
552386
2-9-5
42
1320


 


60
75
ACTGGAGCCACCAGCA
552442
3-9-4
72
1320


 


61
76
AACTGGAGCCACCAGC
552387
2-9-5
43
1321


 


61
76
AACTGGAGCCACCAGC
552443
3-9-4
56
1321


 


62
77
GAACTGGAGCCACCAG
552388
2-9-5
18
86


 


62
77
GAACTGGAGCCACCAG
552444
3-9-4
39
86


 


411
426
GCATAGCAGCAGGATG
552389
2-9-5
24
137


 


411
426
GCATAGCAGCAGGATG
552445
3-9-4
53
137


 


412
427
GGCATAGCAGCAGGAT
552390
2-9-5
40
140


 


412
427
GGCATAGCAGCAGGAT
552446
3-9-4
57
140


 


413
428
AGGCATAGCAGCAGGA
552391
2-9-5
51
143


 


413
428
AGGCATAGCAGCAGGA
552447
3-9-4
53
143


 


414
429
GAGGCATAGCAGCAGG
552392
2-9-5
0
145


 


414
429
GAGGCATAGCAGCAGG
552448
3-9-4
57
145


 


415
430
TGAGGCATAGCAGCAG
552393
2-9-5
52
147


 


415
430
TGAGGCATAGCAGCAG
552449
3-9-4
49
147


 


416
431
ATGAGGCATAGCAGCA
552394
2-9-5
32
149


 


416
431
ATGAGGCATAGCAGCA
552450
3-9-4
44
149


 


417
432
GATGAGGCATAGCAGC
552395
2-9-5
33
151


 


417
432
GATGAGGCATAGCAGC
552451
3-9-4
38
151


 


418
433
AGATGAGGCATAGCAG
552396
2-9-5
46
153


 


418
433
AGATGAGGCATAGCAG
552452
3-9-4
30
153


 


457
473
ACGGGCAACATACCTTG
552130
2-9-6
46
33


 


457
473
ACGGGCAACATACCTTG
552184
3-9-5
34
33


 


457
473
ACGGGCAACATACCTTG
552238
4-9-4
41
33


 


457
473
ACGGGCAACATACCTTG
552292
5-9-3
45
33


 


457
473
ACGGGCAACATACCTTG
552346
6-9-2
0
33


 


457
472
CGGGCAACATACCTTG
552397
2-9-5
37
167


 


457
472
CGGGCAACATACCTTG
552453
3-9-4
45
167


 


458
473
ACGGGCAACATACCTT
552398
2-9-5
42
168


 


458
473
ACGGGCAACATACCTT
552454
3-9-4
39
168


 


670
685
CTAGTAAACTGAGCCA
552399
2-9-5
34
181


 


671
686
ACTAGTAAACTGAGCC
552400
2-9-5
47
1322


 


672
687
CACTAGTAAACTGAGC
552401
2-9-5
53
1323


 


673
688
GCACTAGTAAACTGAG
552402
2-9-5
47
1324


 


687
702
CCACTGAACAAATGGC
552403
2-9-5
70
188


 


688
703
ACCACTGAACAAATGG
552404
2-9-5
44
190


 


689
704
AACCACTGAACAAATG
552405
2-9-5
0
191


 


690
705
GAACCACTGAACAAAT
552406
2-9-5
25
192


 


691
706
CGAACCACTGAACAAA
552407
2-9-5
23
194


 


1261
1276
GCAGTATGGATCGGCA
552408
2-9-5
73
211


 


1262
1277
CGCAGTATGGATCGGC
552409
2-9-5
71
1325


 


1263
1278
CCGCAGTATGGATCGG
552410
2-9-5
52
1326


 


1264
1279
TCCGCAGTATGGATCG
552411
2-9-5
62
1327


 


1265
1280
TTCCGCAGTATGGATC
552412
2-9-5
50
1328


 


1266
1281
GTTCCGCAGTATGGAT
552413
2-9-5
55
1329


 


1267
1282
AGTTCCGCAGTATGGA
552414
2-9-5
64
1330


 


1268
1283
GAGTTCCGCAGTATGG
552415
2-9-5
45
1331


 


1269
1284
GGAGTTCCGCAGTATG
552416
2-9-5
45
1332


 


1270
1285
AGGAGTTCCGCAGTAT
552417
2-9-5
37
1333


 


1577
1592
AGCGAAGTGCACACGG
552418
2-9-5
73
1334


 


1578
1593
AAGCGAAGTGCACACG
552419
2-9-5
68
1335


 


1579
1594
GAAGCGAAGTGCACAC
552420
2-9-5
64
1336


 


1580
1595
TGAAGCGAAGTGCACA
552421
2-9-5
54
1337


 


1581
1596
GTGAAGCGAAGTGCAC
552422
2-9-5
60
1338


 


1582
1597
GGTGAAGCGAAGTGCA
552423
2-9-5
62
1339


 


1583
1598
AGGTGAAGCGAAGTGC
552424
2-9-5
60
1340


 


1584
1599
GAGGTGAAGCGAAGTG
552425
2-9-5
46
1341


 


1585
1600
AGAGGTGAAGCGAAGT
552426
2-9-5
48
1342


 


1586
1601
CAGAGGTGAAGCGAAG
552427
2-9-5
36
1343


 


1587
1602
GCAGAGGTGAAGCGAA
552428
2-9-5
57
1344


 


1588
1603
TGCAGAGGTGAAGCGA
552429
2-9-5
36
1345


 


1589
1604
GTGCAGAGGTGAAGCG
552430
2-9-5
42
1346


 


1590
1605
CGTGCAGAGGTGAAGC
552431
2-9-5
60
1347


 


1591
1606
ACGTGCAGAGGTGAAG
552432
2-9-5
44
1348


 


1778
1793
TATGCCTACAGCCTCC
552433
2-9-5
55
230


 


1779
1794
TTATGCCTACAGCCTC
552434
2-9-5
46
231


 


1780
1795
TTTATGCCTACAGCCT
552435
2-9-5
47
232


 


1781
1796
ATTTATGCCTACAGCC
552436
2-9-5
25
233


 


1782
1797
AATTTATGCCTACAGC
552437
2-9-5
19
234


 


1783
1798
CAATTTATGCCTACAG
552438
2-9-5
25
235


 


1784
1799
CCAATTTATGCCTACA
552439
2-9-5
22
236
















TABLE 41







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















414
429
GAGGCATAGCAGCAGG
509959
3-10-3
49
145


 


58
73
TGGAGCCACCAGCAGG
552496
4-9-3
35
1318


 


59
74
CTGGAGCCACCAGCAG
552497
4-9-3
60
1319


 


60
75
ACTGGAGCCACCAGCA
552498
4-9-3
20
1320


 


61
76
AACTGGAGCCACCAGC
552499
4-9-3
45
1321


 


62
77
GAACTGGAGCCACCAG
552500
4-9-3
53
86


 


411
426
GCATAGCAGCAGGATG
552501
4-9-3
56
137


 


412
427
GGCATAGCAGCAGGAT
552502
4-9-3
50
140


 


413
428
AGGCATAGCAGCAGGA
552503
4-9-3
36
143


 


414
429
GAGGCATAGCAGCAGG
552504
4-9-3
50
145


 


415
430
TGAGGCATAGCAGCAG
552505
4-9-3
53
147


 


416
431
ATGAGGCATAGCAGCA
552506
4-9-3
49
149


 


417
432
GATGAGGCATAGCAGC
552507
4-9-3
35
151


 


418
433
AGATGAGGCATAGCAG
552508
4-9-3
62
153


 


457
472
CGGGCAACATACCTTG
552509
4-9-3
65
167


 


458
473
ACGGGCAACATACCTT
552510
4-9-3
54
168


 


670
685
CTAGTAAACTGAGCCA
552455
3-9-4
60
181


 


670
685
CTAGTAAACTGAGCCA
552511
4-9-3
65
181


 


671
686
ACTAGTAAACTGAGCC
552456
3-9-4
69
1322


 


671
686
ACTAGTAAACTGAGCC
552512
4-9-3
63
1322


 


672
687
CACTAGTAAACTGAGC
552457
3-9-4
4
1323


 


672
687
CACTAGTAAACTGAGC
552513
4-9-3
50
1323


 


673
688
GCACTAGTAAACTGAG
552458
3-9-4
59
1324


 


673
688
GCACTAGTAAACTGAG
552514
4-9-3
53
1324


 


687
702
CCACTGAACAAATGGC
552459
3-9-4
69
188


 


687
702
CCACTGAACAAATGGC
552515
4-9-3
68
188


 


688
703
ACCACTGAACAAATGG
552460
3-9-4
3
190


 


688
703
ACCACTGAACAAATGG
552516
4-9-3
65
190


 


689
704
AACCACTGAACAAATG
552461
3-9-4
37
191


 


689
704
AACCACTGAACAAATG
552517
4-9-3
54
191


 


690
705
GAACCACTGAACAAAT
552462
3-9-4
42
192


 


690
705
GAACCACTGAACAAAT
552518
4-9-3
23
192


 


691
706
CGAACCACTGAACAAA
552463
3-9-4
28
194


 


691
706
CGAACCACTGAACAAA
552519
4-9-3
32
194


 


1261
1276
GCAGTATGGATCGGCA
552464
3-9-4
72
211


 


1261
1276
GCAGTATGGATCGGCA
552520
4-9-3
61
211


 


1262
1277
CGCAGTATGGATCGGC
552465
3-9-4
68
1325


 


1262
1277
CGCAGTATGGATCGGC
552521
4-9-3
68
1325


 


1263
1278
CCGCAGTATGGATCGG
552466
3-9-4
76
1326


 


1263
1278
CCGCAGTATGGATCGG
552522
4-9-3
71
1326


 


1264
1279
TCCGCAGTATGGATCG
552467
3-9-4
72
1327


 


1264
1279
TCCGCAGTATGGATCG
552523
4-9-3
73
1327


 


1265
1280
TTCCGCAGTATGGATC
552468
3-9-4
50
1328


 


1265
1280
TTCCGCAGTATGGATC
552524
4-9-3
49
1328


 


1266
1281
GTTCCGCAGTATGGAT
552469
3-9-4
65
1329


 


1266
1281
GTTCCGCAGTATGGAT
552525
4-9-3
45
1329


 


1267
1282
AGTTCCGCAGTATGGA
552470
3-9-4
58
1330


 


1267
1282
AGTTCCGCAGTATGGA
552526
4-9-3
39
1330


 


1268
1283
GAGTTCCGCAGTATGG
552471
3-9-4
30
1331


 


1268
1283
GAGTTCCGCAGTATGG
552527
4-9-3
39
1331


 


1269
1284
GGAGTTCCGCAGTATG
552472
3-9-4
43
1332


 


1269
1284
GGAGTTCCGCAGTATG
552528
4-9-3
43
1332


 


1270
1285
AGGAGTTCCGCAGTAT
552473
3-9-4
25
1333


 


1270
1285
AGGAGTTCCGCAGTAT
552529
4-9-3
50
1333


 


1577
1592
AGCGAAGTGCACACGG
552474
3-9-4
70
1334


 


1577
1592
AGCGAAGTGCACACGG
552530
4-9-3
73
1334


 


1578
1593
AAGCGAAGTGCACACG
552475
3-9-4
64
1335


 


1578
1593
AAGCGAAGTGCACACG
552531
4-9-3
62
1335


 


1579
1594
GAAGCGAAGTGCACAC
552476
3-9-4
50
1336


 


1580
1595
TGAAGCGAAGTGCACA
552477
3-9-4
66
1337


 


1581
1596
GTGAAGCGAAGTGCAC
552478
3-9-4
68
1338


 


1582
1597
GGTGAAGCGAAGTGCA
552479
3-9-4
60
1339


 


1583
1598
AGGTGAAGCGAAGTGC
552480
3-9-4
58
1340


 


1584
1599
GAGGTGAAGCGAAGTG
552481
3-9-4
54
1341


 


1585
1600
AGAGGTGAAGCGAAGT
552482
3-9-4
44
1342


 


1586
1601
CAGAGGTGAAGCGAAG
552483
3-9-4
17
1343


 


1587
1602
GCAGAGGTGAAGCGAA
552484
3-9-4
64
1344


 


1588
1603
TGCAGAGGTGAAGCGA
552485
3-9-4
56
1345


 


1589
1604
GTGCAGAGGTGAAGCG
552486
3-9-4
26
1346


 


1590
1605
CGTGCAGAGGTGAAGC
552487
3-9-4
42
1347


 


1591
1606
ACGTGCAGAGGTGAAG
552488
3-9-4
35
1348


 


1778
1793
TATGCCTACAGCCTCC
552489
3-9-4
46
230


 


1779
1794
TTATGCCTACAGCCTC
552490
3-9-4
41
231


 


1780
1795
TTTATGCCTACAGCCT
552491
3-9-4
38
232


 


1781
1796
ATTTATGCCTACAGCC
552492
3-9-4
47
233


 


1782
1797
AATTTATGCCTACAGC
552493
3-9-4
49
234


 


1783
1798
CAATTTATGCCTACAG
552494
3-9-4
22
235


 


1784
1799
CCAATTTATGCCTACA
552495
3-9-4
0
236
















TABLE 42







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
5-10-5
56
224







55



 


414
429
GAGGCATAGCAGCAGG
509959
3-10-3
54
145


 


58
73
TGGAGCCACCAGCAGG
552552
5-9-2
32
1355


 


59
74
CTGGAGCCACCAGCAG
552553
5-9-2
53
1319


 


60
75
ACTGGAGCCACCAGCA
552554
5-9-2
48
1320


 


61
76
AACTGGAGCCACCAGC
552555
5-9-2
39
1321


 


62
77
GAACTGGAGCCACCAG
552556
5-9-2
39
86


 


411
426
GCATAGCAGCAGGATG
552557
5-9-2
54
137


 


412
427
GGCATAGCAGCAGGAT
552558
5-9-2
41
140


 


413
428
AGGCATAGCAGCAGGA
552559
5-9-2
56
143


 


414
429
GAGGCATAGCAGCAGG
552560
5-9-2
39
145


 


415
430
TGAGGCATAGCAGCAG
552561
5-9-2
51
147


 


416
431
ATGAGGCATAGCAGCA
552562
5-9-2
56
149


 


417
432
GATGAGGCATAGCAGC
552563
5-9-2
31
151


 


418
433
AGATGAGGCATAGCAG
552564
5-9-2
31
153


 


457
472
CGGGCAACATACCTTG
552565
5-9-2
53
167


 


458
473
ACGGGCAACATACCTT
552566
5-9-2
46
168


 


670
685
CTAGTAAACTGAGCCA
552567
5-9-2
63
181


 


671
686
ACTAGTAAACTGAGCC
552568
5-9-2
66
1322


 


672
687
CACTAGTAAACTGAGC
552569
5-9-2
60
1323


 


673
688
GCACTAGTAAACTGAG
552570
5-9-2
60
1324


 


687
702
CCACTGAACAAATGGC
552571
5-9-2
44
188


 


688
703
ACCACTGAACAAATGG
552572
5-9-2
52
190


 


689
704
AACCACTGAACAAATG
552573
5-9-2
20
191


 


690
705
GAACCACTGAACAAAT
552574
5-9-2
36
192


 


691
706
CGAACCACTGAACAAA
552575
5-9-2
19
194


 


1261
1276
GCAGTATGGATCGGCA
552576
5-9-2
61
211


 


1262
1277
CGCAGTATGGATCGGC
552577
5-9-2
57
1325


 


1263
1278
CCGCAGTATGGATCGG
552578
5-9-2
71
1326


 


1264
1279
TCCGCAGTATGGATCG
552579
5-9-2
59
1327


 


1265
1280
TTCCGCAGTATGGATC
552580
5-9-2
58
1328


 


1266
1281
GTTCCGCAGTATGGAT
552581
5-9-2
51
1329


 


1267
1282
AGTTCCGCAGTATGGA
552582
5-9-2
40
1330


 


1268
1283
GAGTTCCGCAGTATGG
552583
5-9-2
35
1331


 


