OA19828A - RNAi agents for hepatitis B virus infection - Google Patents

RNAi agents for hepatitis B virus infection Download PDF

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Publication number
OA19828A
OA19828A OA1201900396 OA19828A OA 19828 A OA19828 A OA 19828A OA 1201900396 OA1201900396 OA 1201900396 OA 19828 A OA19828 A OA 19828A
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seq
rnai agent
sequence
antisense strand
hbv
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OA1201900396
Inventor
David L. Lewis
David B. Rozema
Darren H. Wakefield
Rui ZHU
Christine I. Wooddell
Bruce D. Given
Tao Pei
Lauren J. Almeida
Zhen Li
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Arrowhead Pharmaceuticals, Inc.
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Publication of OA19828A publication Critical patent/OA19828A/en

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Abstract

Described are compositions and methods for inhibition of Hepatitis B virus gene expression. RNA interference (RNAi) agents for inhibiting the expression of Hepatitis B virus gene are described. The HBV RNAi agents disclosed herein may be targeted to cells, such as hepatocytes, for example, by using conjugated targeting ligands. Pharmaceutical compositions comprising one or more HBV RNAi agents optionally with one or more additional therapeutics arc also described. Delivery of the described HBV RNAi agents to infected liver in vivo provides for inhibition of HBV gene expression and treatment of diseases and conditions associated with HBV infection.

Description

RNAi Agents for Hepatitis B Virus Infection
Cross Reference To Related Applications
This application claims priority from United States Provisional Patent Application Serial No. 62/540,639. filed on August 3, 2017, United States Provisional Patent Application Serial No. 62/534,733, filed on July 20,2017, and United States Provisional Patent Application Serial No. 62/370,754, filed on August 4,2016, the contents of each of which are incorporated herein by reference in their entirety.
Field ofthe Invention
Disclosed herein are RNA interférence (RNAi) agents for inhibition of Hepatitis B Virus gene expression, compositions that include HBV RNAi agents, and methods of use thereof.
Background
The Hepatitis B Virus (HBV) is a strict hepatotrophic, double-stranded DNA containing virus. Although DNA is the genetic material, the réplication cycle involves a reverse transcription step to copy a pregenomic RNA into DNA. Hepatitis B Virus is classified as one member of the Hepadnaviruses and belongs to the family of Hepadnaviridae. The primary infection of adult humans with Hepatitis B Virus causes an acute hepatitis with symptoms of organ inflammation, fever, jaundice and increased liver transaminases in blood. Those patients that are not able to overcome the virus infection suffer a chronic disease progression over many years with increased risk of developing cinhotic liver or liver cancer. Périnatal transmission from Hepatitis B Virus-infected mothers to newboms also leads to chronic hepatitis.
Upon uptake by hépatocytes, the nucleocapsid is transferred to the nucléus and DNA is released. There, the DNA strand synthesis is completed and gaps repaired to give the covalently closed circular (ccc) supercoiled DNA of 3.2kb. The cccDNA serves as a template for transcription of five major viral mRNAs, which are 3.5, 3.5, 2.4, 2.1 and 0.7 kb long. Ail mRNAs are 5'-capped and polyadenylated at the 3'-end. There is sequence overlap at the 3'end between ail five mRNAs.
One 3.5 kb mRNA serves as template for core protein and polymerase production. In addition, the same transcript serves as a pre-genomic réplication intermediate and allows the viral i
polymerase to initiate the reverse transcription into DNA. Core protein is needed for nucleocapsid formation. The other 3.5 kb mRNA encodes pre-core, the secretable e-antigen (HBeAg). In the absence of réplication inhibitors, the abundance of e-antigen in blood correlates with Hepatitis B Virus réplication in liver and serves as an important diagnostic marker for monitoring the disease progression.
The 2.4 and 2.1 kb mRNAs can}- the open reading frames (“ORF”) pre-Sl, pre-S2 and S for expression of viral large, medium and small surface antigen The s-antigen is associated with infectious, complété particles. In addition, blood of infected patients also contain noninfectious particles derived from s-antigen alone, free of genomic DNA or polymerase. The function of these particles is not fully understood. The complété and lasting déplétion of détectable s-antigen in blood is considered as a reliable indicator for Hepatitis B Virus clearance.
The 0.7 kb mRNA encodes the X protein. This gene product is important for efficient transcription of viral genes and also acts as a transactivator on host gene expression. The latter activity seems to be important for hépatocyte transformation during development of liver cancer.
Patients with détectable s-antigen, e-antigen, and/or viral DNA in the blood for more than 6 months are considered chronically infected. Nucleoside analogs as inhibitors of reverse transcriptase activity are typically the first treatment option for many patients. Administration of lamivudine, tenofovir, and/or entecavir has been shown to suppress Hepatitis B Virus réplication, sometimes to undetectable levels, with improvement of liver function and réduction of liver inflammation typically seen as the most important benefîts. However, only few’ patients achieve complété and lasting remission after the end of treatment. Furthermore, the Hepatitis B Virus develops drug résistance with increasing duration of treatment. This is especially difficult for patients co-infected with Hepatitis B and Human Immunodefîciency Virus (HIV). Both viruses are susceptible to nucleoside analogue drugs and may co-develop résistance.
A second treatment option is the administration of interferon-alpha. Here, patients receive high doses of interferon-alpha over a period of 6 months. The Asian génotype B gives very poor response rates. Co-infection with Hepatitis D Virus (HDV) or Human Immunodefîciency Xfîrus has been shown to render interferon-alpha therapy completely ineffective. Patients with strong liver damage and heavy fibrotic conditions are not qualified for interferon-alpha therapy.
Certain Hepatitis B Virus-specific RNA interférence (RNAi) agents hâve been previously shown to inhibit expression of HBV gene expression. For example, U.S. Patent Application Publication No. 2013/0005793, to Chin et al., which is incorporated herein by reference in its entirety, discloses certain double-stranded ribonucleic acid (dsRNA) molécules for inhibiting the expression of Hepatitis B Virus gene.
SUMMARY
There exists a need for novel Hepatitis B Virus (HBV)-specific RNA interférence (RNAi) agents (also herein termed RNAi agent, RNAi trigger, or trigger) that are able to selectively and efficiently inhibit the expression of an Hepatitis B Virus (HBV) gene. Further, there exists a need for combinations of novel HBV-specific RNAi agents for the treatment of HBV infection and prévention of diseases associated with HBV.
Described herein are HBV gene-specific RNAi agents able to selectively and efficiently decrease expression of an HBV gene. The described HBV RNAi agents can be used in methods for therapeutic treatment and/or prévention of symptoms and diseases associated with HBV infection, including but not limited to chronic liver diseases/disorders, inflammations, fibrotic conditions, proliférative disorders (including cancers, such as hepatocellular carcinoma), Hepatitis D Virus (HDV) infection, and acute HBV infection. In some embodiments, the HBV RNAi agents can be used in methods for therapeutic treatment and/or prévention of symptoms and diseases associated with chronic HBV infection and/or HDV infection. Such methods comprise administration of one or more HBV RNAi agents as described herein to a subject, e.g., a human or animal subject.
Additionally, described herein are compositions comprising one or more of the disclosed HBV RNAi agents that are able to selectively and efficiently decrease expression of an HBV gene. The compositions comprising one or more HBV RNAi agents can be administered to a subject, such as a human or animal subject, for the treatment and/or prévention of symptoms and diseases associated with HBV infection.