1269
1284
GGAGTTCCGCAGTATG
552584
5-9-2
50
1332


 


1270
1285
AGGAGTTCCGCAGTAT
552585
5-9-2
48
1333


 


1577
1592
AGCGAAGTGCACACGG
552586
5-9-2
74
1334


 


1578
1593
AAGCGAAGTGCACACG
552587
5-9-2
68
1335


 


1579
1594
GAAGCGAAGTGCACAC
552532
4-9-3
59
1336


 


1579
1594
GAAGCGAAGTGCACAC
552588
5-9-2
67
1336


 


1580
1595
TGAAGCGAAGTGCACA
552533
4-9-3
52
1337


 


1580
1595
TGAAGCGAAGTGCACA
552589
5-9-2
47
1337


 


1581
1596
GTGAAGCGAAGTGCAC
552534
4-9-3
71
1338


 


1581
1596
GTGAAGCGAAGTGCAC
552590
5-9-2
58
1338


 


1582
1597
GGTGAAGCGAAGTGCA
552535
4-9-3
59
1339


 


1582
1597
GGTGAAGCGAAGTGCA
552591
5-9-2
46
1339


 


1583
1598
AGGTGAAGCGAAGTGC
552536
4-9-3
19
1340


 


1583
1598
AGGTGAAGCGAAGTGC
552592
5-9-2
44
1340


 


1584
1599
GAGGTGAAGCGAAGTG
552537
4-9-3
26
1341


 


1584
1599
GAGGTGAAGCGAAGTG
552593
5-9-2
39
1341


 


1585
1600
AGAGGTGAAGCGAAGT
552538
4-9-3
54
1342


 


1585
1600
AGAGGTGAAGCGAAGT
552594
5-9-2
52
1342


 


1586
1601
CAGAGGTGAAGCGAAG
552539
4-9-3
50
1343


 


1586
1601
CAGAGGTGAAGCGAAG
552595
5-9-2
57
1343


 


1587
1602
GCAGAGGTGAAGCGAA
552540
4-9-3
60
1344


 


1587
1602
GCAGAGGTGAAGCGAA
552596
5-9-2
58
1344


 


1588
1603
TGCAGAGGTGAAGCGA
552541
4-9-3
68
1345


 


1588
1603
TGCAGAGGTGAAGCGA
552597
5-9-2
52
1345


 


1589
1604
GTGCAGAGGTGAAGCG
552542
4-9-3
63
1346


 


1589
1604
GTGCAGAGGTGAAGCG
552598
5-9-2
51
1346


 


1590
1605
CGTGCAGAGGTGAAGC
552543
4-9-3
44
1347


 


1590
1605
CGTGCAGAGGTGAAGC
552600
5-9-2
51
1347


 


1591
1606
ACGTGCAGAGGTGAAG
552544
4-9-3
45
1348


 


1591
1606
ACGTGCAGAGGTGAAG
552602
5-9-2
13
1348


 


1778
1793
TATGCCTACAGCCTCC
552545
4-9-3
42
230


 


1778
1793
TATGCCTACAGCCTCC
552604
5-9-2
42
230


 


1779
1794
TTATGCCTACAGCCTC
552546
4-9-3
46
231


 


1779
1794
TTATGCCTACAGCCTC
552606
5-9-2
42
231


 


1780
1795
TTTATGCCTACAGCCT
552547
4-9-3
38
232


 


1780
1795
TTTATGCCTACAGCCT
552608
5-9-2
37
232


 


1781
1796
ATTTATGCCTACAGCC
552548
4-9-3
49
233


 


1781
1796
ATTTATGCCTACAGCC
552610
5-9-2
41
233


 


1782
1797
AATTTATGCCTACAGC
552549
4-9-3
34
234


 


1782
1797
AATTTATGCCTACAGC
552612
5-9-2
23
234


 


1783
1798
CAATTTATGCCTACAG
552550
4-9-3
13
235


 


1783
1798
CAATTTATGCCTACAG
552614
5-9-2
11
235


 


1784
1799
CCAATTTATGCCTACA
552551
4-9-3
8
236


 


1784
1799
CCAATTTATGCCTACA
552616
5-9-2
6
236
















TABLE 43







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
5-10-5
47
224


 


1780
1799
CCAATTTATGCCTACAGCCT
509934
5-10-5
67
50


 


58
77
GAACTGGAGCCACCAGCAGG
552007
6-10-4
53
83


 


58
77
GAACTGGAGCCACCAGCAGG
552039
7-10-3
74
83


 


253
272
AGAGAAGTCCACCACGAGTC
552008
6-10-4
47
103


 


253
272
AGAGAAGTCCACCACGAGTC
552040
7-10-3
57
103


 


411
430
TGAGGCATAGCAGCAGGATG
552009
6-10-4
70
136


 


411
430
TGAGGCATAGCAGCAGGATG
552041
7-10-3
65
136


 


412
431
ATGAGGCATAGCAGCAGGAT
552010
6-10-4
51
139


 


412
431
ATGAGGCATAGCAGCAGGAT
552042
7-10-3
59
139


 


413
432
GATGAGGCATAGCAGCAGGA
552011
6-10-4
47
142


 


413
432
GATGAGGCATAGCAGCAGGA
552043
7-10-3
36
142


 


414
433
AGATGAGGCATAGCAGCAGG
552012
6-10-4
62
20


 


414
433
AGATGAGGCATAGCAGCAGG
552044
7-10-3
82
20


 


415
434
AAGATGAGGCATAGCAGCAG
552013
6-10-4
72
22


 


415
434
AAGATGAGGCATAGCAGCAG
552045
7-10-3
62
22


 


416
435
GAAGATGAGGCATAGCAGCA
552014
6-10-4
73
24


 


416
435
GAAGATGAGGCATAGCAGCA
552046
7-10-3
74
24


 


417
436
AGAAGATGAGGCATAGCAGC
552015
6-10-4
66
26


 


417
436
AGAAGATGAGGCATAGCAGC
552047
7-10-3
60
26


 


418
437
AAGAAGATGAGGCATAGCAG
552016
6-10-4
67
28


 


418
437
AAGAAGATGAGGCATAGCAG
552048
7-10-3
60
28


 


687
706
CGAACCACTGAACAAATGGC
552017
6-10-4
72
39


 


687
706
CGAACCACTGAACAAATGGC
552049
7-10-3
68
39


 


1261
1280
TTCCGCAGTATGGATCGGCA
552018
6-10-4
89
719


 


1261
1280
TTCCGCAGTATGGATCGGCA
552050
7-10-3
86
719


 


1262
1281
GTTCCGCAGTATGGATCGGC
552019
6-10-4
87
212


 


1262
1281
GTTCCGCAGTATGGATCGGC
552051
7-10-3
86
212


 


1263
1282
AGTTCCGCAGTATGGATCGG
551986
4-10-6
64
720


 


1263
1282
AGTTCCGCAGTATGGATCGG
552020
6-10-4
86
720


 


1263
1282
AGTTCCGCAGTATGGATCGG
552052
7-10-3
87
720


 


1264
1283
GAGTTCCGCAGTATGGATCG
551987
4-10-6
76
721


 


1264
1283
GAGTTCCGCAGTATGGATCG
552021
6-10-4
84
721


 


1264
1283
GAGTTCCGCAGTATGGATCG
552053
7-10-3
75
721


 


1265
1284
GGAGTTCCGCAGTATGGATC
551988
4-10-6
5
1349


 


1265
1284
GGAGTTCCGCAGTATGGATC
552005
5-10-5
72
1349


 


1265
1284
GGAGTTCCGCAGTATGGATC
552022
6-10-4
80
1349


 


1265
1284
GGAGTTCCGCAGTATGGATC
552054
7-10-3
83
1349


 


1266
1285
AGGAGTTCCGCAGTATGGAT
551989
4-10-6
64
722


 


1266
1285
AGGAGTTCCGCAGTATGGAT
552023
6-10-4
78
722


 


1266
1285
AGGAGTTCCGCAGTATGGAT
552055
7-10-3
57
722


 


1577
1596
GTGAAGCGAAGTGCACACGG
551990
4-10-6
83
224


 


1577
1596
GTGAAGCGAAGTGCACACGG
552024
6-10-4
89
224


 


1577
1596
GTGAAGCGAAGTGCACACGG
552056
7-10-3
82
224


 


1578
1597
GGTGAAGCGAAGTGCACACG
551991
4-10-6
0
801


 


1578
1597
GGTGAAGCGAAGTGCACACG
552025
6-10-4
89
801


 


1578
1597
GGTGAAGCGAAGTGCACACG
552057
7-10-3
89
801


 


1579
1598
AGGTGAAGCGAAGTGCACAC
551992
4-10-6
67
802


 


1579
1598
AGGTGAAGCGAAGTGCACAC
552026
6-10-4
84
802


 


1579
1598
AGGTGAAGCGAAGTGCACAC
552058
7-10-3
82
802


 


1580
1599
GAGGTGAAGCGAAGTGCACA
551993
4-10-6
78
225


 


1580
1599
GAGGTGAAGCGAAGTGCACA
552027
6-10-4
85
225


 


1580
1599
GAGGTGAAGCGAAGTGCACA
552059
7-10-3
85
225


 


1581
1600
AGAGGTGAAGCGAAGTGCAC
551994
4-10-6
82
804


 


1581
1600
AGAGGTGAAGCGAAGTGCAC
552028
6-10-4
82
804


 


1581
1600
AGAGGTGAAGCGAAGTGCAC
552060
7-10-3
74
804


 


1582
1601
CAGAGGTGAAGCGAAGTGCA
551995
4-10-6
81
805


 


1582
1601
CAGAGGTGAAGCGAAGTGCA
552029
6-10-4
81
805


 


1582
1601
CAGAGGTGAAGCGAAGTGCA
552061
7-10-3
81
805


 


1583
1602
GCAGAGGTGAAGCGAAGTGC
551996
4-10-6
79
226


 


1583
1602
GCAGAGGTGAAGCGAAGTGC
552030
6-10-4
86
226


 


1583
1602
GCAGAGGTGAAGCGAAGTGC
552062
7-10-3
85
226


 


1584
1603
TGCAGAGGTGAAGCGAAGTG
551997
4-10-6
80
806


 


1584
1603
TGCAGAGGTGAAGCGAAGTG
552031
6-10-4
86
806


 


1585
1604
GTGCAGAGGTGAAGCGAAGT
551998
4-10-6
74
807


 


1585
1604
GTGCAGAGGTGAAGCGAAGT
552032
6-10-4
78
807


 


1586
1605
CGTGCAGAGGTGAAGCGAAG
551999
4-10-6
79
227


 


1586
1605
CGTGCAGAGGTGAAGCGAAG
552033
6-10-4
80
227


 


1587
1606
ACGTGCAGAGGTGAAGCGAA
552000
4-10-6
84
1350


 


1587
1606
ACGTGCAGAGGTGAAGCGAA
552006
5-10-5
86
1350


 


1587
1606
ACGTGCAGAGGTGAAGCGAA
552034
6-10-4
81
1350


 


1778
1797
AATTTATGCCTACAGCCTCC
552001
4-10-6
66
46


 


1778
1797
AATTTATGCCTACAGCCTCC
552035
6-10-4
55
46


 


1779
1798
CAATTTATGCCTACAGCCTC
552002
4-10-6
54
48


 


1779
1798
CAATTTATGCCTACAGCCTC
552036
6-10-4
58
48


 


1780
1799
CCAATTTATGCCTACAGCCT
552003
4-10-6
50
50


 


1780
1799
CCAATTTATGCCTACAGCCT
552037
6-10-4
43
50


 


1781
1800
ACCAATTTATGCCTACAGCC
552004
4-10-6
56
52


 


1781
1800
ACCAATTTATGCCTACAGCC
552038
6-10-4
66
52
















TABLE 44







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
5-10-5
61
224


 


411
427
GGCATAGCAGCAGGATG
510100
3-10-4
66
17


 


58
74
CTGGAGCCACCAGCAGG
552168
3-9-5
64
1288


 


58
74
CTGGAGCCACCAGCAGG
552222
4-9-4
76
1288


 


59
75
ACTGGAGCCACCAGCAG
552169
3-9-5
65
1289


 


59
75
ACTGGAGCCACCAGCAG
552223
4-9-4
41
1289


 


60
76
AACTGGAGCCACCAGCA
552170
3-9-5
58
1290


 


60
76
AACTGGAGCCACCAGCA
552224
4-9-4
58
1290


 


61
77
GAACTGGAGCCACCAGC
552171
3-9-5
51
1291


 


61
77
GAACTGGAGCCACCAGC
552225
4-9-4
49
1291


 


253
269
GAAGTCCACCACGAGTC
552172
3-9-5
23
9


 


253
269
GAAGTCCACCACGAGTC
552226
4-9-4
36
9


 


254
270
AGAAGTCCACCACGAGT
552173
3-9-5
44
10


 


254
270
AGAAGTCCACCACGAGT
552227
4-9-4
20
10


 


255
271
GAGAAGTCCACCACGAG
552174
3-9-5
28
11


 


255
271
GAGAAGTCCACCACGAG
552228
4-9-4
29
11


 


256
272
AGAGAAGTCCACCACGA
552175
3-9-5
56
12


 


411
427
GGCATAGCAGCAGGATG
552176
3-9-5
66
17


 


412
428
AGGCATAGCAGCAGGAT
552177
3-9-5
53
18


 


413
429
GAGGCATAGCAGCAGGA
552178
3-9-5
57
19


 


414
430
TGAGGCATAGCAGCAGG
552179
3-9-5
56
21


 


415
431
ATGAGGCATAGCAGCAG
552180
3-9-5
51
23


 


416
432
GATGAGGCATAGCAGCA
552181
3-9-5
51
25


 


417
433
AGATGAGGCATAGCAGC
552182
3-9-5
63
27


 


418
434
AAGATGAGGCATAGCAG
552183
3-9-5
60
29


 


670
686
ACTAGTAAACTGAGCCA
552185
3-9-5
67
1292


 


671
687
CACTAGTAAACTGAGCC
552186
3-9-5
37
1293


 


672
688
GCACTAGTAAACTGAGC
552187
3-9-5
68
1294


 


687
703
ACCACTGAACAAATGGC
552188
3-9-5
71
40


 


688
704
AACCACTGAACAAATGG
552189
3-9-5
51
41


 


689
705
GAACCACTGAACAAATG
552190
3-9-5
47
42


 


690
706
CGAACCACTGAACAAAT
552191
3-9-5
50
43


 


1261
1277
CGCAGTATGGATCGGCA
552192
3-9-5
80
1295


 


1262
1278
CCGCAGTATGGATCGGC
552193
3-9-5
73
1296


 


1263
1279
TCCGCAGTATGGATCGG
552194
3-9-5
58
1297


 


1264
1280
TTCCGCAGTATGGATCG
552195
3-9-5
60
1298


 


1265
1281
GTTCCGCAGTATGGATC
552196
3-9-5
54
1299


 


1266
1282
AGTTCCGCAGTATGGAT
552197
3-9-5
64
1300


 


1267
1283
GAGTTCCGCAGTATGGA
552198
3-9-5
62
1301


 


1268
1284
GGAGTTCCGCAGTATGG
552199
3-9-5
57
1302


 


1269
1285
AGGAGTTCCGCAGTATG
552200
3-9-5
52
1303


 


1577
1593
AAGCGAAGTGCACACGG
552201
3-9-5
73
1304


 


1578
1594
GAAGCGAAGTGCACACG
552202
3-9-5
60
1305


 


1579
1595
TGAAGCGAAGTGCACAC
552203
3-9-5
60
1306


 


1580
1596
GTGAAGCGAAGTGCACA
552204
3-9-5
63
1307


 


1581
1597
GGTGAAGCGAAGTGCAC
552151
2-9-6
71
1308


 


1581
1597
GGTGAAGCGAAGTGCAC
552205
3-9-5
64
1308


 


1582
1598
AGGTGAAGCGAAGTGCA
552152
2-9-6
69
1309


 


1582
1598
AGGTGAAGCGAAGTGCA
552206
3-9-5
71
1309


 


1583
1599
GAGGTGAAGCGAAGTGC
552153
2-9-6
63
1310


 


1583
1599
GAGGTGAAGCGAAGTGC
552207
3-9-5
71
1310


 


1584
1600
AGAGGTGAAGCGAAGTG
552154
2-9-6
56
1311


 


1584
1600
AGAGGTGAAGCGAAGTG
552208
3-9-5
52
1311


 


1585
1601
CAGAGGTGAAGCGAAGT
552155
2-9-6
61
1312


 


1585
1601
CAGAGGTGAAGCGAAGT
552209
3-9-5
50
1312


 


1586
1602
GCAGAGGTGAAGCGAAG
552156
2-9-6
40
1313


 


1586
1602
GCAGAGGTGAAGCGAAG
552210
3-9-5
66
1313


 


1587
1603
TGCAGAGGTGAAGCGAA
552157
2-9-6
45
1314


 


1587
1603
TGCAGAGGTGAAGCGAA
552211
3-9-5
63
1314


 


1588
1604
GTGCAGAGGTGAAGCGA
552158
2-9-6
66
1315


 


1588
1604
GTGCAGAGGTGAAGCGA
552212
3-9-5
62
1315


 


1589
1605
CGTGCAGAGGTGAAGCG
552159
2-9-6
68
1316


 


1589
1605
CGTGCAGAGGTGAAGCG
552213
3-9-5
64
1316


 


1590
1606
ACGTGCAGAGGTGAAGC
552160
2-9-6
78
1317


 


1590
1606
ACGTGCAGAGGTGAAGC
552214
3-9-5
72
1317


 


1778
1794
TTATGCCTACAGCCTCC
552161
2-9-6
57
47


 


1778
1794
TTATGCCTACAGCCTCC
552215
3-9-5
54
47


 


1779
1795
TTTATGCCTACAGCCTC
552162
2-9-6
54
49


 


1779
1795
TTTATGCCTACAGCCTC
552216
3-9-5
49
49


 


1780
1796
ATTTATGCCTACAGCCT
552163
2-9-6
65
51


 


1780
1796
ATTTATGCCTACAGCCT
552217
3-9-5
50
51


 


1781
1797
AATTTATGCCTACAGCC
552164
2-9-6
48
53


 


1781
1797
AATTTATGCCTACAGCC
552218
3-9-5
39
53


 


1782
1798
CAATTTATGCCTACAGC
552165
2-9-6
46
54


 


1782
1798
CAATTTATGCCTACAGC
552219
3-9-5
41
54


 


1783
1799
CCAATTTATGCCTACAG
552166
2-9-6
42
55


 


1783
1799
CCAATTTATGCCTACAG
552220
3-9-5
32
55


 


1784
1800
ACCAATTTATGCCTACA
552167
2-9-6
47
56


 


1784
1800
ACCAATTTATGCCTACA
552221
3-9-5
33
56
















TABLE 45







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
5-10-5
87
224







56



 


1780
1799
CCAATTTATGCCTACAGCCT
509934
5-10-5
56
50


 


411
427
GGCATAGCAGCAGGATG
510100
3-10-4
69
17


 


58
77
GAACTGGAGCCACCAGCAGG
552071
8-10-2
73
83


 


58
74
CTGGAGCCACCAGCAGG
552114
2-9-6
64
1288


 


59
75
ACTGGAGCCACCAGCAG
552115
2-9-6
61
1289


 


60
76
AACTGGAGCCACCAGCA
552116
2-9-6
53
1290


 


61
77
GAACTGGAGCCACCAGC
552117
2-9-6
69
1291


 


253
272
AGAGAAGTCCACCACGAGTC
552072
8-10-2
39
103


 


253
269
GAAGTCCACCACGAGTC
552118
2-9-6
49
9


 


254
270
AGAAGTCCACCACGAGT
552119
2-9-6
49
10


 


255
271
GAGAAGTCCACCACGAG
552120
2-9-6
21
11


 


256
272
AGAGAAGTCCACCACGA
552121
2-9-6
27
12


 


411
430
TGAGGCATAGCAGCAGGATG
552073
8-10-2
73
136


 


411
427
GGCATAGCAGCAGGATG
552122
2-9-6
48
17


 


412
431
ATGAGGCATAGCAGCAGGAT
552074
8-10-2
69
139


 


412
428
AGGCATAGCAGCAGGAT
552123
2-9-6
68
18


 


413
432
GATGAGGCATAGCAGCAGGA
552075
8-10-2
78
142


 


413
429
GAGGCATAGCAGCAGGA
552124
2-9-6
47
19


 


414
433
AGATGAGGCATAGCAGCAGG
552076
8-10-2
63
20


 


414
430
TGAGGCATAGCAGCAGG
552125
2-9-6
72
21


 


415
434
AAGATGAGGCATAGCAGCAG
552077
8-10-2
62
22


 


415
431
ATGAGGCATAGCAGCAG
552126
2-9-6
64
23


 


416
435
GAAGATGAGGCATAGCAGCA
552078
8-10-2
59
24


 


416
432
GATGAGGCATAGCAGCA
552127
2-9-6
65
25


 


417
436
AGAAGATGAGGCATAGCAGC
552079
8-10-2
80
26


 


417
433
AGATGAGGCATAGCAGC
552128
2-9-6
78
27


 


418
437
AAGAAGATGAGGCATAGCAG
552080
8-10-2
74
28


 


418
434
AAGATGAGGCATAGCAG
552129
2-9-6
68
29


 


457
473
ACGGGCAACATACCTTG
552130
2-9-6
46
33


 


670
686
ACTAGTAAACTGAGCCA
552131
2-9-6
61
1292


 


671
687
CACTAGTAAACTGAGCC
552132
2-9-6
66
1293


 


672
688
GCACTAGTAAACTGAGC
552133
2-9-6
78
1294


 


687
706
CGAACCACTGAACAAATGGC
552081
8-10-2
69
39


 


687
703
ACCACTGAACAAATGGC
552134
2-9-6
68
40


 


688
704
AACCACTGAACAAATGG
552135
2-9-6
59
41


 


689
705
GAACCACTGAACAAATG
552136
2-9-6
39
42


 


690
706
CGAACCACTGAACAAAT
552137
2-9-6
36
43


 


1261
1280
TTCCGCAGTATGGATCGGCA
552082
8-10-2
86
719


 


1261
1277
CGCAGTATGGATCGGCA
552138
2-9-6
80
1295


 


1262
1281
GTTCCGCAGTATGGATCGGC
552083
8-10-2
85
212


 


1262
1278
CCGCAGTATGGATCGGC
552139
2-9-6
80
1296


 


1263
1282
AGTTCCGCAGTATGGATCGG
552084
8-10-2
86
720


 


1263
1279
TCCGCAGTATGGATCGG
552140
2-9-6
70
1297


 


1264
1283
GAGTTCCGCAGTATGGATCG
552085
8-10-2
83
721


 


1264
1280
TTCCGCAGTATGGATCG
552141
2-9-6
72
1298


 


1265
1284
GGAGTTCCGCAGTATGGATC
552086
8-10-2
83
1349


 


1265
1281
GTTCCGCAGTATGGATC
552142
2-9-6
58
1299


 


1266
1285
AGGAGTTCCGCAGTATGGAT
552087
8-10-2
77
722


 


1266
1282
AGTTCCGCAGTATGGAT
552143
2-9-6
70
1300


 


1267
1283
GAGTTCCGCAGTATGGA
552144
2-9-6
66
1301


 


1268
1284
GGAGTTCCGCAGTATGG
552145
2-9-6
78
1302


 


1269
1285
AGGAGTTCCGCAGTATG
552146
2-9-6
63
1303


 


1577
1596
GTGAAGCGAAGTGCACACGG
552088
8-10-2
90
224


 


1577
1593
AAGCGAAGTGCACACGG
552147
2-9-6
80
1304


 


1578
1597
GGTGAAGCGAAGTGCACACG
552089
8-10-2
87
801


 


1578
1594
GAAGCGAAGTGCACACG
552148
2-9-6
74
1305


 


1579
1598
AGGTGAAGCGAAGTGCACAC
552090
8-10-2
85
802


 


1579
1595
TGAAGCGAAGTGCACAC
552149
2-9-6
79
1306


 


1580
1599
GAGGTGAAGCGAAGTGCACA
552091
8-10-2
84
225


 


1581
1600
AGAGGTGAAGCGAAGTGCAC
552092
8-10-2
86
804


 


1582
1601
CAGAGGTGAAGCGAAGTGCA
552093
8-10-2
82
805


 


1583
1602
GCAGAGGTGAAGCGAAGTGC
552094
8-10-2
84
226


 


1584
1603
TGCAGAGGTGAAGCGAAGTG
552063
7-10-3
79
806


 


1584
1603
TGCAGAGGTGAAGCGAAGTG
552095
8-10-2
85
806


 


1585
1604
GTGCAGAGGTGAAGCGAAGT
552064
7-10-3
83
807


 


1585
1604
GTGCAGAGGTGAAGCGAAGT
552096
8-10-2
88
807


 


1586
1605
CGTGCAGAGGTGAAGCGAAG
552065
7-10-3
86
227


 


1586
1605
CGTGCAGAGGTGAAGCGAAG
552097
8-10-2
90
227


 


1587
1606
ACGTGCAGAGGTGAAGCGAA
552066
7-10-3
35
1350


 


1587
1606
ACGTGCAGAGGTGAAGCGAA
552098
8-10-2
86
1350


 


1778
1797
AATTTATGCCTACAGCCTCC
552067
7-10-3
53
46


 


1778
1797
AATTTATGCCTACAGCCTCC
552099
8-10-2
66
46


 


1779
1798
CAATTTATGCCTACAGCCTC
552068
7-10-3
70
48


 


1779
1798
CAATTTATGCCTACAGCCTC
552100
8-10-2
67
48


 


1780
1799
CCAATTTATGCCTACAGCCT
552069
7-10-3
68
50


 


1780
1799
CCAATTTATGCCTACAGCCT
552101
8-10-2
65
50


 


1781
1800
ACCAATTTATGCCTACAGCC
552070
7-10-3
64
52


 


1781
1800
ACCAATTTATGCCTACAGCC
552102
8-10-2
54
52
















TABLE 46







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
5-10-5
69
224







57



 


411
427
GGCATAGCAGCAGGATG
510100
3-10-4
59
17


 


58
74
CTGGAGCCACCAGCAGG
552330
6-9-2
50
1288


 


59
75
ACTGGAGCCACCAGCAG
552331
6-9-2
46
1289


 


60
76
AACTGGAGCCACCAGCA
552332
6-9-2
50
1290


 


61
77
GAACTGGAGCCACCAGC
552333
6-9-2
48
1291


 


253
269
GAAGTCCACCACGAGTC
552334
6-9-2
42
9


 


254
270
AGAAGTCCACCACGAGT
552335
6-9-2
30
10


 


255
271
GAGAAGTCCACCACGAG
552336
6-9-2
23
11


 


256
272
AGAGAAGTCCACCACGA
552337
6-9-2
42
12


 


411
427
GGCATAGCAGCAGGATG
552338
6-9-2
40
17


 


412
428
AGGCATAGCAGCAGGAT
552339
6-9-2
50
18


 


413
429
GAGGCATAGCAGCAGGA
552340
6-9-2
45
19


 


414
430
TGAGGCATAGCAGCAGG
552341
6-9-2
44
21


 


415
431
ATGAGGCATAGCAGCAG
552342
6-9-2
51
23


 


416
432
GATGAGGCATAGCAGCA
552343
6-9-2
44
25


 


417
433
AGATGAGGCATAGCAGC
552344
6-9-2
24
27


 


418
434
AAGATGAGGCATAGCAG
552345
6-9-2
41
29


 


457
473
ACGGGCAACATACCTTG
552346
6-9-2
0
33


 


670
686
ACTAGTAAACTGAGCCA
552347
6-9-2
75
1292


 


671
687
CACTAGTAAACTGAGCC
552348
6-9-2
72
1293


 


672
688
GCACTAGTAAACTGAGC
552349
6-9-2
65
1294


 


687
703
ACCACTGAACAAATGGC
552350
6-9-2
42
40


 


688
704
AACCACTGAACAAATGG
552351
6-9-2
45
41


 


689
705
GAACCACTGAACAAATG
552352
6-9-2
43
42


 


690
706
CGAACCACTGAACAAAT
552353
6-9-2
20
43


 


1261
1277
CGCAGTATGGATCGGCA
552354
6-9-2
70
1295


 


1262
1278
CCGCAGTATGGATCGGC
552355
6-9-2
66
1296


 


1263
1279
TCCGCAGTATGGATCGG
552356
6-9-2
62
1297


 


1264
1280
TTCCGCAGTATGGATCG
552357
6-9-2
53
1298


 


1265
1281
GTTCCGCAGTATGGATC
552358
6-9-2
57
1299


 


1266
1282
AGTTCCGCAGTATGGAT
552359
6-9-2
46
1300


 


1267
1283
GAGTTCCGCAGTATGGA
552360
6-9-2
45
1301


 


1268
1284
GGAGTTCCGCAGTATGG
552361
6-9-2
44
1302


 


1269
1285
AGGAGTTCCGCAGTATG
552308
5-9-3
38
1303


 


1269
1285
AGGAGTTCCGCAGTATG
552362
6-9-2
51
1303


 


1577
1593
AAGCGAAGTGCACACGG
552309
5-9-3
76
1304


 


1577
1593
AAGCGAAGTGCACACGG
552363
6-9-2
73
1304


 


1578
1594
GAAGCGAAGTGCACACG
552310
5-9-3
58
1305


 


1578
1594
GAAGCGAAGTGCACACG
552364
6-9-2
66
1305


 


1579
1595
TGAAGCGAAGTGCACAC
552311
5-9-3
38
1306


 


1579
1595
TGAAGCGAAGTGCACAC
552365
6-9-2
64
1306


 


1580
1596
GTGAAGCGAAGTGCACA
552150
2-9-6
68
1307


 


1580
1596
GTGAAGCGAAGTGCACA
552312
5-9-3
75
1307


 


1580
1596
GTGAAGCGAAGTGCACA
552366
6-9-2
55
1307


 


1581
1597
GGTGAAGCGAAGTGCAC
552313
5-9-3
66
1308


 


1581
1597
GGTGAAGCGAAGTGCAC
552367
6-9-2
67
1308


 


1582
1598
AGGTGAAGCGAAGTGCA
552314
5-9-3
56
1309


 


1582
1598
AGGTGAAGCGAAGTGCA
552368
6-9-2
41
1309


 


1583
1599
GAGGTGAAGCGAAGTGC
552315
5-9-3
46
1310


 


1583
1599
GAGGTGAAGCGAAGTGC
552369
6-9-2
52
1310


 


1584
1600
AGAGGTGAAGCGAAGTG
552316
5-9-3
55
1311


 


1584
1600
AGAGGTGAAGCGAAGTG
552370
6-9-2
35
1311


 


1585
1601
CAGAGGTGAAGCGAAGT
552317
5-9-3
53
1312


 


1585
1601
CAGAGGTGAAGCGAAGT
552371
6-9-2
58
1312


 


1586
1602
GCAGAGGTGAAGCGAAG
552318
5-9-3
59
1313


 


1586
1602
GCAGAGGTGAAGCGAAG
552372
6-9-2
68
1313


 


1587
1603
TGCAGAGGTGAAGCGAA
552319
5-9-3
56
1314


 


1587
1603
TGCAGAGGTGAAGCGAA
552373
6-9-2
63
1314


 


1588
1604
GTGCAGAGGTGAAGCGA
552320
5-9-3
62
1315


 


1588
1604
GTGCAGAGGTGAAGCGA
552374
6-9-2
70
1315


 


1589
1605
CGTGCAGAGGTGAAGCG
552321
5-9-3
63
1316


 


1589
1605
CGTGCAGAGGTGAAGCG
552375
6-9-2
64
1316


 


1590
1606
ACGTGCAGAGGTGAAGC
552322
5-9-3
52
1317


 