Each HBV RNAi agent disclosed herein includes at least a sense strand and an antisense strand. The sense strand and the antisense strand can be partially, substantially, or fully complementaiy to each other. The length of the RNAi agent sense and antisense strands described herein each can be 16 to 30 nucléotides in length. In some embodiments, the sense and antisense strands are independently 17 to 26 nucléotides in length. In some embodiments, the sense and antisense strands are independently 19 to 26 nucléotides in length. In some embodiments, the sense and antisense strands are independently 21 to 26 nucléotides in length. In some embodiments, the sense and antisense strands are independently 21 to 24 nucléotides in length. The sense and antisense strands can be either the same length or different lengths. The HBV RNAi agents disclosed herein hâve been designed to include antisense strand sequences that are at least partially complementary to a sequence in the HBV genome that is conserved across the majority of known serotypes ofHBV. The RNAi agents described herein, upon delivery to a cell expressing HBV, inhibit the expression of one or more HBV genes in vivo or in vitro.
An HBV RNAi agent includes a sense strand (also referred to as a passenger strand) that includes a first sequence, and an antisense strand (also referred to as a guide strand) that includes a second sequence. A sense strand of the HBV RNAi agents described herein includes a core stretch having at least about 85% identity to a nucléotide sequence of at least 16 consecutive nucléotides in an HBV mRNA. In some embodiments, the sense strand core nucléotide stretch having at least about 85% identity to a sequence in an HBV mRNA is 16, 17,18,19, 20,21,22, or 23 nucléotides in length. An antisense strand of an HBV RNAi agent comprises a nucléotide sequence having at least about 85% complementary over a core stretch of at least 16 consecutive nucléotides to a sequence in an HBV mRNA and the corresponding sense strand. In some embodiments, the antisense strand core nucléotide sequence having at least about 85% complementarity to a sequence in an HBV mRNA or the corresponding sense strand is 16,17,18,19,20,21,22, or 23 nucléotides in length.
Examples of HBV RNAi agent sense strands and antisense strands that can be used in HBV RNAi agents are provided in Tables 3 and 4. Examples of HBV RNAi agent duplexes are provided in Table 5. Examples of 19-nucleotide core stretch sequences that consist of or are included in the sense strands and antisense strands of HBV RNAi agents disclosed herein, are provided in Table 2.
In some embodiments, one or more HBV RNAi agents are delivered to target cells or tissues using any oligonucleotide delivery technology known in the art. Nucleic acid delivery methods include, but are not limited to, by encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodégradable nanocapsules, and bioadhesive microspheres, proteinaceous vectors or Dynamic Polyconjugates (DPCs) (see, for example WO 2000/053722, WO 2008/0022309, WO 2011/104169, and WO 2012/083185, each of which is incorporated herein by reference). In some embodiments, an HBV RNAi agent is delivered to target cells or tissues by covalently linking the RNAi agent to a targeting group. In some embodiments, the targeting group can include a cell receptor ligand, such as an asialoglycoprotein receptor (ASGPr) ligand. In some embodiments, an ASGPr ligand includes or consists of a galactose dérivative cluster. In some embodiments, a galactose dérivative cluster includes an N-acetyl-galactosamine trimer or an N-acetyl-galactosamine tetramer. In some embodiments, a galactose dérivative cluster is an N-acetyl-galactosamine trimer or an N-acetyl-galactosamine tetramer.
A targeting group can be linked to the 3' or 5' end of a sense strand or an antisense strand of an HBV RNAi agent. In some embodiments, a targeting group is linked to the 3' or 5' end of the sense strand. In some embodiments, a targeting group is linkedto the 5’ end ofthe sense strand. In some embodiments, a targeting group is linked to the RNAi agent via a linker.
A targeting group, with or without a linker, can be linked to the 5' or 3' end of any of the sense and/or antisense strands disclosed in Tables 2,3, and 4. A linker, with or without a targeting group, can be attached to the 5' or 3' end of any of the sense and/or antisense strands disclosed in Tables 2,3, and 4.
In some embodiments, described herein are compositions that include one or more HBV RNAi agents having the duplex sequences disclosed in Table 5.
In some embodiments, described herein are compositions that include a combination or cocktail of at least two HBV RNAi agents having different nucléotide sequences. In some embodiments, the two or more different HBV RNAi agents are each separately and independently linked to targeting groups. In some embodiments, the two or more different HBV RNAi agents are each linked to targeting groups comprised of N-acetyl-galactosamines. In some embodiments, when two or more RNAi agents are included in a composition, each of the RNAi agents is linked to the same targeting group. In some embodiments, when two or more RNAi agents are included in a composition, each of the RNAi agents is linked to different targeting groups, such as targeting groups having different Chemical structures.
In some embodiments, targeting groups are linked to the HBV RNAi agents without the use of an additional linker. In some embodiments, the targeting group is designed having a linker readily présent to facilitate the linkage to an HBV RNAi agent. In some embodiments, when two or more RNAi agents are included in a composition, the two or more RNAi agents may be linked to the targeting groups using the same linkers. In some embodiments, when two or more RNAi agents are included in a composition, the two or more RNAi agents are linked to the targeting groups using different linkers.
In some embodiments, described herein are compositions that include a combination of at least two HBV RNAi agents having different sequences, wherein each HBV RNAi agent targets a different location or different région of an HBV gene. In some embodiments, described herein are compositions that include a combination of at least two HBV RNAi agents, wherein each HBV RNAi agent is designed to target a different HBV transcript (for example, a composition that includes two HBV RNAi agents, wherein the first HBV RNAi agent includes an antisense strand that is at least partially complementary to a nucléotide sequence located in the S ORF of an HBV gene, while the second HBV RNAi agent includes an antisense strand that is at least partially complementary' to a nucléotide sequence located in the X ORF of an HBV gene). As used herein, an RNAi agent that includes an antisense strand at least partially complementary' to a nucléotide sequence located in the S ORF targets a portion of the HBV genome of SEQ ID NO:1 between positions 1-1307 and 3185-3221. As used herein, an RNAi agent that includes an antisense strand at least partially complementary to a nucléotide sequence located in the X ORF targets a portion of the HBV genome of SEQ ID NO: 1 between positions 1308-1930.
HBV mRNA is known to be polycistronic, resulting in the translation of multiple polypeptides, and separate mRNAs overlap in RNA sequence, therefore a single RNAi agent targeting an HBV gene may resuit in inhibition of most or ail HBV transcripts. However, while not wishing to be bound to any theory, it is hypothesized that a composition that includes two or more HBV RNAi agents targeting different locations or régions of an HBV gene (and, in particular, two or more HBV RNAi agents wherein one HBV RNAi agent targets the S ORF and a second HBV RNAi agent targets the X ORF) may provide for additional advantages over a composition that includes only a single ΗΒλζ RNAi agent, such as (a) ensuring that ail HBV viral transcripts are targeted (i.e., 3.5 kb pre-genomic RNA; 3.5 kb pre-core mRNA; 2.4 kb pre-Sl mRNA; 2.1 kb pre-S2/S mRNA; 0.7 kb X mRNA; as well as any S-antigen expressing mRNAs produced from integrated HBV DNA); (b) serving to expand the génotype coverage to potentially address a larger patient population; and/or (c) potentially decreasing the viral résistance due to mutations in the siRNA binding site.
In some embodiments, described herein are compositions that include a combination of one HBV RNAi agent that largets the S ORF of an HBV RNA (i.e., having an antisense strand that targets the S transcripts (S, pre-Sl, and pre-S2), the pregenomic RNA (core and polymerase), and the pre-core transcripts (HBeAg) of an HBV genome), and one HBV RNAi agent tirât targets the X ORF of an HBV RNA (i.e., having an antisense strand that targets tire X transcript of an HBV genome, the S transcripts (S, pre-Sl, and pre-S2), the pregenomic RNA (core and polymerase), and the pre-core transcripts (HBeAg) of an ΗΒλζ genome). In some embodiments, the compositions described herein include at least one HBV RNAi agent that contains a sequence that targets the S ORF of an HBV gene, and a second HBV RNAi agent that contains a sequence that targets the X ORF of an HBV gene.
Disclosed herein are methods for inhibiting expression of an HBV gene, the method comprising administering one or more HBV RNAi agents having an antisense strand comprising the sequence of any of the sequences in Table 3.
Disclosed herein are methods for inhibiting expression of an HBV gene, the method comprising administering one or more HBV RNAi agents having a sense strand comprising the sequence of any of the sequences in Table 4.
Disclosed herein are methods for inhibiting expression of an HBV gene, the method comprising administering one or more HBV RNAi agents having an antisense strand comprising the sequence of any of the sequences in Table 3, and a sense strand comprising the sequence of any of the sequences in Table 4 that is at least partially complementary to the antisense strand.
Disclosed herein are methods for inhibiting expression of an HBV gene, the method comprising administering one or more HBV RNAi agents having an antisense strand that consists of the sequence of any of the sequences in Table 3, and a sense strand that consists of the sequence of any of the sequences in Table 4 that is at least partially complementary to the antisense strand.
Disclosed herein are methods for inhibiting expression of an HBV gene in a cell. the method comprising administering one or more HBV RNAi agents having the duplex structure of Table 5.
Disclosed herein are methods of treatment of an HBV infection or prévention of disease or symptoms caused by an HBV infection, the method comprising administering one or more HBV RNAi agents having an antisense strand comprising the sequence of any of the sequences in Table 3.
Disclosed herein are methods of treatment of an HBV infection or prévention of disease or symptoms caused by an HBV infection, the method comprising administering one or more HBV RNAi agents having a sense strand comprising the sequence of any of the sequences in Table 4.
Disclosed herein are methods of treatment of an HBV infection or prévention of disease or symptoms caused by an HBV infection, the method comprising administering one or more HBV RNAi agents having an antisense strand comprising the sequence of any of the sequences in Table 3, and a sense strand comprising the sequence of any of the sequences in Table 4 that is at least partially complementary to the antisense strand.
Disclosed herein are methods of treatment of an HBV infection or prévention of disease or symptoms caused by an HBV infection, the method comprising administering one or more HBV RNAi agents having an antisense strand that consists of the sequence of any of the sequences in Table 3, and a sense strand that consists of the sequence of any of the sequences in Table 4 that is at least partially complementary to the antisense strand.
Disclosed herein are methods of treatment of an HBV infection or prévention of disease or symptoms caused by an HBV infection, the method comprising administering one or more HBV RNAi agents having the duplex structure of Table 5.
s
Disclosed herein are methods for inhibiting expression of an HBV gene, the method comprising administering (i) an HBV RNAi agent having an antisense strand comprising or consisting of 1he sequence of any of the sequences in Table 2 or Table 3, and (ii) a second HBV RNAi agent having an antisense strand comprising or consisting of the sequence of any of the sequences in Table 2 or Table 3.
Disclosed herein are methods of treatment of an HBV infection or prévention of disease or symptoms caused by an HBV infection, the method comprising administering (i) an HBV RNAi agent having an antisense strand comprising or consisting of the sequence of any of the sequences in Table 2 or Table 3, and (ii) a second HBV RNAi agent having an antisense strand comprising or consisting of the sequence of any of the sequences in Table 2 or Table 3.
Disclosed herein are methods for inhibiting expression of an HBV gene, the method comprising administering (i) a first HBV RNAi agent having an antisense strand comprising or consisting of the sequence of any of the sequences in Table 2 or Table 3 and a sense strand comprising or consisting of the sequence of any of the sequences in Table 2 or Table 4 that is at least partially complementaiy to the antisense strand of the first HBV RNAi agent, and (ii) a second HBV RNAi agent having an antisense strand comprising or consisting of the sequence of any of the sequences in Table 2 or Table 3 and a sense strand comprising or consisting ofthe sequence of any of the sequences in Table 2 or Table 4 that is at least partially complementary to the antisense strand ofthe second HBV RNAi agent.
Disclosed herein are methods of treatment of an HBV infection or prévention of disease or symptoms caused by an HBV infection, the method comprising administering (i) a first HBV RNAi agent having an antisense strand comprising or consisting of the sequence of any of the sequences in Table 2 or Table 3 and a sense strand comprising or consisting ofthe sequence of any of the sequences in Table 2 or Table 4 that is at least partially complementaiy to the antisense strand of the first HBV RNAi agent, and (ii) a second HBV RNAi agent having an antisense strand comprising or consisting of the sequence of any of the sequences in Table 2 or Table 3 and a sense strand comprising or consisting of the sequence of any of the sequences in Table 2 or Table 4 that is at least partially complementary’ to the antisense strand of the second HBV RNAi agent.
In some embodiments, an HBV RNAi agent disclosed herein comprises:
a. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-> 3 ') AUUGAGAGAAGUCCACCAC (SEQ ID NO: 7), and a sense strand that comprises the nucleobase sequence differing by 0.1,2 or 3 nucleobases from the sequence (5'->3') GUGGUGGACUUCUCUCAAU (SEQ ID NO: 34); or
b. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^ 3') UUUGAGAGAAGUCCACCAC (SEQ ID NO: S), and a sense strand that comprises the nucleobase sequence differing by 0,1,2 or 3 nucleobases from the sequence (5'~^3') GUGGUGGACUUCUCUCAAA (SEQ ID NO: 35); or
c. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') AAUUGAGAGAAGUCCACCA (SEQ ID NO: 12), and a sense strand that comprises the nucleobase sequence differing by 0,1, 2 or 3 nucleobases from the sequence (5'-^ 3') UGGUGGACUUCUCUCAAUU (SEQ IDNO: 39); or
d. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UAUUGAGAGAAGUCCACCA (SEQ ID NO: 13), and a sense strand that comprises the nucleobase sequence differing by 0,1, 2 or 3 nucleobases from the sequence (5'-^3') UGGUGGACUUCUCUCAAUA (SEQ IDNO: 40);or
e. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') AGAAAAUUGAGAGAAGUCC (SEQ ID NO: 17), and a sense strand that comprises the nucleobase sequence differing by 0,1, 2 or 3 nucleobases from the sequence (5'-^3') GGACUUCUCUCAAUUUUCU (SEQ ED NO: 44); or
f. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-à3') UGAAAAUUGAGAGAAGUCC (SEQ ID NO: 18), and a sense strand that comprises the nucleobase sequence differing by 0,1, 2 or 3 nucleobases from the sequence (53') GGACUUCUCUCAAUUUUCA (SEQ IDNO: 45);or
g. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') ACCAAUUUAUGCCUACAGC (SEQ ID NO: 22), and a sense strand that comprises the nucleobase sequence differing by 0,1,2 or 3 nucleobases from the sequence (5'-^3') GCUGUAGGCAUAAAUUGGU (SEQ ID NO: 49); or
h. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UCCAAUUUAUGCCUACAGC (SEQ IDNO: 23), and a sense strand thaï comprises the nucleobase sequence diiTering by 0,1,2 or 3 nucleobases from the sequence (5'·ά3') GCUGUAGGCAUAAAUUGGA (SEQ ID NO: 50); or
i. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^ 3') GACCAAUUUAUGCCUACAG (SEQ ID NO: 27), and a sense strand that comprises the nucleobase sequence differing by 0,1,2 or 3 nucleobases from the sequence (5'-^3') CUGUAGGCAUAAAUUGGUC (SEQ ID NO: 54); or
j. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^ 3') AACCAAUUUAUGCCUACAG (SEQ ID NO: 28), and a sense strand that comprises the nucleobase sequence differing by 0,1,2 or 3 nucleobases from the sequence (5'-^3') CUGUAGGCAUAAAUUGGUU (SEQ ID NO: 55); or
k. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (51-^ 3 ') UACCAAUUUAUGCCUACAG (SEQ ID NO: 29), and a sense strand that comprises the nucleobase sequence differing by 0,1,2 or 3 nucleobases from the sequence (5'-^3') CUGUAGGCAUAAAUUGGUA (SEQ ID NO: 56).
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising an HBV RNAi agent.