1590
1606
ACGTGCAGAGGTGAAGC
552376
6-9-2
58
1317


 


1778
1794
TTATGCCTACAGCCTCC
552323
5-9-3
45
47


 


1778
1794
TTATGCCTACAGCCTCC
552377
6-9-2
42
47


 


1779
1795
TTTATGCCTACAGCCTC
552324
5-9-3
49
49


 


1779
1795
TTTATGCCTACAGCCTC
552378
6-9-2
37
49


 


1780
1796
ATTTATGCCTACAGCCT
552325
5-9-3
48
51


 


1780
1796
ATTTATGCCTACAGCCT
552379
6-9-2
57
51


 


1781
1797
AATTTATGCCTACAGCC
552326
5-9-3
50
53


 


1781
1797
AATTTATGCCTACAGCC
552380
6-9-2
48
53


 


1782
1798
CAATTTATGCCTACAGC
552327
5-9-3
13
54


 


1782
1798
CAATTTATGCCTACAGC
552381
6-9-2
22
54


 


1783
1799
CCAATTTATGCCTACAG
552328
5-9-3
9
55


 


1783
1799
CCAATTTATGCCTACAG
552382
6-9-2
20
55


 


1784
1800
ACCAATTTATGCCTACA
552329
5-9-3
18
56


 


1784
1800
ACCAATTTATGCCTACA
552383
6-9-2
18
56









Example 15
Antisense Inhibition of HBV Viral mRNA in HepG2 Cells by Deoxy, MOE and (S)-cEt Gapmers
Antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. ISIS 146786 and ISIS 509934, which were described in an earlier application (U.S. Provisional Application No. 61/478,040 filed on Apr. 21, 2011), were also included in these studies for comparison. Cultured HepG2 cells at a density of 28,000 cells per well were transfected using LipofectAMINE2000® with 70 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 (forward sequence CTTGGTCATGGGCCATCAG, designated herein as SEQ ID NO: 1; reverse sequence CGGCTAGGAGTTCCGCAGTA, designated herein as SEQ ID NO: 2; probe sequence TGCGTGGAACCTTTTCGGCTCC, designated herein as SEQ ID NO: 3) was used to measure mRNA levels. Levels were also measured using primer probe set RTS3371 (forward sequence CCAAACCTTCGGACGGAAA, designated herein as SEQ ID NO: 311; reverse sequence TGAGGCCCACTCCCATAGG, designated herein as SEQ ID NO: 312; probe sequence CCCATCATCCTGGGCTTTCGGAAAAT, designated herein as SEQ ID NO: 313). HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in Tables below were designed as deoxy, MOE and (S)-cEt gapmers. The gapmers are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the number indicates the number of deoxynucleosides; and ‘e’ indicates a MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
“Viral Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. Each gapmer listed in the Tables is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1). The potency of the newly designed oligonucleotides was compared with ISIS 146786, 509934, ISIS 509959, and ISIS 510100.







TABLE 47







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1780
1799
CCAATTTATGCCTACAGCCT
509934
eeeee-10-eeeee
30
50


 


58
73
TGGAGCCACCAGCAGG
552787
ekk-10-kke
57
1318


 


59
74
CTGGAGCCACCAGCAG
552788
ekk-10-kke
60
1319


 


60
75
ACTGGAGCCACCAGCA
552789
ekk-10-kke
67
1320


 


61
76
AACTGGAGCCACCAGC
552790
ekk-10-kke
67
1321


 


62
77
GAACTGGAGCCACCAG
552791
ekk-10-kke
65
86


 


245
260
CACGAGTCTAGACTCT
552792
ekk-10-kke
44
93


 


246
261
CCACGAGTCTAGACTC
552793
ekk-10-kke
0
95


 


250
265
TCCACCACGAGTCTAG
552794
ekk-10-kke
54
98


 


251
266
GTCCACCACGAGTCTA
552795
ekk-10-kke
55
100


 


252
267
AGTCCACCACGAGTCT
552796
ekk-10-kke
62
102


 


253
268
AAGTCCACCACGAGTC
552797
ekk-10-kke
59
104


 


254
269
GAAGTCCACCACGAGT
552798
ekk-10-kke
59
106


 


255
270
AGAAGTCCACCACGAG
552799
ekk-10-kke
58
109


 


256
271
GAGAAGTCCACCACGA
552800
ekk-10-kke
62
112


 


258
273
GAGAGAAGTCCACCAC
552801
ekk-10-kke
65
115


 


259
274
TGAGAGAAGTCCACCA
552802
ekk-10-kke
53
117


 


411
426
GCATAGCAGCAGGATG
552803
ekk-10-kke
67
137


 


412
427
GGCATAGCAGCAGGAT
552804
ekk-10-kke
75
140


 


413
428
AGGCATAGCAGCAGGA
552805
ekk-10-kke
72
143


 


414
429
GAGGCATAGCAGCAGG
552806
ekk-10-kke
64
145


 


415
430
TGAGGCATAGCAGCAG
552807
ekk-10-kke
68
147


 


416
431
ATGAGGCATAGCAGCA
552808
ekk-10-kke
65
149


 


417
432
GATGAGGCATAGCAGC
552809
ekk-10-kke
60
151


 


418
433
AGATGAGGCATAGCAG
552810
ekk-10-kke
59
153


 


419
434
AAGATGAGGCATAGCA
552811
ekk-10-kke
64
155


 


420
435
GAAGATGAGGCATAGC
552812
ekk-10-kke
69
157


 


421
436
AGAAGATGAGGCATAG
552813
ekk-10-kke
64
159


 


422
437
AAGAAGATGAGGCATA
552814
ekk-10-kke
62
161


 


457
472
CGGGCAACATACCTTG
552815
ekk-10-kke
61
167


 


458
473
ACGGGCAACATACCTT
552816
ekk-10-kke
63
168


 


639
654
GGCCCACTCCCATAGG
552817
ekk-10-kke
42
176


 


641
656
GAGGCCCACTCCCATA
552818
ekk-10-kke
44
177


 


642
657
TGAGGCCCACTCCCAT
552819
ekk-10-kke
56
178


 


643
658
CTGAGGCCCACTCCCA
552820
ekk-10-kke
59
179


 


670
685
CTAGTAAACTGAGCCA
552821
ekk-10-kke
76
181


 


671
686
ACTAGTAAACTGAGCC
552822
ekk-10-kke
77
1322


 


672
687
CACTAGTAAACTGAGC
552823
ekk-10-kke
73
1323


 


673
688
GCACTAGTAAACTGAG
552824
ekk-10-kke
73
1324


 


678
693
AAATGGCACTAGTAAA
552825
ekk-10-kke
51
1364


 


679
694
CAAATGGCACTAGTAA
552826
ekk-10-kke
55
1365


 


680
695
ACAAATGGCACTAGTA
552827
ekk-10-kke
67
1366


 


681
696
AACAAATGGCACTAGT
552828
ekk-10-kke
78
1367


 


682
697
GAACAAATGGCACTAG
552829
ekk-10-kke
72
1368


 


683
698
TGAACAAATGGCACTA
552830
ekk-10-kke
71
1369


 


684
699
CTGAACAAATGGCACT
552831
ekk-10-kke
69
1370


 


685
700
ACTGAACAAATGGCAC
552832
ekk-10-kke
67
1371


 


686
701
CACTGAACAAATGGCA
552833
ekk-10-kke
65
1372


 


687
702
CCACTGAACAAATGGC
552834
ekk-10-kke
78
188


 


688
703
ACCACTGAACAAATGG
552835
ekk-10-kke
70
190


 


689
704
AACCACTGAACAAATG
552836
ekk-10-kke
64
191


 


690
705
GAACCACTGAACAAAT
552837
ekk-10-kke
65
192


 


691
706
CGAACCACTGAACAAA
552838
ekk-10-kke
64
194


 


738
753
CCACATCATCCATATA
552839
ekk-10-kke
60
199


 


739
754
ACCACATCATCCATAT
552840
ekk-10-kke
35
201


 


1176
1191
CAGCAAACACTTGGCA
552841
ekk-10-kke
62
208


 


1177
1192
TCAGCAAACACTTGGC
552842
ekk-10-kke
67
209


 


1261
1276
GCAGTATGGATCGGCA
552843
ekk-10-kke
77
211


 


1262
1277
CGCAGTATGGATCGGC
552844
ekk-10-kke
81
1325


 


1263
1278
CCGCAGTATGGATCGG
552845
ekk-10-kke
63
1326


 


1264
1279
TCCGCAGTATGGATCG
552846
ekk-10-kke
79
1327


 


1265
1280
TTCCGCAGTATGGATC
552847
ekk-10-kke
47
1328


 


1266
1281
GTTCCGCAGTATGGAT
552848
ekk-10-kke
69
1329


 


1267
1282
AGTTCCGCAGTATGGA
552849
ekk-10-kke
59
1330


 


1268
1283
GAGTTCCGCAGTATGG
552850
ekk-10-kke
83
1331


 


1269
1284
GGAGTTCCGCAGTATG
552851
ekk-10-kke
90
1332


 


1270
1285
AGGAGTTCCGCAGTAT
552852
ekk-10-kke
89
1333


 


1577
1592
AGCGAAGTGCACACGG
552853
ekk-10-kke
83
1334


 


1578
1593
AAGCGAAGTGCACACG
552854
ekk-10-kke
80
1335


 


1579
1594
GAAGCGAAGTGCACAC
552855
ekk-10-kke
75
1336


 


1580
1595
TGAAGCGAAGTGCACA
552856
ekk-10-kke
69
1337


 


1581
1596
GTGAAGCGAAGTGCAC
552857
ekk-10-kke
68
1338


 


1582
1597
GGTGAAGCGAAGTGCA
552858
ekk-10-kke
79
1339


 


1583
1598
AGGTGAAGCGAAGTGC
552859
ekk-10-kke
79
1340


 


1584
1599
GAGGTGAAGCGAAGTG
552860
ekk-10-kke
71
1341


 


1585
1600
AGAGGTGAAGCGAAGT
552861
ekk-10-kke
68
1342


 


1586
1601
CAGAGGTGAAGCGAAG
552862
ekk-10-kke
65
1343


 


1587
1602
GCAGAGGTGAAGCGAA
552863
ekk-10-kke
70
1344


 


1588
1603
TGCAGAGGTGAAGCGA
552864
ekk-10-kke
71
1345
















TABLE 48







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















58
73
TGGAGCCACCAGCAGG
552787
ekk-10-kke
53
1318


 


59
74
CTGGAGCCACCAGCAG
552788
ekk-10-kke
45
1319


 


60
75
ACTGGAGCCACCAGCA
552789
ekk-10-kke
75
1320


 


61
76
AACTGGAGCCACCAGC
552790
ekk-10-kke
68
1321


 


62
77
GAACTGGAGCCACCAG
552791
ekk-10-kke
51
86


 


245
260
CACGAGTCTAGACTCT
552792
ekk-10-kke
38
93


 


246
261
CCACGAGTCTAGACTC
552793
ekk-10-kke
0
95


 


250
265
TCCACCACGAGTCTAG
552794
ekk-10-kke
44
98


 


251
266
GTCCACCACGAGTCTA
552795
ekk-10-kke
56
100


 


252
267
AGTCCACCACGAGTCT
552796
ekk-10-kke
45
102


 


253
268
AAGTCCACCACGAGTC
552797
ekk-10-kke
46
104


 


254
269
GAAGTCCACCACGAGT
552798
ekk-10-kke
53
106


 


255
270
AGAAGTCCACCACGAG
552799
ekk-10-kke
48
109


 


256
271
GAGAAGTCCACCACGA
552800
ekk-10-kke
54
112


 


258
273
GAGAGAAGTCCACCAC
552801
ekk-10-kke
63
115


 


259
274
TGAGAGAAGTCCACCA
552802
ekk-10-kke
49
117


 


411
426
GCATAGCAGCAGGATG
552803
ekk-10-kke
71
137


 


412
427
GGCATAGCAGCAGGAT
552804
ekk-10-kke
64
140


 


413
428
AGGCATAGCAGCAGGA
552805
ekk-10-kke
70
143


 


414
429
GAGGCATAGCAGCAGG
552806
ekk-10-kke
67
145


 


415
430
TGAGGCATAGCAGCAG
552807
ekk-10-kke
61
147


 


416
431
ATGAGGCATAGCAGCA
552808
ekk-10-kke
83
149


 


417
432
GATGAGGCATAGCAGC
552809
ekk-10-kke
59
151


 


418
433
AGATGAGGCATAGCAG
552810
ekk-10-kke
56
153


 


419
434
AAGATGAGGCATAGCA
552811
ekk-10-kke
62
155


 


420
435
GAAGATGAGGCATAGC
552812
ekk-10-kke
66
157


 


421
436
AGAAGATGAGGCATAG
552813
ekk-10-kke
63
159


 


422
437
AAGAAGATGAGGCATA
552814
ekk-10-kke
65
161


 


457
472
CGGGCAACATACCTTG
552815
ekk-10-kke
63
167


 


458
473
ACGGGCAACATACCTT
552816
ekk-10-kke
88
168


 


639
654
GGCCCACTCCCATAGG
552817
ekk-10-kke
94
176


 


641
656
GAGGCCCACTCCCATA
552818
ekk-10-kke
82
177


 


642
657
TGAGGCCCACTCCCAT
552819
ekk-10-kke
80
178


 


643
658
CTGAGGCCCACTCCCA
552820
ekk-10-kke
84
179


 


670
685
CTAGTAAACTGAGCCA
552821
ekk-10-kke
71
181


 


671
686
ACTAGTAAACTGAGCC
552822
ekk-10-kke
85
1322


 


672
687
CACTAGTAAACTGAGC
552823
ekk-10-kke
71
1323


 


673
688
GCACTAGTAAACTGAG
552824
ekk-10-kke
81
1324


 


678
693
AAATGGCACTAGTAAA
552825
ekk-10-kke
51
1364


 


679
694
CAAATGGCACTAGTAA
552826
ekk-10-kke
64
1365


 


680
695
ACAAATGGCACTAGTA
552827
ekk-10-kke
61
1366


 


681
696
AACAAATGGCACTAGT
552828
ekk-10-kke
76
1367


 


682
697
GAACAAATGGCACTAG
552829
ekk-10-kke
61
1368


 


683
698
TGAACAAATGGCACTA
552830
ekk-10-kke
59
1369


 


684
699
CTGAACAAATGGCACT
552831
ekk-10-kke
58
1370


 


685
700
ACTGAACAAATGGCAC
552832
ekk-10-kke
64
1371


 


686
701
CACTGAACAAATGGCA
552833
ekk-10-kke
75
1372


 


687
702
CCACTGAACAAATGGC
552834
ekk-10-kke
84
188


 


688
703
ACCACTGAACAAATGG
552835
ekk-10-kke
57
190


 


689
704
AACCACTGAACAAATG
552836
ekk-10-kke
51
191


 


690
705
GAACCACTGAACAAAT
552837
ekk-10-kke
53
192


 


691
706
CGAACCACTGAACAAA
552838
ekk-10-kke
48
194


 


738
753
CCACATCATCCATATA
552839
ekk-10-kke
50
199


 


739
754
ACCACATCATCCATAT
552840
ekk-10-kke
54
201


 


1176
1191
CAGCAAACACTTGGCA
552841
ekk-10-kke
61
208


 


1177
1192
TCAGCAAACACTTGGC
552842
ekk-10-kke
71
209


 


1261
1276
GCAGTATGGATCGGCA
552843
ekk-10-kke
75
211


 


1262
1277
CGCAGTATGGATCGGC
552844
ekk-10-kke
78
1325


 


1263
1278
CCGCAGTATGGATCGG
552845
ekk-10-kke
52
1326


 


1264
1279
TCCGCAGTATGGATCG
552846
ekk-10-kke
76
1327


 


1265
1280
TTCCGCAGTATGGATC
552847
ekk-10-kke
61
1328


 


1266
1281
GTTCCGCAGTATGGAT
552848
ekk-10-kke
72
1329


 


1267
1282
AGTTCCGCAGTATGGA
552849
ekk-10-kke
87
1330


 


1268
1283
GAGTTCCGCAGTATGG
552850
ekk-10-kke
76
1331


 


1269
1284
GGAGTTCCGCAGTATG
552851
ekk-10-kke
76
1332


 


1270
1285
AGGAGTTCCGCAGTAT
552852
ekk-10-kke
79
1333


 


1577
1592
AGCGAAGTGCACACGG
552853
ekk-10-kke
82
1334


 


1578
1593
AAGCGAAGTGCACACG
552854
ekk-10-kke
85
1335


 


1579
1594
GAAGCGAAGTGCACAC
552855
ekk-10-kke
78
1336


 


1580
1595
TGAAGCGAAGTGCACA
552856
ekk-10-kke
77
1337


 


1581
1596
GTGAAGCGAAGTGCAC
552857
ekk-10-kke
75
1338


 


1582
1597
GGTGAAGCGAAGTGCA
552858
ekk-10-kke
75
1339


 


1583
1598
AGGTGAAGCGAAGTGC
552859
ekk-10-kke
79
1340


 


1584
1599
GAGGTGAAGCGAAGTG
552860
ekk-10-kke
71
1341


 


1585
1600
AGAGGTGAAGCGAAGT
552861
ekk-10-kke
74
1342


 


1586
1601
CAGAGGTGAAGCGAAG
552862
ekk-10-kke
66
1343


 


1587
1602
GCAGAGGTGAAGCGAA
552863
ekk-10-kke
70
1344


 


1588
1603
TGCAGAGGTGAAGCGA
552864
ekk-10-kke
73
1345
















TABLE 49







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371













Viral
Viral







Target
Target




SEQ


Start
Stop



%
ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
eeeee-10-eeeee
60
224


 


58
73
TGGAGCCACCAGCAGG
552889
ek-10-keke
59
1318


 


59
74
CTGGAGCCACCAGCAG
552890
ek-10-keke
56
1319


 


60
75
ACTGGAGCCACCAGCA
552891
ek-10-keke
67
1320


 


61
76
AACTGGAGCCACCAGC
552892
ek-10-keke
65
1321


 


62
77
GAACTGGAGCCACCAG
552893
ek-10-keke
68
86


 


250
265
TCCACCACGAGTCTAG
552894
ek-10-keke
71
98


 


251
266
GTCCACCACGAGTCTA
552895
ek-10-keke
51
100


 


252
267
AGTCCACCACGAGTCT
552896
ek-10-keke
51
102


 


253
268
AAGTCCACCACGAGTC
552897
ek-10-keke
43
104


 


254
269
GAAGTCCACCACGAGT
552898
ek-10-keke
43
106


 


255
270
AGAAGTCCACCACGAG
552899
ek-10-keke
55
109


 


256
271
GAGAAGTCCACCACGA
552900
ek-10-keke
34
112


 


258
273
GAGAGAAGTCCACCAC
552901
ek-10-keke
42
115


 


259
274
TGAGAGAAGTCCACCA
552902
ek-10-keke
60
117


 


411
426
GCATAGCAGCAGGATG
552903
ek-10-keke
76
137


 


412
427
GGCATAGCAGCAGGAT
552904
ek-10-keke
74
140


 


413
428
AGGCATAGCAGCAGGA
552905
ek-10-keke
66
143


 


415
430
TGAGGCATAGCAGCAG
552907
ek-10-keke
69
147


 


416
431
ATGAGGCATAGCAGCA
552908
ek-10-keke
63
149


 


417
432
GATGAGGCATAGCAGC
552909
ek-10-keke
70
151


 


418
433
AGATGAGGCATAGCAG
552910
ek-10-keke
72
153


 


457
472
CGGGCAACATACCTTG
552911
ek-10-keke
72
167


 


458
473
ACGGGCAACATACCTT
552912
ek-10-keke
67
168


 


670
685
CTAGTAAACTGAGCCA
552913
ek-10-keke
74
181


 


682
697
GAACAAATGGCACTAG
552914
ek-10-keke
75
1368


 


684
699
CTGAACAAATGGCACT
552915
ek-10-keke
58
1370


 


686
701
CACTGAACAAATGGCA
552916
ek-10-keke
74
1372


 


687
702
CCACTGAACAAATGGC
552917
ek-10-keke
76
188


 


688
703
ACCACTGAACAAATGG
552918
ek-10-keke
75
190


 


689
704
AACCACTGAACAAATG
552919
ek-10-keke
55
191


 


690
705
GAACCACTGAACAAAT
552920
ek-10-keke
49
192


 


691
706
CGAACCACTGAACAAA
552921
ek-10-keke
45
194


 


1261
1276
GCAGTATGGATCGGCA
552922
ek-10-keke
83
211


 


1262
1277
CGCAGTATGGATCGGC
552923
ek-10-keke
83
1325


 


1263
1278
CCGCAGTATGGATCGG
552924
ek-10-keke
0
1326


 


1264
1279
TCCGCAGTATGGATCG
552925
ek-10-keke
85
1327


 


1265
1280
TTCCGCAGTATGGATC
552926
ek-10-keke
50
1328


 


1266
1281
GTTCCGCAGTATGGAT
552927
ek-10-keke
76
1329


 


1267
1282
AGTTCCGCAGTATGGA
552928
ek-10-keke
78
1330


 


1268
1283
GAGTTCCGCAGTATGG
552929
ek-10-keke
75
1331


 


1269
1284
GGAGTTCCGCAGTATG
552930
ek-10-keke
78
1332


 


1270
1285
AGGAGTTCCGCAGTAT
552931
ek-10-keke
74
1333


 


1577
1592
AGCGAAGTGCACACGG
552932
ek-10-keke
86
1334


 


1578
1593
AAGCGAAGTGCACACG
552933
ek-10-keke
82
1335


 


1579
1594
GAAGCGAAGTGCACAC
552934
ek-10-keke
74
1336


 


1580
1595
TGAAGCGAAGTGCACA
552935
ek-10-keke
76
1337


 


1581
1596
GTGAAGCGAAGTGCAC
552936
ek-10-keke
81
1338


 


1582
1597
GGTGAAGCGAAGTGCA
552937
ek-10-keke
80
1339


 


1583
1598
AGGTGAAGCGAAGTGC
552938
ek-10-keke
78
1340


 


1584
1599
GAGGTGAAGCGAAGTG
552939
ek-10-keke
75
1341


 


1585
1600
AGAGGTGAAGCGAAGT
552940
ek-10-keke
63
1342


 


1586
1601
CAGAGGTGAAGCGAAG
552941
ekk-10-kke
78
1343


 


1587
1602
GCAGAGGTGAAGCGAA
552942
ek-10-keke
80
1344


 


1589
1604
GTGCAGAGGTGAAGCG
552865
ekk-10-kke
67
1346


 


1590
1605
CGTGCAGAGGTGAAGC
552866
ekk-10-kke
68
1347


 


1778
1793
TATGCCTACAGCCTCC
552868
ekk-10-kke
55
230


 


1779
1794
TTATGCCTACAGCCTC
552869
ekk-10-kke
48
231


 


1780
1795
TTTATGCCTACAGCCT
552870
ekk-10-kke
55
232


 


1781
1796
ATTTATGCCTACAGCC
552871
ekk-10-kke
57
233


 


1782
1797
AATTTATGCCTACAGC
552872
ekk-10-kke
70
234


 


1783
1798
CAATTTATGCCTACAG
552873
ekk-10-kke
49
235


 


1784
1799
CCAATTTATGCCTACA
552874
ekk-10-kke
42
236


 


1785
1800
ACCAATTTATGCCTAC
552875
ekk-10-kke
41
237


 


1822
1837
GGCAGAGGTGAAAAAG
552876
ekk-10-kke
50
244


 


1823
1838
AGGCAGAGGTGAAAAA
552877
ek-10-keke
39
245


 


1824
1839
TAGGCAGAGGTGAAAA
552878
ekk-10-kke
31
247


 


1865
1880
AGCTTGGAGGCTTGAA
552879
ekk-10-kke
5
252


 


1866
1881
CAGCTTGGAGGCTTGA
552880
ekk-10-kke
5
254


 


1867
1882
ACAGCTTGGAGGCTTG
552881
ekk-10-kke
10
256


 


1868
1883
CACAGCTTGGAGGCTT
552882
ekk-10-kke
11
258


 


1869
1884
GCACAGCTTGGAGGCT
552883
ekk-10-kke
27
260


 


1870
1885
GGCACAGCTTGGAGGC
552884
ekk-10-kke
36
262


 


1871
1886
AGGCACAGCTTGGAGG
552885
ekk-10-kke
12
264


 


1872
1887
AAGGCACAGCTTGGAG
552886
ekk-10-kke
32
266


 


1874
1889
CCAAGGCACAGCTTGG
552888
ekk-10-kke
1
271
















TABLE 50







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371













Viral
Viral







Target
Target




SEQ


Start
Stop




ID


Site
Site
Sequence
ISIS No
Motif
% inhibition
NO
















1577
1596
GTGAAGCGAAGTGCACACGG
146786
eeeee-10-eeeee
59
224


 


58
73
TGGAGCCACCAGCAGG
552955
eee-10-kkk
60
1318


 


59
74
CTGGAGCCACCAGCAG
552956
eee-10-kkk
60
1319


 


60
75
ACTGGAGCCACCAGCA
552957
eee-10-kkk
64
1320


 


61
76
AACTGGAGCCACCAGC
552958
eee-10-kkk
56
1321


 


62
77
GAACTGGAGCCACCAG
552959
eee-10-kkk
59
86


 


250
265
TCCACCACGAGTCTAG
552960
eee-10-kkk
42
98


 


251
266
GTCCACCACGAGTCTA
552961
eee-10-kkk
41
100


 


252
267
AGTCCACCACGAGTCT
552962
eee-10-kkk
35
102


 


253
268
AAGTCCACCACGAGTC
552963
eee-10-kkk
19
104


 


254
269
GAAGTCCACCACGAGT
552964
eee-10-kkk
34
106


 


255
270
AGAAGTCCACCACGAG
552965
eee-10-kkk
42
109


 


256
271
GAGAAGTCCACCACGA
552966
eee-10-kkk
60
112


 


258
273
GAGAGAAGTCCACCAC
552967
eee-10-kkk
38
115


 


259
274
TGAGAGAAGTCCACCA
552968
eee-10-kkk
35
117


 


411
426
GCATAGCAGCAGGATG
552969
eee-10-kkk
67
137


 


412
427
GGCATAGCAGCAGGAT
552970
eee-10-kkk
56
140


 


413
428
AGGCATAGCAGCAGGA
552971
eee-10-kkk
69
143


 


414
429
GAGGCATAGCAGCAGG
552972
eee-10-kkk
75
145


 


415
430
TGAGGCATAGCAGCAG
552973
eee-10-kkk
59
145


 


416
431
ATGAGGCATAGCAGCA
552974
eee-10-kkk
71
149


 


417
432
GATGAGGCATAGCAGC
552975
eee-10-kkk
56
151


 


418
433
AGATGAGGCATAGCAG
552976
eee-10-kkk
50
153


 


457
472
CGGGCAACATACCTTG
552977
eee-10-kkk
56
167


 


458
473
ACGGGCAACATACCTT
552978
eee-10-kkk
43
168


 


670
685
CTAGTAAACTGAGCCA
552979
eee-10-kkk
71
181


 


682
697
GAACAAATGGCACTAG
552980
eee-10-kkk
80
1368


 


684
699
CTGAACAAATGGCACT
552981
eee-10-kkk
64
1370


 


686
701
CACTGAACAAATGGCA
552982
ek-10-keke
61
1372


 


687
702
CCACTGAACAAATGGC
552983
eee-10-kkk
77
188


 


688
703
ACCACTGAACAAATGG
552984
eee-10-kkk
65
190


 


689
704
AACCACTGAACAAATG
552985
eee-10-kkk
41
191


 


690
705
GAACCACTGAACAAAT
552986
eee-10-kkk
30
192


 


691
706
CGAACCACTGAACAAA
552987
eee-10-kkk
41
194


 


1261
1276
GCAGTATGGATCGGCA
552988
eee-10-kkk
74
211


 


1262
1277
CGCAGTATGGATCGGC
552989
eee-10-kkk
85
1325


 


1263
1278
CCGCAGTATGGATCGG
552990
eee-10-kkk
72
1326


 


1264
1279
TCCGCAGTATGGATCG
552991
eee-10-kkk
73
1327


 


1265
1280
TTCCGCAGTATGGATC
552992
eee-10-kkk
60
1328


 


1266
1281
GTTCCGCAGTATGGAT
552993
eee-10-kkk
52
1329


 


1267
1282
AGTTCCGCAGTATGGA
552994
eee-10-kkk
58
1330


 


1268
1283
GAGTTCCGCAGTATGG
552995
eee-10-kkk
70
1331


 


1269
1284
GGAGTTCCGCAGTATG
552996
eee-10-kkk
74
1332


 


1270
1285
AGGAGTTCCGCAGTAT
552997
eee-10-kkk
59
1333


 


1577
1592
AGCGAAGTGCACACGG
552998
eee-10-kkk
82
1334


 


1578
1593
AAGCGAAGTGCACACG
552999
eee-10-kkk
70
1335


 


1579
1594
GAAGCGAAGTGCACAC
553000
eee-10-kkk
67
1336


 


1580
1595
TGAAGCGAAGTGCACA
553001
eee-10-kkk
67
1337


 


1581
1596
GTGAAGCGAAGTGCAC
553002
eee-10-kkk
74
1338


 


1582
1597
GGTGAAGCGAAGTGCA
553003
eee-10-kkk
72
1339


 


1583
1598
AGGTGAAGCGAAGTGC
553004
eee-10-kkk
73
1340


 


1584
1599
GAGGTGAAGCGAAGTG
553005
eee-10-kkk
67
1341


 


1585
1600
AGAGGTGAAGCGAAGT
553006
eee-10-kkk
69
1342


 


1586
1601
CAGAGGTGAAGCGAAG
553007
eee-10-kkk
60
1343


 


1587
1602
GCAGAGGTGAAGCGAA
553008
eee-10-kkk
71
1344


 


1588
1603
TGCAGAGGTGAAGCGA
552943
ek-10-keke
77
1345


 


1588
1603
TGCAGAGGTGAAGCGA
553009
eee-10-kkk
78
1345


 


1589
1604
GTGCAGAGGTGAAGCG
552944
ek-10-keke
74
1346


 


1589
1604
GTGCAGAGGTGAAGCG
553010
eee-10-kkk
78
1346


 


1590
1605
CGTGCAGAGGTGAAGC
552945
ek-10-keke
76
1347


 


1590
1605
CGTGCAGAGGTGAAGC
553011
eee-10-kkk
72
1347


 


1591
1606
ACGTGCAGAGGTGAAG
552946
ek-10-keke
71
1348


 


1591
1606
ACGTGCAGAGGTGAAG
553012
eee-10-kkk
74
1348


 


1778
1793
TATGCCTACAGCCTCC
552947
ek-10-keke
54
230


 


1778
1793
TATGCCTACAGCCTCC
553013
eee-10-kkk
39
230


 


1779
1794
TTATGCCTACAGCCTC
552948
ek-10-keke
50
231


 


1779
1794
TTATGCCTACAGCCTC
553014
eee-10-kkk
37
231


 


1780
1795
TTTATGCCTACAGCCT
552949
ek-10-keke
8
232


 


1780
1795
TTTATGCCTACAGCCT
553015
eee-10-kkk
45
232


 


1781
1796
ATTTATGCCTACAGCC
552950
ek-10-keke
44
233


 


1781
1796
ATTTATGCCTACAGCC
553016
eee-10-kkk
47
233


 


1782
1797
AATTTATGCCTACAGC
552951
ek-10-keke
60
234


 


1782
1797
AATTTATGCCTACAGC
553017
eee-10-kkk
47
234


 


1783
1798
CAATTTATGCCTACAG
552952
ek-10-keke
35
235


 