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two or more HBV RNAi agents, wherein a first HBV RNAi agent comprises:
i) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') AAUUGAGAGAAGUCCACCA (SEQ ID NO: 12), and a sense strand that comprises the nucleobase sequence differing by 0,1, 2 or 3 nucleobases from the sequence (5'->3') UGGUGGACUUCUCUCAAUU (SEQ IDNO: 39);or ii) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UAUUGAGAGAAGUCCACCA (SEQ ID NO: 13), and a sense strand that comprises the nucleobase sequence differing by 0,1, 2 or 3 nucleobases from the sequence (5'^3') UGGUGGACUUCUCUCAAUA (SEQ ID NO: 40);
and wherein a second HBV RNAi agent comprises:
i) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') GACCAAUUUAUGCCUACAG (SEQ IDNO: 27), and a sense strand that comprises the nucleobase sequence differing by 0,1,2 or 3 nucleobases from the sequence (5'·ά3') CUGUAGGCAUAAAUUGGUC (SEQ ID NO: 54); or ii) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') AACCAAUUUAUGCCUACAG (SEQ ID NO: 28), and a sense strand that comprises the nucleobase sequence differing by 0,1,2 or 3 nucleobases from the sequence (5'^3') CUGUAGGCAUAAAUUGGUU (SEQ ID NO: 55); or iii) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3‘) UACCAAUUUAUGCCUACAG (SEQ ID NO: 29), and a sense strand that comprises the nucleobase sequence differing by 0,1,2 or 3 nucleobases from the sequence (5'-^3') CUGUAGGCAUAAAUUGGUA (SEQ ID NO: 56).
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two or more HBV RNAi agents, wherein a first HBV RNAi agent comprises:
i) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5,-^3') AGAAAAUUGAGAGAAGUCC (SEQ ID NO: 17), and a sense strand that comprises the nucleobase sequence differing 1¾7 0,1, 2 or 3 nucleobases from the sequence (5'->3') GGACUUCUCUCAAUUUUCU (SEQ IDNO: 44); or ii) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-> 3') UGAAAAUUGAGAGAAGUCC (SEQ ID NO: 18), and a sense strand that comprises the nucleobase sequence differing by 0, 1, or 3 nucleobases from the sequence (5'-^3') GGACUUCUCUCAAUUUUCA (SEQ IDNO: 45);
and wherein a second HBV RNAi agent comprises:
i) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^ 3 ') GACCAAUUUAUGCCUACAG (SEQ ID NO: 27), and a sense strand that comprises the nucleobase sequence differing by 0,1,2 or 3 nucleobases from the sequence (5'*^3') CUGUAGGCAUAAAUUGGUC (SEQ ID NO: 54); or ii) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') AACCAAUUUAUGCCUACAG (SEQ ID NO: 28), and a sense strand that comprises the nucleobase sequence differing by 0,1,2 or 3 nucleobases from the sequence (5'^3') CUGUAGGCAUAAAUUGGUU (SEQ ID NO: 55); or iii) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3‘) UACCAAUUUAUGCCUACAG (SEQ ID NO: 29), and a sense strand that comprises the nucleobase sequence differing by 0,1,2 or 3 nucleobases from the sequence (5,-^3') CUGUAGGCAUAAAUUGGUA (SEQ ID NO: 56).
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two or more HBV RNAi agents, wherein a first HBV RNAi agent comprises:
i) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') AAUUGAGAGAAGUCCACCA (SEQ ID NO: 12), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') UGGUGGACUUCUCUCAAUU (SEQ IDNO: 39);or ii) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UAUUGAGAGAAGUCCACCA (SEQ ID NO: 13), and a sense strand that comprises the nucleobase sequence differing by 0,1, 2 or 3 nucleobases from the sequence (5'-^3') UGGUGGACUUCUCUCAAUA (SEQ IDNO: 40);
and wherein a second HBV RNAi agent comprises an antisense strand having a sequence that is at least partially complementary' to a portion of the X ORF bf an HBV mRNA.
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two or more HBV RNAi agents, wherein a first HBV RNAi agent comprises:
i) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') AGAAAAUUGAGAGAAGUCC (SEQ ID NO: 17), and a sense strand that comprises the nucleobase sequence differing by 0,1, 2 or 3 nucleobases from the sequence (5'->3') GGACUUCUCUCAAUUUUCU (SEQ ID NO: 44); or ii) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-à3') UGAAAAUUGAGAGAAGUCC (SEQ ID NO: 18), and a sense strand that comprises the nucleobase sequence differing by 0,1, 2 or 3 nucleobases from the sequence (5'->3') GGACUUCUCUCAAUUUUCA (SEQ IDNO: 45);
and wherein a second HBV RNAi agent comprises an antisense strand having a sequence that is at least partially complementary’ to a portion of the X ORF of an HBV mRNA:
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two or more HBV RNAi agents, wherein a first HBV RNAi agent comprises an antisense strand having a sequence that is at least partially complementary7 to a portion of the S ORF of an HBV mRNA, and wherein a second HBV RNAi agent comprises:
i) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'·^ 3') GACCAAUUUAUGCCUACAG (SEQ IDNO: 27), and a sense strand that comprises the nucleobase sequence differing by 0,1,2 or 3 nucleobases from the sequence (5'~>3*) CUGUAGGCAUAAAUUGGUC (SEQ ID NO: 54); or ii) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') AACCAAUUUAUGCCUACAG (SEQ ID NO: 28), and a sense strand that comprises the nucleobase sequence differing by 0,1,2 or 3 nucleobases from the sequence (5'->3') CUGUAGGCAUAAAUUGGUU (SEQ ID NO: 55); or iii) an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-> 3') UACCAAUUUAUGCCUACAG (SEQ IDNO:
29), and a sense strand that comprises the nucleobase sequence differing by 0,1,2 or 3 nucleobases from the sequence (5'->3j CUGUAGGCAUAAAUUGGUA (SEQ ID NO: 56).
In some embodiments, an HBV RNAi agent disclosed herein comprises:
a. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UACCAAUUUAUGCCUACAGGCCUUAU (SEQ IDNO: 149); or
b. an antisense strand that comprises the nucleobase sequence dilfering by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UACCAAUUUAUGCCUACAGGCCU (SEQ IDNO: 150): or
c. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UACCAAUUUAUGCCUACAGGC (SEQ ID NO: 151); or
d. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5l-^3') UGAAAAUUGAGAGAAGUCCUU (SEQ ID NO: 152); or
e. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') UACCAAUUUAUGCCUACAGUU (SEQ ID NO: 154); or
f. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-> 3') UAUUGAGAGAAGUCCACCACG (SEQ ID NO: 160); or
g. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UACCAAUUUAUGCCUACAGCC (SEQ ID NO: 162); or
h. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3 j UACCAAUUUAUGCCUACAGCCUU (SEQ IDNO: 163); or
i. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') UAUUGAGAGAAGUCCACCACGA (SEQ IDNO: 170); or
j. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') AGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 171); or
k. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UACCAAUUUAUGCCUACAGCUU (SEQ IDNO: 172): or
1. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UACCAAUUUAUGCCUACAGCCU (SEQ ID NO: 173); or
m. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') UAUUGAGAGAAGUCCACCAUU (SEQ ID NO: 174); or
n. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UAUUGAGAGAAGUCCACCACUU (SEQ IDNO: 175); or
o. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'·^3') AGAAAAUUGAGAGAAGUCCUU (SEQ ID NO: 178); or
p. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5,-^3') AGAAAAUUGAGAGAAGUCCACUU (SEQ IDNO: 179); or
q. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') AGAAAAUUGAGAGAAGUCCACC (SEQ IDNO: 180); or
r. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'·^3') UGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 181); or
s. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') ACCAAUUUAUGCCUACAGCUU (SEQ ID NO: 182); or
t. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^ 3') ACCAAUUUAUGCCUACAGCCUU (SEQ IDNO: 183); or
u. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') ACCAAUUUAUGCCUACAGCCUC (SEQ IDNO: 184); or
v. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UCCAAUUUAUGCCUACAGCUU (SEQ ID NO: 185); or
w. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UCCAAUUUAUGCCUACAGCCUU (SEQ IDNO: 186); or
x. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^ 3') UACCAAUUUAUGCCUACAGCU (SEQ ID NO: 187); or
y. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^ 3') UACCAAUUUAUGCCUACAGCG (SEQ ID NO: 188); or
z. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') AACCAAUUUAUGCCUACAGCC (SEQ ID NO: 189); or aa. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5^31) ACCAAUUUAUGCCUACAGCCU (SEQ ID NO: 190); or bb. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UCCAAUUUAUGCCUACAGCCU (SEQ ID NO: 191); or cc. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5,_à3') ACCAAUUUAUGCCUACAGCCG (SEQ ID NO: 192); or dd. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5*->3') UCCAAUUUAUGCCUACAGCCG (SEQ ID NO: 193); or ee. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-> 3') UACCAAUUUAUGCCUACAGGG (SEQ ID NO: 194);
and wherein the HBV RNAi agent further comprises a sense strand at least partially complementary' to the respective antisense strand.