1783
1798
CAATTTATGCCTACAG
553018
eee-10-kkk
30
235


 


1784
1799
CCAATTTATGCCTACA
552953
ek-10-keke
37
236


 


1784
1799
CCAATTTATGCCTACA
553019
eee-10-kkk
37
236


 


1785
1800
ACCAATTTATGCCTAC
552954
ek-10-keke
40
237


 


1785
1800
ACCAATTTATGCCTAC
553020
eee-10-kkk
24
237
















TABLE 51







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















58
73
TGGAGCCACCAGCAGG
552889
ek-10-keke
42
1318


 


59
74
CTGGAGCCACCAGCAG
552890
ek-10-keke
56
1319


 


60
75
ACTGGAGCCACCAGCA
552891
ek-10-keke
55
1320


 


61
76
AACTGGAGCCACCAGC
552892
ek-10-keke
53
1321


 


62
77
GAACTGGAGCCACCAG
552893
ek-10-keke
56
86


 


250
265
TCCACCACGAGTCTAG
552894
ek-10-keke
53
98


 


251
266
GTCCACCACGAGTCTA
552895
ek-10-keke
38
100


 


252
267
AGTCCACCACGAGTCT
552896
ek-10-keke
43
102


 


253
268
AAGTCCACCACGAGTC
552897
ek-10-keke
40
104


 


254
269
GAAGTCCACCACGAGT
552898
ek-10-keke
50
106


 


255
270
AGAAGTCCACCACGAG
552899
ek-10-keke
37
109


 


256
271
GAGAAGTCCACCACGA
552900
ek-10-keke
43
112


 


258
273
GAGAGAAGTCCACCAC
552901
ek-10-keke
56
115


 


259
274
TGAGAGAAGTCCACCA
552902
ek-10-keke
43
117


 


411
426
GCATAGCAGCAGGATG
552903
ek-10-keke
78
137


 


412
427
GGCATAGCAGCAGGAT
552904
ek-10-keke
75
140


 


413
428
AGGCATAGCAGCAGGA
552905
ek-10-keke
52
143


 


415
430
TGAGGCATAGCAGCAG
552907
ek-10-keke
75
147


 


416
431
ATGAGGCATAGCAGCA
552908
ek-10-keke
57
149


 


417
432
GATGAGGCATAGCAGC
552909
ek-10-keke
66
151


 


418
433
AGATGAGGCATAGCAG
552910
ek-10-keke
60
153


 


457
472
CGGGCAACATACCTTG
552911
ek-10-keke
65
167


 


458
473
ACGGGCAACATACCTT
552912
ek-10-keke
37
168


 


670
685
CTAGTAAACTGAGCCA
552913
ek-10-keke
76
181


 


682
697
GAACAAATGGCACTAG
552914
ek-10-keke
79
1368


 


684
699
CTGAACAAATGGCACT
552915
ek-10-keke
71
1370


 


686
701
CACTGAACAAATGGCA
552916
ek-10-keke
82
1372


 


687
702
CCACTGAACAAATGGC
552917
ek-10-keke
78
188


 


688
703
ACCACTGAACAAATGG
552918
ek-10-keke
64
190


 


689
704
AACCACTGAACAAATG
552919
ek-10-keke
38
191


 


690
705
GAACCACTGAACAAAT
552920
ek-10-keke
43
192


 


691
706
CGAACCACTGAACAAA
552921
ek-10-keke
49
194


 


1261
1276
GCAGTATGGATCGGCA
552922
ek-10-keke
90
211


 


1262
1277
CGCAGTATGGATCGGC
552923
ek-10-keke
92
1325


 


1263
1278
CCGCAGTATGGATCGG
552924
ek-10-keke
30
1326


 


1264
1279
TCCGCAGTATGGATCG
552925
ek-10-keke
81
1327


 


1265
1280
TTCCGCAGTATGGATC
552926
ek-10-keke
39
1328


 


1266
1281
GTTCCGCAGTATGGAT
552927
ek-10-keke
53
1329


 


1267
1282
AGTTCCGCAGTATGGA
552928
ek-10-keke
48
1330


 


1268
1283
GAGTTCCGCAGTATGG
552929
ek-10-keke
68
1331


 


1269
1284
GGAGTTCCGCAGTATG
552930
ek-10-keke
87
1332


 


1270
1285
AGGAGTTCCGCAGTAT
552931
ek-10-keke
87
1333


 


1577
1592
AGCGAAGTGCACACGG
552932
ek-10-keke
88
1334


 


1578
1593
AAGCGAAGTGCACACG
552933
ek-10-keke
75
1335


 


1579
1594
GAAGCGAAGTGCACAC
552934
ek-10-keke
76
1336


 


1580
1595
TGAAGCGAAGTGCACA
552935
ek-10-keke
71
1337


 


1581
1596
GTGAAGCGAAGTGCAC
552936
ek-10-keke
80
1338


 


1582
1597
GGTGAAGCGAAGTGCA
552937
ek-10-keke
81
1339


 


1583
1598
AGGTGAAGCGAAGTGC
552938
ek-10-keke
85
1340


 


1584
1599
GAGGTGAAGCGAAGTG
552939
ek-10-keke
82
1341


 


1585
1600
AGAGGTGAAGCGAAGT
552940
ek-10-keke
76
1342


 


1586
1601
CAGAGGTGAAGCGAAG
552941
ekk-10-kke
72
1343


 


1587
1602
GCAGAGGTGAAGCGAA
552942
ek-10-keke
85
1344


 


1589
1604
GTGCAGAGGTGAAGCG
552865
ekk-10-kke
70
1346


 


1590
1605
CGTGCAGAGGTGAAGC
552866
ekk-10-kke
65
1347


 


1778
1793
TATGCCTACAGCCTCC
552868
ekk-10-kke
36
230


 


1779
1794
TTATGCCTACAGCCTC
552869
ekk-10-kke
23
231


 


1780
1795
TTTATGCCTACAGCCT
552870
ekk-10-kke
49
232


 


1781
1796
ATTTATGCCTACAGCC
552871
ekk-10-kke
46
233


 


1782
1797
AATTTATGCCTACAGC
552872
ekk-10-kke
73
234


 


1783
1798
CAATTTATGCCTACAG
552873
ekk-10-kke
41
235


 


1784
1799
CCAATTTATGCCTACA
552874
ekk-10-kke
18
236


 


1785
1800
ACCAATTTATGCCTAC
552875
ekk-10-kke
0
237


 


1822
1837
GGCAGAGGTGAAAAAG
552876
ekk-10-kke
49
244


 


1823
1838
AGGCAGAGGTGAAAAA
552877
ek-10-keke
37
245


 


1824
1839
TAGGCAGAGGTGAAAA
552878
ekk-10-kke
28
247


 


1865
1880
AGCTTGGAGGCTTGAA
552879
ekk-10-kke
0
252


 


1866
1881
CAGCTTGGAGGCTTGA
552880
ekk-10-kke
12
254


 


1867
1882
ACAGCTTGGAGGCTTG
552881
ekk-10-kke
0
256


 


1868
1883
CACAGCTTGGAGGCTT
552882
ekk-10-kke
0
258


 


1869
1884
GCACAGCTTGGAGGCT
552883
ekk-10-kke
12
260


 


1870
1885
GGCACAGCTTGGAGGC
552884
ekk-10-kke
39
262


 


1871
1886
AGGCACAGCTTGGAGG
552885
ekk-10-kke
37
264


 


1872
1887
AAGGCACAGCTTGGAG
552886
ekk-10-kke
15
266


 


1874
1889
CCAAGGCACAGCTTGG
552888
ekk-10-kke
0
271
















TABLE 52







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370













Viral
Viral







Target
Target







Start
Stop



%
SEQ ID


Site
Site
Sequence
ISIS No
Motif
inhibition
NO
















58
73
TGGAGCCACCAGCAGG
552955
eee-10-kkk
67
1318


 


59
74
CTGGAGCCACCAGCAG
552956
eee-10-kkk
60
1319


 


60
75
ACTGGAGCCACCAGCA
552957
eee-10-kkk
73
1320


 


61
76
AACTGGAGCCACCAGC
552958
eee-10-kkk
63
1321


 


62
77
GAACTGGAGCCACCAG
552959
eee-10-kkk
58
86


 


250
265
TCCACCACGAGTCTAG
552960
eee-10-kkk
67
98


 


251
266
GTCCACCACGAGTCTA
552961
eee-10-kkk
78
100


 


252
267
AGTCCACCACGAGTCT
552962
eee-10-kkk
29
102


 


253
268
AAGTCCACCACGAGTC
552963
eee-10-kkk
25
104


 


254
269
GAAGTCCACCACGAGT
552964
eee-10-kkk
33
106


 


255
270
AGAAGTCCACCACGAG
552965
eee-10-kkk
55
109


 


256
271
GAGAAGTCCACCACGA
552966
eee-10-kkk
71
112


 


258
273
GAGAGAAGTCCACCAC
552967
eee-10-kkk
23
115


 


259
274
TGAGAGAAGTCCACCA
552968
eee-10-kkk
41
117


 


411
426
GCATAGCAGCAGGATG
552969
eee-10-kkk
76
137


 


412
427
GGCATAGCAGCAGGAT
552970
eee-10-kkk
44
140


 


413
428
AGGCATAGCAGCAGGA
552971
eee-10-kkk
77
143


 


414
429
GAGGCATAGCAGCAGG
552972
eee-10-kkk
74
145


 


415
430
TGAGGCATAGCAGCAG
552973
eee-10-kkk
61
145


 


416
431
ATGAGGCATAGCAGCA
552974
eee-10-kkk
73
149


 


417
432
GATGAGGCATAGCAGC
552975
eee-10-kkk
66
151


 


418
433
AGATGAGGCATAGCAG
552976
eee-10-kkk
70
153


 


457
472
CGGGCAACATACCTTG
552977
eee-10-kkk
65
167


 


458
473
ACGGGCAACATACCTT
552978
eee-10-kkk
40
168


 


670
685
CTAGTAAACTGAGCCA
552979
eee-10-kkk
79
181


 


682
697
GAACAAATGGCACTAG
552980
eee-10-kkk
81
64


 


684
699
CTGAACAAATGGCACT
552981
eee-10-kkk
74
66


 


686
701
CACTGAACAAATGGCA
552982
ek-10-keke
52
68


 


687
702
CCACTGAACAAATGGC
552983
eee-10-kkk
78
188


 


688
703
ACCACTGAACAAATGG
552984
eee-10-kkk
71
190


 


689
704
AACCACTGAACAAATG
552985
eee-10-kkk
38
191


 


690
705
GAACCACTGAACAAAT
552986
eee-10-kkk
48
192


 


691
706
CGAACCACTGAACAAA
552987
eee-10-kkk
54
194


 


1261
1276
GCAGTATGGATCGGCA
552988
eee-10-kkk
85
211


 


1262
1277
CGCAGTATGGATCGGC
552989
eee-10-kkk
84
1325


 


1263
1278
CCGCAGTATGGATCGG
552990
eee-10-kkk
79
1326


 


1264
1279
TCCGCAGTATGGATCG
552991
eee-10-kkk
53
1327


 


1265
1280
TTCCGCAGTATGGATC
552992
eee-10-kkk
68
1328


 


1266
1281
GTTCCGCAGTATGGAT
552993
eee-10-kkk
67
1329


 


1267
1282
AGTTCCGCAGTATGGA
552994
eee-10-kkk
69
1330


 


1268
1283
GAGTTCCGCAGTATGG
552995
eee-10-kkk
62
1331


 


1269
1284
GGAGTTCCGCAGTATG
552996
eee-10-kkk
82
1332


 


1270
1285
AGGAGTTCCGCAGTAT
552997
eee-10-kkk
58
1333


 


1577
1592
AGCGAAGTGCACACGG
552998
eee-10-kkk
86
1334


 


1578
1593
AAGCGAAGTGCACACG
552999
eee-10-kkk
63
1335


 


1579
1594
GAAGCGAAGTGCACAC
553000
eee-10-kkk
67
1336


 


1580
1595
TGAAGCGAAGTGCACA
553001
eee-10-kkk
70
1337


 


1581
1596
GTGAAGCGAAGTGCAC
553002
eee-10-kkk
84
1338


 


1582
1597
GGTGAAGCGAAGTGCA
553003
eee-10-kkk
83
1339


 


1583
1598
AGGTGAAGCGAAGTGC
553004
eee-10-kkk
68
1340


 


1584
1599
GAGGTGAAGCGAAGTG
553005
eee-10-kkk
57
1341


 


1585
1600
AGAGGTGAAGCGAAGT
553006
eee-10-kkk
74
1342


 


1586
1601
CAGAGGTGAAGCGAAG
553007
eee-10-kkk
62
1343


 


1587
1602
GCAGAGGTGAAGCGAA
553008
eee-10-kkk
50
1344


 


1588
1603
TGCAGAGGTGAAGCGA
552943
ek-10-keke
86
1345


 


1588
1603
TGCAGAGGTGAAGCGA
553009
eee-10-kkk
79
1345


 


1589
1604
GTGCAGAGGTGAAGCG
552944
ek-10-keke
83
1346


 


1589
1604
GTGCAGAGGTGAAGCG
553010
eee-10-kkk
74
1346


 


1590
1605
CGTGCAGAGGTGAAGC
552945
ek-10-keke
79
1347


 


1590
1605
CGTGCAGAGGTGAAGC
553011
eee-10-kkk
60
1347


 


1591
1606
ACGTGCAGAGGTGAAG
552946
ek-10-keke
68
1348


 


1591
1606
ACGTGCAGAGGTGAAG
553012
eee-10-kkk
78
1348


 


1778
1793
TATGCCTACAGCCTCC
552947
ek-10-keke
51
230


 


1778
1793
TATGCCTACAGCCTCC
553013
eee-10-kkk
45
230


 


1779
1794
TTATGCCTACAGCCTC
552948
ek-10-keke
56
231


 


1779
1794
TTATGCCTACAGCCTC
553014
eee-10-kkk
53
231


 


1780
1795
TTTATGCCTACAGCCT
552949
ek-10-keke
1
232


 


1780
1795
TTTATGCCTACAGCCT
553015
eee-10-kkk
55
232


 


1781
1796
ATTTATGCCTACAGCC
552950
ek-10-keke
52
233


 


1781
1796
ATTTATGCCTACAGCC
553016
eee-10-kkk
65
233


 


1782
1797
AATTTATGCCTACAGC
552951
ek-10-keke
59
234


 


1782
1797
AATTTATGCCTACAGC
553017
eee-10-kkk
36
234


 


1783
1798
CAATTTATGCCTACAG
552952
ek-10-keke
34
235


 


1783
1798
CAATTTATGCCTACAG
553018
eee-10-kkk
20
235


 


1784
1799
CCAATTTATGCCTACA
552953
ek-10-keke
55
236


 


1784
1799
CCAATTTATGCCTACA
553019
eee-10-kkk
34
236


 


1785
1800
ACCAATTTATGCCTAC
552954
ek-10-keke
51
237


 


1785
1800
ACCAATTTATGCCTAC
553020
eee-10-kkk
28
237









Example 16
Dose-Dependent Antisense Inhibition of HBV mRNA in HepG2 Cells by MOE Gapmers
Antisense oligonucleotides from the study described in Example 14 exhibiting in vitro inhibition of HBV mRNA were selected and tested at various doses in HepG2 cells. Cells were plated at a density of 28,000 cells per well and transfected using LipofectAMINE2000® with 9.26 nM, 27.78 nM, 83.33 nM, and 250.00 nM concentrations of antisense oligonucleotide, as specified in Table 53. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. HBV primer probe set RTS3371 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.
As illustrated in Table 53, HBV mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. ‘n/a’ indicates that the data for that dosage is not available.