In some embodiments, an HBV RNAi agent disclosed herein comprises:
a. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') UACCAAUUUAUGCCUACAGGCCUUAU (SEQID NO: 149); or
b. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'·^3') UACCAAUUUAUGCCUACAGGCCU (SEQ IDNO: 150); or
c. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5^31) UACCAAUUUAUGCCUACAGGC (SEQ ID NO: 151); or
d. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') UGAAAAUUGAGAGAAGUCCUU (SEQ ID NO: 152); or
e. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') UACCAAUUUAUGCCUACAGUU (SEQ ID NO: 154); or
f. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UAUUGAGAGAAGUCCACCACG (SEQ ID NO: 160); or
g. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UACCAAUUUAUGCCUACAGGC (SEQ ID NO: 162); or
h. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^ 3 ') UACCAAUUUAUGCCUACAGCCUU (SEQ IDNO: 163); or
i. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UAUUGAGAGAAGUCCACCACGA (SEQ IDNO: 170); or
j. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') AGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 171); or
k. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UACCAAUUUAUGCCUACAGCUU (SEQ IDNO: 172); or
1. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'·^3') UACCAAUUUAUGCCUACAGCCU (SEQ
IDNO: 173); or
m. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-à3') UAUUGAGAGAAGUCCACCAUU (SEQ ID NO: 174); or
n. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') UAUUGAGAGAAGUCCACCACUU (SEQ IDNO: 175); or
o. an antisense strand that consists of the nucleobase sequence differing by 0. 1, 2 or 3 nucleobases from the sequence (5'-> 3') AGAAAAUUGAGAGAAGUCCUU (SEQ ID
NO: 178); or
p. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') AGAAAAUUGAGAGAAGUCCACUU (SEQ IDNO: 179); or
q. an antisense strand that consists of the nucleobase sequence differing by 0, 1,2 or 3 nucleobases from the sequence (5'-^3') AGAAAAUUGAGAGAAGUCCACC (SEQ IDNO: 180); or
r. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') UGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 181); or
s. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') ACCAAUUUAUGCCUACAGCUU (SEQ ID NO: 182); or
t. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') ACCAAUUUADGCCUACAGCCUU (SEQ
IDNO: 183); or
u. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') ACCAAUUUAUGCCUACAGCCUC (SEQ IDNO: 184); or
v. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') UCCAAUUUAUGCCUACAGCUU (SEQ ID NO: 185); or
w. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'·^3') UCCAAUUUAUGCCUACAGCCUU (SEQ IDNO: 186); or
x. an antisense strand that consists of the nucleobase sequence differing by 0. 1, 2 or 3 nucleobases from the sequence (5'-^3') UACCAAUUUAUGCCUACAGCU (SEQ ID NO: 187); or
y. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UACCAAUUUAUGCCUACAGCG (SEQ ID NO: 188); or
z. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') AACCAAUUUAUGCCUACAGCC (SEQ ID NO: 189); or aa. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^ 3') ACCAAUUUAUGCCUACAGCCU (SEQ ID NO: 190); or bb. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') UCCAAUUUAUGCCUACAGCCU (SEQ ID NO: 191); or cc. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') ACCAAUUUAUGCCUACAGCCG (SEQ ID NO: 192); or dd. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-à3') UCCAAUUUAUGCCUACAGCCG (SEQ ID NO: 193); or.
ee. an antisense strand that consists of the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UACCAAUUUAUGCCUACAGCG (SEQ ID NO: 194);
and wherein the HBV RNAi agent further comprises a sense strand at least partially complementaiy to the respective antisense strand.
In some embodiments, an HBV RNAi agent disclosed herein comprises:
i. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-à 3') usAfscCfaAfuUfuAfuGfcCfuAfcAfgGfccsusuAu (SEQ IDNO: 61); or ii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-^3') usAfscCfaAfuUfuAfuGfcCruAfcAfgGfcscsu (SEQ ID NO: 62); or iii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-^ 3') usAfscsCfaAfuUfuAfuGfcCfuAfcAfgGfccsu (SEQ ID NO: 63); or iv. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'^3') usAfscsCfaAfuUfuAfuGfcCfuAfcAfgGfsc (SEQ ID NO: 64); or
v. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-^ 3') us AfscsCfaAfuUfuAfuGfcCfuAfcAfgusu (SEQ ID NO: 68); or vi. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-^3') usAfscscaauUfuAfuGfcCfuacagcsc (SEQ ID NO: 85); or vii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'->3') usAfsusugagAfgAfaGfuCfcaccacsg (SEQ ID NO: 94); or viii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'->3') usAfsusUfgAfgAfgAfaGfuCfcAfcCfaCfgsa (SEQ ID NO: 98); or ix. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-à3') usAfscsCfaAfuuuauGfcCfuAfcAfgcsc (SEQ ID NO: 102); or
x. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'->3') usAfscsCfaAfuuuauGfcCfuAfcAfgcusu (SEQ ID NO: 103); or xi. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'^3') usAfscsCfaAfuuuauGfcCfuAfcAfgccsu (SEQ ID NO: 104); or xii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-^3') usAfscsCfaAfuuuauGfcCfuAfcAfgccusu (SEQ ID NO: 105); or xiii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'->3') cPrpusAfscsCfaAfuUfuAfuGfcCfuAfcAfgusu (SEQ ID NO: 107); or xiv. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-^3') cPrpusAfsusUfgAfgAfgAfaGfuCfcAfcCfaCfsg (SEQ IDNO: 108); or xv. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'->3') usAfsusUfgAfgagaaGfuCfcAfcCfausu (SEQ ID NO: 109); or xvi. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-^3') usAfsusUfgAfgagaaGfuCfcAfcCfacsg (SEQ ID NO: 110); or xvii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-^3') usAfsusUfgAfgagaaGfuCfcAfcCfacsusu (SEQ ID NO: 111); or xviii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-^3') usAfsusUfgAfgagaaGfuCfcAfcCfacsgsa (SEQ ID NO: 112); or xix. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-^3') usAfsusUfgAfgagaaGfuCfcAfcCfacusu (SEQ ID NO: 120); or xx. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-^3') asGfsasAfaAfuUfgAfgAfgAfaGfuCfcusu (SEQ ID NO: 125);
xxi. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'->3') asGfsasAfaAfuUfgAfgAfgAfaGfuCfcasc (SEQ ID NO: 126); or xxii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-^3') asGfsasAfaAfuUfgAfgAfgAfaGfuCfcacusu (SEQ ID NO: 127); or xxiii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5’-> 3') asGfsasAfaAfuUfgAfgAfgAfaGfuCfcacsc (SEQ ID NO: 128); or xxiv. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-^3') usGfsasAfaAfuUfgAfgAfgAfaGfuCfcusu (SEQ ID NO: 129); or xxv. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5' 3') usGfsasAfaAfuUfgAfgAfgAfaGfuCfcasc (SEQ ID NO: 130); or xxvi. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'->3') asCfscsAfaUfuUfaUfgCfcUfaCfaGfcusu (SEQ ID NO: 131); or xxvii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-^ 3') asCfscsAfaUfuUfaUfgCfcUfaCfaGfccusu (SEQ IDNO: 132); or xxviii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-^ 3 ') asCfscs AfaUfuUfaUfgCfcUfaCfaGfccusc (SEQ ID NO: 133); or xxix. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'~> 3 ') usCfecsAfaUfuUfaUfgCfcUfaCfaGfcusu (SEQ ID NO: 134); or xxx. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5,-^3') usCfscsAfaUfuUfaUfgCfcUfaCfaGfccusu (SEQ ID NO: 135); or xxxi. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'->3') cPrpusAfscsCfaAfuUfuAfuGfcCfuAfcAfgcsc (SEQ ID NO: 136); or xxxii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'^3') usAfscsCfaAfuUfuAfuGfcCfuAfcAfgscsc (SEQ ID NO: 137); or xxxiii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'~> 3') cPrpusAfscsCfaAfuUfuAfuGfcCfuAfcAfgscsc (SEQ ID NO: 138); or xxxiv. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-> 3') usAfscsCfaAfuUfuAfuGfcCfuAfcAfgcsu (SEQ ID NO: 139); or xxxv. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'->3') usAfscsCfaAfuUfuAfuGfcCfuAfcAfgcsg (SEQ ID NO: 140); or xxxvi. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'->3') asAfscsCfaAfuUfuAfuGfcCfuAfcAfgcsc (SEQ IDNO: 141); or xxxvii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'->3') usAfscsCfaAfuUfUfAfuGfcCfuAfcAfgusu (SEQ ID NO: 142); or xxxviii. an antisense strand that comprises the sequence differing by 0, 1, 2 or 3 nucléotides from the sequence (5'-^ 3') usAfscsCfaAfuUfuAfuGfcCfuAfcAfgCfsc (SEQ ID NO: 143); or xxxix. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'·^ 3') asCfscAfaUfuUfaUfgCfcUfaCfaGfcCfsu (SEQ ID NO: 144); or xl. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'-> 3') usCfscAfaUfuUfaUfgCfcUfaCfaGfcCfsu (SEQ ID NO: 145); or xli. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'->3') asCfscAfaUfuUfaUfgCfcüfaCfaGfccsg (SEQ ID NO: 146); or xlii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'->3') usCfscAfaUfuUfaUfgCfcUfaCfaGfccsg (SEQ ID NO: 147); or xliii. an antisense strand that comprises the sequence differing by 0,1,2 or 3 nucléotides from the sequence (5'^3') usAfscsCfaAfuUfuAfuGfcCfuAfcAfggsg (SEQ ID NO: 148);
wherein a, g, c and u are 2'-O-methyl (2'-OMe) modified nucléotides; Af Cf, Gf, and Uf are 2'-fluoro modified nucléotides; s is a phosphorothioate intemucleoside linkage and the remaining nucléotide monomers are linked by phosphodiester bonds; and cPrpu is 5'cyclopropyl phosphonate-2'-O-methyl modified nucléotide; and wherein the HBV RNAi agent further comprises a sense strand at least partially complementary’ to the respective antisense strand.
In some embodiments, an HBV RNAi agent disclosed herein comprises:
5 i. an antisense strand that consists of the sequence (5'^3')
ii. usAfscCfaAfuUfuAfuGfcCfuAfcAfgGfccsusuAu (SEQ ID NO: 61); or an antisense strand that consists of the sequence usAfscCfaAfuUfuAfuGfcCfuAfcAfgGfcscsu (SEQ ID NO: 62); or (5'^3')
iii. an antisense strand that consists of the sequence (5'^3')
10 iv. usAfscsCfaAfuUfuAfuGfcCfuAfcAfgGfccsu (SEQ ID NO: 63); or an antisense strand that consists of the sequence usAfscsCfaAfuUfuAfuGfcCfuAfcAfgGfsc (SEQ ID NO: 64); or (5'^3')
v. an antisense strand that consists of the sequence usAfscsCfaAfuUfuAfuGfcCfuAfcAfgusu (SEQ ID NO: 68); or (5'^3')
15 vi. an antisense strand that consists of the sequence (5'^3')
vii. usAfscscaauUfuAfuGfcCfuacagcsc (SEQ ID NO: 85); or an antisense strand that consists of the sequence usAfsusugagAfgAfaGfuCfcaccacsg (SEQ ID NO: 94); or (5'^3')
viii. an antisense strand that consists of the sequence (5'^3')
20 ix. usAfsusUfgAfgAfgAfaGfuCfcAfcCfaCfgsa (SEQ ID NO: 98); or an antisense strand that consists of the sequence usAfscsCfaAfuuuauGfcCfuAfcAfgcsc (SEQ ID NO: 102); or (5'^3')
X. an antisense strand that consists of the sequence usAfscsCfaAfuuuauGfcCfuAfcAfgcusu (SEQ ID NO: 103); or (5'-»3')
25 xi. an antisense strand that consists of the sequence (5'^3')
xii. usAfscsCfaAfuuuauGfcCfuAfcAfgccsu (SEQ IDNO: 104); or an antisense strand that consists of the sequence usAfscsCfaAfuuuauGfcCfuAfcAfgccusu (SEQ ID NO: 105); or (5^31)
xiii. an antisense strand that consists of the sequence (5'^3')
30 xiv. cPrpusAfscsCfaAfuUfuAfuGfcCfuAfcAfgusu (SEQ ID NO: 107); or an antisense strand that consists of the sequence cPrpusAfsusUfgAfgAfgAfaGfuCfcAfcCfaCfsg (SEQ ID NO: 108); or (5'^3·)
XV. an antisense strand that consists of the sequence usAfsusUfgAfgagaaGfuCfcAfcCfausu (SEQ ID NO: 109); or (5'->3')
xvi. an antisense strand that consists of the sequence usAfsusUfgAfgagaaGfuCfcAfcCfacsg (SEQ ID NO: 110); or (5’->3’>
xvii. an antisense strand that consists of the sequence usAfsusUfgAfgagaaGfuCfcAfcCfacsusu (SEQ ID NO: 111); or (5'-»3')
5 xviii. an antisense strand that consists of the sequence usAfsusUfgAfgagaaGfuCfcAfcCfacsgsa (SEQ ID NO: 112); or (5'^3')
xix. an antisense strand that consists of the sequence (5'^3')
XX. usAfsusUfgAfgagaaGfuCfcAfcCfacusu (SEQ IDNO: 120); or an antisense strand that consists of the sequence (5'^3')
10 xxi. asGfsasAfaAfuUfgAfgAfgAfaGfuCfcusu (SEQ IDNO: 125); an antisense strand that consists of the sequence asGfsasAfaAfuUfgAfgAfgAfaGfuCfcasc (SEQ ID NO: 126); or (5'->3')
xxii. an antisense strand that consists of the sequence asGfsasAfaAfuUfgAfgAfgAfaGfuCfcacusu (SEQ ID NO: 127); or (5'^3')
15 xxiii. an antisense strand that consists of the sequence asGfsasAfaAfuUfgAfgAfgAfaGfuCfcacsc (SEQ ID NO: 128); or (5'^3')
xxiv. an antisense strand that consists of the sequence usGfsasAfaAfuUfgAfgAfgAfaGfuCfcusu (SEQ ID NO: 129); or (5'->3')