TABLE 53







Dose-dependent antisense inhibition


of human HBV in HepG2 cells











ISIS No
9.2593 nM
27.7778 nM
83.3333 nM
250.0 nM














146786
10
43
74
89


509934
12
31
52
79


509959
4
24
49
67


510100
11
28
60
77


510124
3
11
13
41


551926
1
26
51
76


551958
15
17
56
82


551987
4
40
65
81


551990
7
55
78
91


551993
15
30
70
80


551994
0
30
39
58


551995
6
41
73
85


551996
13
47
71
85


551997
16
38
68
89


551998
4
36
69
85


551999
10
31
67
86


552000
0
17
61
78


552006
6
37
74
89


552009
1
5
39
60


552013
0
28
3
72


552014
0
26
32
77


552018
6
27
63
81


552019
15
34
65
90


552020
2
35
65
91


552021
4
11
53
82


552022
6
35
57
79


552023
11
33
59
81


552024
15
43
69
91


552025
17
35
69
87


552026
14
26
66
86


552027
3
46
62
88


552028
9
43
58
78


552029
8
40
72
89


552030
18
48
77
92


552031
0
38
66
89


552032
42
48
80
88


552033
2
40
64
84


552034
6
40
70
81


552039
2
33
56
83


552044
19
30
63
84


552046
4
21
47
77


552050
15
44
70
92


552051
8
33
69
90


552052
17
38
71
91


552053
0
40
59
86


552054
7
15
58
75


552056
19
62
86
92


552057
11
33
69
86


552058
30
55
79
90


552059
11
25
69
90


552060
9
32
61
86


552061
6
40
69
88


552062
22
48
75
89


552064
23
49
69
90


552065
10
8
69
86


552069
11
4
28
60


552073
9
31
62
78


552075
21
18
33
65


552077
0
17
40
72


552079
1
12
44
70


552080
3
12
34
69


552082
13
29
66
87


552083
24
54
69
88


552084
10
25
48
82


552085
28
35
64
85


552086
0
24
65
84


552088
33
53
77
93


552089
0
41
69
92


552090
17
35
70
87


552091
13
31
69
89


552092
6
23
66
89


552093
0
17
61
89


552094
12
38
65
88


552095
20
42
73
88


552096
n/a
39
66
91


552097
24
43
67
88


552098
0
24
56
85


552101
3
13
28
61


552147
11
27
58
80


552160
20
25
69
89


552163
0
21
22
53


552176
16
11
40
66


552192
7
38
78
89


552222
0
24
65
79


552247
0
38
69
86


552255
5
27
69
81


552301
5
38
65
86


552309
8
26
62
85


552312
0
4
32
62


552347
2
15
38
75


552348
12
40
42
65


552354
10
35
44
76


552361
2
25
55
74


552363
20
36
54
76


552374
7
4
38
76


552379
0
12
24
46


552403
8
27
54
76


552408
2
25
44
77


552409
6
31
56
80


552418
0
30
72
84


552420
9
34
53
81


552442
4
23
46
56


552466
0
23
56
79


552474
11
34
66
87


552477
11
22
44
64


552530
25
37
73
87


552559
9
13
29
51









Example 17
Dose-Dependent Antisense Inhibition of HBV mRNA in HepG2 Cells by Deoxy, MOE and (S)-cEt Gapmers
Antisense oligonucleotides from the study described in Example 15 exhibiting in vitro inhibition of HBV mRNA were selected and tested at various doses in HepG2 cells. Cells were plated at a density of 28,000 cells per well and transfected using LipofectAMINE2000® with 9.26 nM, 27.78 nM, 83.33 nM, and 250.00 nM concentrations of antisense oligonucleotide, as specified in Table 54. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. HBV primer probe set RTS3371 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.
As illustrated in Table 54, HBV mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.







TABLE 54







Dose-dependent antisense inhibition


of human HBV in HepG2 cells











ISIS No
9.2593 nM
27.7778 nM
83.3333 nM
250.0 nM














146786
10
43
74
89


552808
13
14
55
70


552816
38
73
87
92


552818
29
63
87
85


552820
58
83
90
90


552821
33
49
71
88


552822
24
55
74
88


552824
8
24
65
87


552834
11
28
68
89


552849
12
25
73
84


552851
13
42
74
89


552852
4
35
70
87


552853
19
52
86
93


552854
28
57
80
89


552916
5
26
64
82


552922
25
44
77
89


552923
22
49
82
91


552925
33
56
80
92


552930
12
49
79
89


552931
12
40
62
82


552932
24
62
84
91


552933
20
40
75
89


552936
18
36
75
88


552937
22
51
82
88


552938
12
36
67
80


552939
17
40
65
79


552942
21
48
74
88


552943
5
39
70
85


552944
14
33
70
77


552980
15
40
69
86


552988
4
36
58
84


552989
0
50
74
81


552996
0
25
53
72


552998
17
49
79
90


553002
0
32
68
86


553003
15
42
67
88









Example 18
Antisense Inhibition of HBV Viral mRNA in HepG2 Cells by Deoxy, MOE and (S)-cEt Gapmers
Additional antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. ISIS 5808 and ISIS 9591, disclosed in U.S. Pat. No. 5,985,662, as well as ISIS 146781, ISIS 146786, 524518, ISIS 552859, and ISIS 552870 were also included in these studies for comparison and are distinguished with an asterisk. Cultured HepG2 cells at a density of 28,000 cells per well were transfected using LipofectAMINE2000® with 100 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe sets RTS3370 and RTS3371 and were used to separately measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in Table below were designed as MOE gapmers or deoxy, MOE and (S)-cEt gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleosides each. The deoxy, MOE and (S)-cEt gapmers are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the number indicates the number of deoxynucleosides; otherwise, ‘d’ indicates a deoxynucleoside; and ‘e’ indicates a MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
“Viral Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. Each gapmer listed in Table 55 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1).







TABLE 55







Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with


RTS3370 or RTS3371














Viral
Viral








Target
Target


%
%

SEQ


Start
Stop


inhibition
inhibition

ID


Site
Site
ISIS No
Motif
(RTS3370)
(RTS3371)
Sequence
NO

















156
176
  5808*
Uniform deoxy
57
64
CCTGATGTGATGTTCTCCATG
1373


 


303
322
524518*
eeeee-10-eeeee
62
72
GGGACTGCGAATTTTGGCCA
428


 


376
395
146781*
eeeee-10-eeeee
72
93
AAACGCCGCAGACACATCCA
1374


 


380
399
582665
eeeee-10-eeeee
57
59
GATAAAACGCCGCAGACACA
1375


 


382
401
582666
eeeee-10-eeeee
49
92
ATGATAAAACGCCGCAGACA
1376


 


411
426
566831
kdkdk-9-ee
96
73
GCATAGCAGCAGGATG
137


 


411
427
577123
eekk-9-ekee
84
96
GGCATAGCAGCAGGATG
17


 


411
427
577124
kdkdk-8-eeee
92
96
GGCATAGCAGCAGGATG
17


 


411
426
577126
kkk-8-eeeee
87
90
GCATAGCAGCAGGATG
137


 


413
428
566830
kdkdk-9-ee
93
95
AGGCATAGCAGCAGGA
143


 


415
430
577130
eek-10-kke
87
94
TGAGGCATAGCAGCAG
147


 


415
430
577131
kdkdk-9-ee
83
93
TGAGGCATAGCAGCAG
147


 


1263
1278
566828
kdkdk-9-ee
97
90
CCGCAGTATGGATCGG
1236


 


1577
1596
146786*
eeeee-10-eeeee
93
71
GTGAAGCGAAGTGCACACGG
224


 


1577
1592
566829
kdkdk-9-ee
98
84
AGCGAAGTGCACACGG
1334


 


1577
1596
577120
kdkdk-10-eeeee
94
93
GTGAAGCGAAGTGCACACGG
224


 


1577
1592
577127
kkk-8-eeeee
95
70
AGCGAAGTGCACACGG
1334


 


1577
1592
577134
kek-8-eeeee
94
89
AGCGAAGTGCACACGG
1334


 


1577
1592
577135
kek-10-kek
96
94
AGCGAAGTGCACACGG
1334


 


1583
1598
552859*
ekk-10-kke
92
91
AGGTGAAGCGAAGTGC
1340


 


1583
1602
577121
kdkdk-10-eeeee
91
74
GCAGAGGTGAAGCGAAGTGC
226


 


1583
1598
577128
kkk-8-eeeee
92
85
AGGTGAAGCGAAGTGC
1340


 


1583
1598
577132
kdkdk-9-ee
97
81
AGGTGAAGCGAAGTGC
1340


 


1583
1598
577136
kek-10-kek
95
95
AGGTGAAGCGAAGTGC
1340


 


1588
1603
566832
kdkdk-9-ee
95
78
TGCAGAGGTGAAGCGA
1345


 


1780
1795
552870*
ekk-10-kke
71
93
TTTATGCCTACAGCCT
232


 


1780
1799
577122
kdkdk-10-eeeee
70
96
CCAATTTATGCCTACAGCCT
50


 


1780
1796
577125
kdkdk-8-eeee
70
94
ATTTATGCCTACAGCCT
51


 


1780
1795
577129
kkk-8-eeeee
76
51
TTTATGCCTACAGCCT
232


 


1780
1795
577133
kdkdk-9-ee
80
52
TTTATGCCTACAGCCT
232


 


1873
1892
  9591*
Uniform deoxy
30
14
CACCCAAGGCACAGCTTGG
1377









Example 19
Efficacy of Gapmers Targeting HBV in Transgenic Mice
Transgenic mice were treated with ISIS antisense oligonucleotides in a number of studies to evaluate the efficacy of the gapmers. HBV DNA and RNA levels were assessed.
Study 1
Groups of 12 mice each were injected subcutaneously twice a week for 4 weeks with 50 mg/kg of ISIS 510106, ISIS 510116, ISIS 505347, or ISIS 509934. A control group of 12 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and livers were harvested for further analysis.
DNA and RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe sets RTS3370, RTS3371, and RTS3372. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe sets RTS3370 and RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 56, expressed as percent inhibition compared to the control group. As shown in Table 56, most of the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The Chemistry column indicates the gap-wing motif of each gapmer.







TABLE 56







Percent inhibition of HBV RNA and DNA in the liver of transgenic mice
















%
%
%
%
%
%




inhibition
inhibition
inhibition
inhibition
inhibition
inhibition


ISIS

DNA
DNA
DNA
RNA
RNA
RNA


No
Chemistry
(RTS3370)
(RTS3371)
(RTS3372)
(RTS3370)
(RTS3371)
(RTS3372)

















505347
5-10-5 MOE
72
79
75
54
28
30


509934
5-10-5 MOE
93
95
94
72
75
92


510106
3-10-4 MOE
0
0
51
0
0
12


510116
3-10-4 MOE
68
79
68
49
54
66









Study 2

Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 50 mg/kg of ISIS 146779, ISIS 505358, ISIS 146786, ISIS 509974, ISIS 509958, or ISIS 509959. A control group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and livers were harvested for further analysis.
DNA and RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe sets RTS3370. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe sets RTS3370 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 57, expressed as percent inhibition compared to the control group. As shown in Table 57, most of the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The Chemistry column indicates the gap-wing motif of each gapmer.







TABLE 57







Percent inhibition of HBV RNA and


DNA in the liver of transgenic mice












% inhibition
% inhibition


ISIS No
Chemistry
DNA
RNA













146779
5-10-5 MOE
39
5


146786
5-10-5 MOE
83
73


505358
5-10-5 MOE
84
77


509958
3-10-3 MOE
82
29


509959
3-10-3 MOE
54
30


509974
3-10-3 MOE
56
28









Study 3

Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 50 mg/kg of ISIS 509960, ISIS 505329, ISIS 146786, ISIS 505339, or ISIS 509927. Another group of 6 mice was administered Entecavir, an oral antiviral drug used to treat Hepatitis B infection, at 1 mg/kg daily for two weeks. A control group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and livers were harvested for further analysis.
DNA and RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe sets RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe sets RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 58, expressed as percent inhibition compared to the control group. As shown in Table 58, most of the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The Chemistry column indicates the gap-wing motif of each gapmer.







TABLE 58







Percent inhibition of HBV RNA and


DNA in the liver of transgenic mice












% inhibition
% inhibition



Oligo Chemistry
DNA
RNA














entecavir
—
94
0


ISIS 146786
5-10-5 MOE
97
92


ISIS 505329
5-10-5 MOE
70
63


ISIS 505339
5-10-5 MOE
74
63


ISIS 509927
5-10-5 MOE
80
57


ISIS 509960
3-10-3 MOE
86
60









Study 4

Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786, ISIS 552176, and ISIS 552073. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
DNA and RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 59. As shown in Table 59, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The Chemistry column indicates the gap-wing motif of each gapmer.







TABLE 59







Percent inhibition of HBV RNA and DNA in transgenic mice












% inhibition of
% inhibition of


ISIS No
Chemistry
RNA
DNA













146786
5-10-5 MOE
81
91


552073
8-10-2 MOE
39
22


552176
3-9-5 MOE
55
56









Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of ALT were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.) (Nyblom, H. et al., Alcohol & Alcoholism 39: 336-339, 2004; Tietz N W (Ed): Clinical Guide to Laboratory Tests, 3rd ed. W. B. Saunders, Philadelphia, Pa., 1995). The results are presented in Table 60 expressed in IU/L. Both the ISIS oligonucleotides were considered tolerable in the mice, as demonstrated by their liver transaminase profile.







TABLE 60







ALT levels (IU/L) of transgenic mice









ALT














PBS
77



ISIS 146786
21



ISIS 552073
19



ISIS 552176
27










Study 5

Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786, ISIS 552056, ISIS 552088, and ISIS 552309. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
DNA and RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 61, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The Chemistry column indicates the gap-wing motif of each gapmer.







TABLE 61







Percent inhibition of HBV DNA and RNA in transgenic mice












% inhibition
% inhibition



Chemistry
(RNA)
(DNA)
















ISIS 146786
5-10-5 MOE
60
90



ISIS 552056
7-10-3 MOE
25
58



ISIS 552088
8-10-2 MOE
8
0



ISIS 552309
5-9-3 MOE
35
84










Study 6

Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786, ISIS 505330, ISIS 509932, ISIS 552032, ISIS 552057, ISIS 552075, ISIS 552092, and ISIS 552255. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
DNA and RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 62, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The Chemistry column indicates the gap-wing motif of each gapmer.







TABLE 62







Percent inhibition of HBV DNA and RNA in transgenic mice












% inhibition
% inhibition


ISIS No
Chemistry
(RNA)
(DNA)













146786
5-10-5 MOE
52
95


505330
5-10-5 MOE
7
61


509932
5-10-5 MOE
83
98


552032
6-10-4 MOE
54
97


552057
7-10-3 MOE
19
62


552075
8-10-2 MOE
12
18


552092
8-10-2 MOE
25
74


552255
4-9-4 MOE
41
89









Study 7

Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 20 mg/kg of ISIS 552859, ISIS 577121, ISIS 577122, ISIS 577123, ISIS 577132, ISIS 577133, and ISIS 577134. These gapmers have deoxy, MOE and (S)-cEt chemistry. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
DNA and RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 63, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the number indicates the number of deoxynucleosides; otherwise, ‘d’ indicates a deoxynucleoside; and ‘e’ indicates a MOE modification.







TABLE 63







Percent inhibition of HBV DNA and RNA in transgenic mice












% inhibition
% inhibition


ISIS No
Chemistry
(RNA)
(DNA)













552859
ekk-10-kke
60
86


577121
kdkdk-10-eeeee
59
93


577122
kdkdk-10-eeeee
42
68


577123
eekk-9-ekee
0
77


577132
kdkdk-9-ee
4
24


577133
kdkdk-9-ee
46
64


577134
kek-8-eeeee
0
17









Study 8

A group of 6 mice was injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786, the 5-10-5 MOE gapmer. Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 10 mg/kg of ISIS 552803, ISIS 552903, ISIS 552817, ISIS 552822, and ISIS 552907. These gapmers all had deoxy, MOE, and (S)-cEt chemistry. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
DNA and RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 64. As shown in Table 64, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the number indicates the number of deoxynucleosides; otherwise, ‘d’ indicates a deoxynucleoside; and ‘e’ indicates a MOE modification; in case of the MOE gapmers, the Chemistry column defines the gap-wing structure.