XXV. an antisense strand that consists of the sequence (5’-»3·)
20 xxvi. usGfsasAfaAfuUfgAfgAfgAfaGfuCfcasc (SEQ IDNO: 130); or an antisense strand that consists of the sequence asCfscsAfaUfuUfaUfgCfcUfaCfaGfcusu(SEQ IDNO: 131); or (5'-^3')
xxvii. an antisense strand that consists of the sequence asCfscsAfaUfuUfaUfgCfcUfaCfaGfccusu (SEQ ID NO: 132); or (5'^3')
25 xxviii. an antisense strand that consists of the sequence asCfscsAfaUfuUfaUfgCfcUfaCfaGfccusc (SEQ ID NO: 133); or (5'^3')
xxix. an antisense strand that consists of the sequence usCfscsAfaUfuUfaUfgCfcUfaCfaGfcusu(SEQ IDNO: 134); or (5'->3')
XXX. an antisense strand that consists of the sequence (5‘->3·)
30 xxxi. usCfscsAfaUfuUfaUfgCfcUfaCfaGfccusu (SEQ ID NO: 135); or an antisense strand that consists of the sequence cPrpusAfscsCfaAfuUfuAfuGfcCfuAfcAfgcsc (SEQ ID NO: 136); or (5'^3')
xxxii. an antisense strand that consists of the sequence usAfscsCfaAfuUfuAfuGfcCfuAfcAfgscsc (SEQ ID NO: 137); or (5'->3')
xxxiii. an antisense strand that consists of the sequence cPrpusAfscsCfaAfuUfuAfuGfcCfuAfcAfgscsc (SEQ IDNO: 138); or xxxiv. an antisense strand that consists of the sequence (5’->3') (5'->3')
usAfscsCfaAfuUfuAfuGfcCfuAfcAfgcsu (SEQ ID NO: 139); or
XXXV. an antisense strand that consists of the sequence usAfscsCfaAfuUfuAfuGfcCfuAfcAfgcsg (SEQ ID NO: 140); or (5'^3')
xxx vi. an antisense strand that consists of the sequence asAfscsCfaAfuUfuAfuGfcCfuAfcAfgcsc (SEQ ID NO: 141); or (5'^3')
xxxvii. an antisense strand that consists of the sequence (5'^3')
usAfscsCfaAfuUfUfAfuGfcCfuAfcAfgusu(SEQ IDNO: 142); or xxxviii. an antisense strand that consists of the sequence (5'->3') usAfscsCfaAfuUfuAfuGfcCfuAfcAfgCfsc (SEQ ID NO: 143); or xxxix. an antisense strand that consists of the sequence (5'~>3') asCfscAfaUfuUfaUfgCfcUfaCfaGfcCfsu (SEQ IDNO: 144); or xl. an antisense strand that consists of the sequence (5'^3') usCfscAfaUfuUfaUfgCfcUfaCfaGfcCfsu (SEQ ID NO: 145); or xli. an antisense strand that consists of the sequence (5'->3') asCfscAfaUfuUfaUfgCfcUfaCfaGfccsg (SEQ ID NO: 146); or xlii. an antisense strand that consists of the sequence (5'->3') usCfecAfaUfuUfaUfgCfcUfaCfaGfccsg (SEQ ID NO: 147); or xliii. an antisense strand that consists of the sequence (5'->3') usAfscsCfaAfuUfuAfuGfcCfuAfcAfggsg (SEQ IDNO: 148);
wherein a, g, c and u are 2'-O-methyI (2'-0Me) modified nucléotides; Af, Cf, Gf, and Uf are 2'-fluoro modified nucléotides; s is a phosphorothioate intemucleoside linkage and the remaining nucléotide monomers are linked by phosphodiester bonds; and cPrpu is 5'cyclopropyl phosphonate-2'-O-methyl modified nucléotide; and wherein the HBV RNAi agent further comprises a sense strand at least partially complementary to the respective antisense strand.
In some embodiments, an HBV RNAi agent disclosed herein comprises:
a. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') UUGCCUGUAGGCAUAAAUUGGUAUT (SEQ IDNO: 275); or
b. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5Ύ3') UAUAUGCCUGUAGGCAUAAAUUGGUA (SEQ IDNO: 276); or
c. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') CUGUAGGCAUAAAUUGGUAUU (SEQ ID NO: 278); or
d. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') CGUGGUGGACUUCUCUCAAUU (SEQ ID NO: 285); or
e. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') CGUGGUGGACUUCUCUCAAUA (SEQ ID NO: 289); or
f. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-à3') CUGUAGGCAUAAAUUGGUA (SEQ ID NO: 292); or
g. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'·^3') GGCUGUAGGCAUAAAUUGGUA (SEQ ID NO: 294); or
h. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UCGUGGUGGACUUCUCUCAAUU (SEQ IDNO: 300); or
i. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') GUGGACUUCUCUCAAUUUUCU (SEQ ID NO: 302); or
j. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-à3') GCUGUAGGCAUAAAUUGGUAUU (SEQ IDNO: 303); or
k. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') GGCUGUAGGCAUAAAUUGGUAUU (SEQ IDNO: 304); or
1. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') UGGUGGACUUCUCUCAAUAUU (SEQ ID NO: 306); or
m. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5^31) GUGGUGGACUUCUCUCAAUAUU (SEQ IDNO: 307); or
n. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-à3') AAUGGUGGACUUCUCUCAAUAUU (SEQ IDNO: 308); or
o. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') GGACUUCUCUCAAUUUUCU (SEQ IDNO: 318); or
p. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') GGUGGACUUCUCUCAAUUUUCU (SEQ IDNO: 319); or
q. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5 3 ') GGACUUCUCUCAAUUUUC A (SEQ ID NO: 320); or
r. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') GUGGACUUCUCUCAAUUUUCA (SEQ ID NO: 321); or
s. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') GCUGUAGGCAUAAAUUGGU (SEQ ID NO: 322); or
t. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') GGCUGUAGGCAUAAAUUGGU (SEQ ID NO: 323); or
u. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') GAGGCUGUAGGCAUAAAUUGGU (SEQ IDNO: 324); or
v. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') GCUGUAGGCAUAAAUUGGA (SEQ ID NO: 325); or
w. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') GGCUGUAGGCAUAAAUUGGA (SEQ ID NO: 326); or .
x. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-à3') AGCUGUAGGCAUAAAUUGGUA (SEQ ID NO: 327); or
y. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') CGCUGUAGGCAUAAAUUGGUA (SEQ ID NO: 328); or
z. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') GGCUGUAGGCAUAAAUUGGUU (SEQ ID NO: 329); or aa. an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') AGGCUGUAGGCAUAAAUUGGU (SEQ ID NO: 330); or bb. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5*~> 3') AGGCUGUAGGCAUAAAUUGGA (SEQ ID NO: 331); or cc. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') CGGCUGUAGGCAUAAAUUGGU (SEQ ID NO: 332); or dd. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^ 3') CGGCUGUAGGCAUAAAUUGGA (SEQ ID NO: 333); or ee. a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-à3') CCCUGUAGGCAUAAAUUGGUA (SEQ ID NO: 334);
and wherein the HBV RNAi agent further comprises an antisense strand at least partially complementary’ to the respective antisense strand.