TABLE 64







Percent inhibition of HBV RNA and DNA in transgenic mice













Dose
% inhibition
% inhibition


ISIS No
Chemistry
(mg/kg/wk)
of RNA
of DNA














146786
5-10-5 MOE
50
81
91


552803
ekk-10-kke
20
71
95


552817
ekk-10-kke
20
86
51


552822
ekk-10-kke
20
90
89


552903
ek-10-keke
20
56
82


552907
ek-10-keke
20
41
45









Study 9

A group of 6 mice was injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786. Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 10 mg/kg of ISIS 552853, ISIS 552854, ISIS 552932, and ISIS 552938. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
DNA and RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 65, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the number indicates the number of deoxynucleosides; otherwise, ‘d’ indicates a deoxynucleoside; and ‘e’ indicates a MOE modification; in case of the MOE gapmers, the Chemistry column defines the gap-wing structure.







TABLE 65







Percent inhibition of HBV DNA and RNA in transgenic mice













Dose
% inhibition
% inhibition



Chemistry
(mg/kg/wk)
(DNA)
(RNA)















ISIS 146786
5-10-5 MOE
50
90
60


ISIS 552853
ekk-10-kke
20
94
60


ISIS 552854
ekk-10-kke
20
61
23


ISIS 552932
ek-10-keke
20
75
70


ISIS 552938
ek-10-keke
20
67
56









Study 10

A group of 6 mice was injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786. Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 10 mg/kg of ISIS 552922, ISIS 552923, ISIS 552942, ISIS 552872, ISIS 552925, ISIS 552937, and ISIS 552939. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
DNA and RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 66, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the number indicates the number of deoxynucleosides; otherwise, ‘d’ indicates a deoxynucleoside; and ‘e’ indicates a MOE modification; in case of the MOE gapmers, the Chemistry column defines the gap-wing structure.







TABLE 66







Percent inhibition of HBV DNA and RNA in transgenic mice













Dose
% inhibition
% inhibition


ISIS No
Chemistry
(mg/kg/wk)
(DNA)
(RNA)














146786
5-10-5 MOE
50
52
57


552922
ekk-10-kke
20
61
50


552923
ek-10-keke
20
89
76


552942
ek-10-keke
20
58
52


552872
ek-10-keke
20
77
46


552925
ek-10-keke
20
89
65


552937
ek-10-keke
20
59
35


552939
ek-10-keke
20
57
19









Example 20
Efficacy of Gapmers Targeting HBV in Transgenic Mice
Mice harboring a HBV gene fragment (Guidotti, L. G. et al., J. Virol. 1995, 69, 6158-6169) were used. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model. HBV DNA, RNA, and antigen levels were assessed.
Groups of 10 mice each were injected subcutaneously twice a week for the first with 50 mg/kg and, subsequently, twice a week for the next 3 weeks with 25 mg/kg of ISIS 146786 or ISIS 510100. Control groups of 10 mice each were treated in a similar manner with ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, SEQ ID NO: 320; 5-10-5 MOE gapmer with no known murine target) or ISIS 459024 (CGGTCCTTGGAGGATGC, SEQ ID NO: 1351; 3-10-4 MOE gapmer with no known murine target). Mice were euthanized 48 hours after the last dose, and organs and serum were harvested for further analysis.
DNA and RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe sets RTS3370, RTS3371, or RTS3372 (forward sequence ATCCTATCAACACTTCCGGAAACT, designated SEQ ID NO: 314; reverse sequence CGACGCGGCGATTGAG, designated SEQ ID NO: 315; probe sequence AAGAACTCCCTCGCCTCGCAGACG, designated SEQ ID NO: 316). The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe sets RTS3370 and RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 67. Serum DNA samples were analyzed after the study period. The data is presented in Table 68, expressed relative to the levels measured in the control group. As shown in Tables 67 and 68, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The Chemistry column defines the gap-wing structure of each gapmer.







TABLE 67







Percent inhibition of HBV RNA and DNA in the liver of transgenic mice
















%
%
%
%
%
%




inhibition 
inhibition
inhibition 
inhibition
inhibition
inhibition


ISIS

DNA
DNA
DNA
RNA
RNA
RNA


No
Chemistry
(RTS3370)
(RTS3371)
(RTS3372)
(RTS3370)
(RTS3371)
(RTS3372)

















146786
5-10-5 MOE
97
97
95
86
85
89


510100
3-10-4 MOE
95
94
94
56
64
77


141923
5-10-5 MOE
2
0
13
0
7
31


459024
3-10-4 MOE
19
0
8
0
0
0
















TABLE 68







Percent inhibition of HBV DNA in the serum of transgenic mice










% inhibition
% inhibition


ISIS No
(RTS3370)
(RTS3371)












146786
98
98


510100
99
98


141923
0
0


459024
0
0









HBV Antigen Analysis

HBV antigens in the supernatants were detected with the ELISA technique. HBs antigen (HBsAg) levels were detected by ELISA from Abazyme LLC, Mass. As presented in Table 57, treatment with ISIS oligonucleotides 146786 or 510100 caused reduction in HBsAg levels. HBe antigen (HBeAg) levels were detected by ELISA from International Immuno-diagnostics, CA. As presented in Table 69, treatment with ISIS oligonucleotides 146786 or 510100 also caused reduction in HBeAg levels.







TABLE 69







HBV antigen levels (PEI U/mL) in transgenic mice










HBsAg
HBeAg













PBS
40
80


146786
3
15


510100
15
22


141923
32
80


459024
44
51









Example 21
Antisense Inhibition of HBV Viral mRNA in HepG2 Cells by Deoxy, MOE and (S)-cEt Gapmers
Additional antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. ISIS 146786, ISIS 505358, ISIS 509932, and ISIS 510100, disclosed in U.S. Provisional Application No. 61/478,040 filed on Apr. 21, 2011; ISIS 552859 disclosed in U.S. Provisional Application No. 61/596,692 filed on Feb. 8, 2012; ISIS 577121, ISIS 577122, ISIS 577123, ISIS 577132, ISIS 577133, and ISIS 577134, disclosed in the study described above, were also included in the assay. Cultured HepG2 cells at a density of 28,000 cells per well were transfected using Cytofectin with 9.375 nM, 18.75 nM, 37.50 nM, 75.00 nM, 150.00 nM, or 300.00 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3371 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in Tables below were designed as deoxy, MOE and (S)-cEt gapmers. The deoxy, MOE and (S)-cEt gapmers are 16, 17, or 18 nucleosides in length wherein the nucleosides have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the number indicates the number of deoxynucleosides; otherwise, ‘d’ indicates a deoxynucleoside; and ‘e’ indicates a MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
“Viral Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. Each gapmer listed in Table 70 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1).







TABLE 70







Chimeric antisense oligonucleotides targeting SEQ ID NO: 1












Viral
Viral






Target
Target



SEQ ID


Start Site
Stop Site
ISIS No
Motif
Sequence
NO















411
427
585163
eeekk-8-eeee
GGCATAGCAGCAGGATG
17


 


414
430
585164
eeekk-7-kkeee
TGAGGCATAGCAGCAGG
21


 


414
430
585165
eeek-9-keee
TGAGGCATAGCAGCAGG
21


 


1577
1593
585170
eeekk-7-kkeee
AAGCGAAGTGCACACGG
1304


 


1577
1593
585171
eeek-9-keee
AAGCGAAGTGCACACGG
1304


 


1577
1593
585172
eeeekk-7-eeee
AAGCGAAGTGCACACGG
1304


 


1577
1593
585173
ekek-9-eeee
AAGCGAAGTGCACACGG
1304


 


1577
1593
585174
ekekdk-7-eeee
AAGCGAAGTGCACACGG
1304


 


1583
1599
585166
eeekk-7-kkeee
GAGGTGAAGCGAAGTGC
1310


 


1583
1599
585167
eeek-9-keee
GAGGTGAAGCGAAGTGC
1310


 


1780
1797
577119
kdkdk-8-eeeee
AATTTATGCCTACAGCCT
1379


 


1780
1796
585168
eeekk-7-kkeee
ATTTATGCCTACAGCCT
51


 


1780
1796
585169
eeek-9-keee
ATTTATGCCTACAGCCT
51
















TABLE 71







Dose dependent inhibition of HBV mRNA


levels by chimeric antisense oligonucleotides















ISIS 
9.375
18.75
37.5
75.0
150.0 
300.0



No
nM
nM
nM
nM
nM
nM



















146786
37
37
58
70
81
93



505358
30
26
28
57
74
85



510100
42
30
43
61
77
91



552859
21
30
39
61
79
91



577119
42
43
46
66
74
75



577121
10
15
42
64
82
89



577122
21
30
53
66
78
84



577123
27
29
45
56
78
84



577132
14
21
42
61
80
92



577133
12
14
32
47
62
77



577134
37
39
59
72
86
90



585174
31
28
48
61
80
90

















TABLE 72







Dose dependent inhibition of HBV mRNA


levels by chimeric antisense oligonucleotides















ISIS
9.375
18.75 
37.5
75.0
150.0
300.0



No
nM
nM
nM
nM
nM
nM



















146786
25
34
57
71
85
92



509932
9
28
59
62
70
74



585163
17
32
52
68
77
81



585164
23
4
29
31
36
56



585165
6
31
42
58
66
82



585166
19
27
35
48
50
63



585167
22
25
50
69
76
88



585168
4
30
44
52
67
76



585169
32
32
42
62
76
80



585170
23
19
39
49
66
75



585171
28
27
42
59
81
88



585172
26
29
30
64
80
91



585173
29
30
41
71
86
88










Example 22
Analysis of the Potency of Uniform Deoxyoligonucleotides in Inhibition of HBV mRNA in HepG2 Cells
Additional antisense oligonucleotides were tested for their effects on HBV mRNA in vitro. ISIS 5808 and ISIS 9591, disclosed in U.S. Pat. No. 5,985,662 were also included in the assay. ISIS 146786 was included in the assay as the benchmark. Cultured HepG2 cells at a density of 28,000 cells per well were transfected using LipofectAMINE2000® with 18.75 nM, 37.50 nM, 75.00 nM, 150.00 nM, or 300.00 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3371 was used to measure mRNA and DNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. S antigen and E antigen levels were also measured by ELISA. Results are presented as percent inhibition, relative to untreated control cells.
The antisense oligonucleotides tested, ISIS 582699, ISIS 582700, and ISIS 582701, were designed according to the sequences and chemistries disclosed in Korba and Gerin, Antiviral Research, 1995, Vol. 28, 225-242; the corresponding names for the oligonucleotides in the reference are S1, C1, and L2c, respectively. The antisense oligonucleotides in Tables below were designed as uniform deoxy oligonucleotides, 16 or 21 nucleosides in length wherein the nucleosides have deoxy modifications. “Viral Target start site” indicates the 5′-most nucleotide to which the oligonucleotide is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the oligonucleotide is targeted viral gene sequence. Each oligonucleotide listed in Table 73 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1). The results indicate that the deoxy oligonucleotides had negligible effect on HBV mRNA expression levels DNA levels and HBV antigen levels.







TABLE 73







Uniform deoxy oligonucleotides targeting SEQ ID


NO: 1











Viral
Viral





Target
Target





Start
Stop


SEQ ID


Site
Site
ISIS No
Sequence
NO














160
180
582699
GAATCCTGATGTGATGTTCTC
1378


 


1884
1899
582701
CCAAAGCCACCCAAGG
1380


 


1910
1930
582700
CAAATTCTTTATAAGGGTCGA
1381
















TABLE 74







Dose dependent inhibition of HBV mRNA levels


after treatment with oligonucleotides












ISIS No
18.75 nM
37.5 nM
75.0 nM
150.0 nM
300.0 nM















5808
38
23
29
40
54


9591
35
20
32
26
40


146786
11
5
45
66
92


582699
32
28
27
39
52


582700
18
12
20
16
23


582701
4
0
0
3
13
















TABLE 75







Dose dependent inhibition of HBV DNA levels in


HepG2 cells after treatment with oligonucleotides











ISIS No
18.75 nM
37.5 nM
75.0 nM
150.0 nM














5808
20
17
0
0


9591
0
0
0
0


146786
32
50
77
83


582699
0
44
0
17


582700
0
0
0
0


582701
0
0
0
0
















TABLE 76







HBV S antigen levels after treatment


with oligonucleotides (arbitrary units)











ISIS No
18.75 nM
37.5 nM
75.0 nM
150.0 nM














5808
9,254
8,228
4,168
2,540


9591
10,924
8,683
9,334
12,142


146786
12,501
7,265
3,408
1,017


582699
9,340
9,325
7,589
4,712


582700
9,697
8,350
11,168
10,703


582701
15,283
18,209
14,632
15,299
















TABLE 77







HBV E antigen levels after treatment


with oligonucleotides (arbitrary units)











ISIS No
18.75 nM
37.5 nM
75.0 nM
150.0 nM














5808
8,075
8,587
5,036
3,286


9591
9,242
8,093
8,257
6,944


146786
8,532
4,034
2,301
449


582699
7,815
7,191
7,026
5,278


582700
8,690
9,304
7,941
6,315


582701
8,847
8,257
8,211
6,276

## Claims

1. An antisense compound comprising a modified oligonucleotide consisting of 18-20 linked nucleosides having a nucleobase sequence comprising an at least 18 contiguous nucleobase portion 100% complementary to nucleotides 1827-1846 of SEQ ID NO: 1, wherein the 5′ end of the nucleobase sequence has a non-complementary nucleobase with respect to SEQ ID NO: 1.

2. The antisense compound of claim 1, wherein at least one internucleoside linkage of said modified oligonucleotide is a modified internucleoside linkage.

3. The antisense compound of claim 2, wherein the modified internucleoside linkage is a phosphorothioate linkage.

4. The antisense compound of claim 2, wherein at least one nucleoside of said modified oligonucleotide comprises a modified sugar.

5. The antisense compound of claim 4, wherein the modified sugar comprises 2′-O—CH3.

6. The antisense compound of claim 5, wherein the modified sugar comprises 2′-F.

7. The antisense compound of claim 1, wherein the compound consists of a single-stranded modified oligonucleotide.

8. The antisense compound of claim 1, wherein the compound comprises a double-stranded RNA oligonucleotide.

9. The antisense compound of claim 8, wherein at least one internucleoside linkage of said modified oligonucleotide is a modified internucleoside linkage.

10. The antisense compound of claim 9, wherein the modified internucleoside linkage is a phosphorothioate linkage.

11. The antisense compound of claim 8, wherein at least one nucleoside of said modified oligonucleotide comprises a modified sugar.

12. The antisense compound of claim 11, wherein the modified sugar comprises 2′-O—CH3.

13. The antisense compound of claim 11, wherein the modified sugar comprises 2′-F.

14. The antisense compound of claim 8, wherein the modified oligonucleotide comprises at least one phosphorothioate linkage and at least one modified sugar.

15. The antisense compound of claim 14, wherein the modified sugar comprises 2′-O—CH3.

16. The antisense compound of claim 15, wherein the modified sugar comprises 2′-F.

17. A method of preventing, treating, ameliorating, or slowing progression of a HBV-related disease, disorder or condition in an animal comprising administering to the animal the compound of claim 1, thereby preventing, treating, ameliorating, or slowing progression of a HBV-related disease, disorder or condition in the animal.

18. A method of preventing, treating, ameliorating, or slowing progression of a HBV-related disease, disorder or condition in an animal comprising administering to the animal the compound of claim 8, thereby preventing, treating, ameliorating, or slowing progression of a HBV-related disease, disorder or condition in the animal.

19. A method of preventing, treating, ameliorating, or slowing progression of a HBV-related disease, disorder or condition in an animal comprising administering to the animal the compound of claim 14, thereby preventing, treating, ameliorating, or slowing progression of a HBV-related disease, disorder or condition in the animal.

20. A method of preventing, treating, ameliorating, or slowing progression of a HBV-related disease, disorder or condition in an animal comprising administering to the animal the compound of claim 15 or 16, thereby preventing, treating, ameliorating, or slowing progression of a HBV-related disease, disorder or condition in the animal.