In some embodiments, an HBV RNAi agent disclosed herein comprises:
a. a sense strand that consists of the nucleobase sequence (5'->3') UUGCCUGUAGGCAUAAAUUGGUAUT (SEQ ID NO: 275); or
b. a sense strand that consists of the nucleobase sequence (5'->3')
UAUAUGCCUGUAGGCAUAAAUUGGUA (SEQ ID NO: 276); or
c. a sense strand that consists of the nucleobase sequence (5'->3')
CUGUAGGCAUAAAUUGGUAUU (SEQ ID NO: 278); or
d. a sense strand that consists of the nucleobase CGUGGUGGACUUCUCUCAAUU (SEQ ID NO: 285); or sequence (5'->3')
e. a sense strand that consists of the nucleobase CGUGGUGGACUUCUCUCAAUA (SEQ ID NO: 289); or sequence (5'->3')
f. a sense strand that consists of the nucleobase CUGUAGGCAUAAAUUGGUA (SEQ ID NO: 292); or sequence (5'->3')
g. a sense strand that consists of the nucleobase GGCUGUAGGCAUAAAUUGGUA (SEQ ID NO: 294); or sequence (5'->3')
h. a sense strand that consists of the nucleobase UCGUGGUGGACUUCUCUCAAUU (SEQ ID NO: 300); or sequence (5'->3')
i. a sense strand that consists of the nucleobase GUGGACUUCUCUCAAUUUUCU (SEQ ID NO: 302); or sequence (5' -à 3')
j. a sense strand that consists of the nucleobase GCUGUAGGCAUAAAUUGGUAUU (SEQ IDNO: 303); or sequence (5'-à3')
k. a sense strand that consists of the nucleobase GGCUGUAGGCAUAAAUUGGUAUU (SEQ ID NO: 304); or sequence (5F3')
1. a sense strand that consists of the nucleobase UGGUGGACUUCUCUCAAUAUU (SEQ ID NO: 306); or sequence (5'->3')
m. a sense strand that consists of the nucleobase GUGGUGGACUUCUCUCAAUAUU(SEQ IDNO: 307); or sequence (5'->3')
n. a sense strand that consists of the nucleobase AAUGGUGGACUUCUCUCAAUAUU (SEQ ID NO: 308); or sequence (5'->3')
o. a sense strand that comprises the nucleobase GGACUUCUCUCAAUUUUCU (SEQ ID NO: 318); or sequence (5'->3')
p. a sense strand that consists of the nucleobase GGUGGACUUCUCUCAAUUUUCU (SEQ ID NO: 319); or sequence (5'^3')
q. a sense strand that consists of the nucleobase GGACUUCUCUCAAUUUUCA (SEQ ID NO: 320); or sequence (5'->3')
r. a sense strand that consists of the nucleobase GUGGACUUCUCUCAAUUUUCA (SEQ ID NO: 321); or sequence (5'-^3')
s. a sense strand that consists of the nucleobase GCUGUAGGCAUAAAUUGGU (SEQ ID NO: 322); or sequence (5'->3')
t. a sense strand that consists of the nucleobase GGCUGUAGGCAUAAAUUGGU (SEQ IDNO: 323); or sequence (5'->3')
u. a sense strand that consists of the nucleobase
GAGGCUGUAGGCAUAAAUUGGU (SEQ IDNO: 324); or
v. a sense strand that consists of the nucleobase
GCUGUAGGCAUAAAUUGGA (SEQ IDNO: 325); or.
w. a sense strand that consists of the nucleobase
GGCUGUAGGCAUAAAUUGGA (SEQ IDNO: 326): or
x. a sense strand that consists of the nucleobase
AGCUGUAGGCAUAAAUUGGUA (SEQ ID NO: 327); or
y. a sense strand that consists of the nucleobase
CGCUGUAGGCAUAAAUUGGUA (SEQ ID NO: 328); or
z. a sense strand that consists of the nucleobase
GGCUGUAGGCAUAAAUUGGUU (SEQ ID NO: 329); or aa. an antisense strand that comprises the nucleobase AGGCUGUAGGCAUAAAUUGGU (SEQ ID NO: 330); or bb. a sense strand that consists of the nucleobase AGGCUGUAGGCAUAAAUUGGA (SEQ ID NO: 331); or cc. a sense strand that consists of the nucleobase
CGGCUGUAGGCAUAAAUUGGU (SEQ ID NO: 332); or dd. a sense strand that consists of the nucleobase
CGGCUGUAGGCAUAAAUUGGA (SEQ ID NO: 333); or ee. a sense strand that consists of the nucleobase
sequence (5’^3')
sequence (5'^3')
sequence (5'^3')
sequence (5'-»3·)
sequence (5'-»3')
sequence (5'^3')
sequence (5'^3')
sequence (5'^3')
sequence (5’^3')
sequence (5'-»3·)
sequence (5'^3')
CGCUGUAGGCAUAAAUUGGUA(SEQ IDNO: 334);
and wherein the HBV RNAi agent further comprises an antisense strand at least partially complementaiy to the respective antisense strand.
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein a first HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0,1, 2 or 3 nucleobases from the sequence (5'->3') UAUUGAGAGAAGUCC ACCACUU (SEQ ID NO: 175), and a sense strand that comprises the nucleobase sequence differing by 0, 1,2 or 3 nucleobases from the sequence (5'^3') GUGGUGGACUUCUCUCAAUAUU (SEQ ID NO: 307); and wherein a second HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') UACCAAUUUAUGCCUACAGUU (SEQ ID NO: 154), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') CUGUAGGCAUAAAUUGGUA (SEQ ID NO: 292).
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein a first HBV RNAi agent comprises an antisense strand that consists of the nucleobase sequence (5'-^3') UAUUGAGAGAAGUCCACCACUU (SEQ ID NO: 175), and a sense strand that consists of the nucleobase sequence (5'^3') GUGGUGGACUUCUCUCAAUAUU (SEQ ID NO: 307); and wherein a second HBV RNAi agent comprises an antisense strand that consists of the nucleobase sequence (5'^3') UACCAAUUUAUGCCUACAGUU (SEQ ID NO: 154), and a sense strand that consists of the nucleobase sequence (5’~>3*) CUGUAGGCAUAAAUUGGUA (SEQ ID NO: 292).
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein a first HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0,1, 2 or 3 nucleobases from the sequence (5'-^ 3') AGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 171), and a sense strand that comprises the nucleobase sequence differing by 0,1,2 or 3 nucleobases from the sequence (5'->3') GUGGACUUCUCUCAAUUUUCU (SEQ ID NO: 302); and wherein a second HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') UACCAAUUUAUGCCUACAGCG (SEQ ID NO: 188), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') CGCUGUAGGCAUAAAUUGGUA (SEQ IDNO: 328).
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein a first HBV RNAi agent comprises an antisense strand that consists of the nucleobase sequence (5'^3') AGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 171), and a sense strand that consists of the nucleobase sequence (5'»3') GUGGACUUCUCUCAAUUUUCU (SEQ ID NO: 302); and wherein a second HBV RNAi agent comprises an antisense strand that consists of the nucleobase sequence (5'^3') UACCAAUUUAUGCCUACAGCG (SEQ ID NO: 188), and a sense strand that consists of the nucleobase sequence (5'->3') CGCUGUAGGCAUAAAUUGGUA (SEQ ID NO: 328).
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein a first HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0,1, 2 or 3 nucleobases from the sequence (5'-^3') AGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 171), and a sense strand that comprises the nucleobase sequence differing by 0, 1,2 or 3 nucleobases from the sequence (5'->3') GUGGACUUCUCUCAAUUUUCU (SEQ ID NO: 302); and wherein a second HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5*->3') UACCAAUUUAUGCCUACAGCC (SEQ ID NO: 162), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') GGCUGUAGGCAUAAAUUGGUA (SEQ ID NO: 294).
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein a first HBV RNAi agent comprises an antisense strand that consists of the nucleobase sequence (5'->3') AGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 171), and a sense strand that consists of the nucleobase sequence (5'^3') GUGGACUUCUCUCAAUUUUCU (SEQ IDNO: 302); and wherein a second HBV RNAi agent comprises an antisense strand that consists of the nucleobase sequence (5'-»3') UACCAAUUUAUGCCUACAGCC (SEQ ID NO: 162), and a sense strand that consists of the nucleobase sequence (5'-^3') GGCUGUAGGCAUAAAUUGGUA (SEQ ID NO: 294).
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein a first HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0,1, 2 or 3 nucleobases from the sequence (5'-à3') AGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 171), and a sense strand that comprises the nucleobase sequence differing by 0, 1,2‘or 3 nucleobases from the sequence (5'^3') GUGGACUUCUCUCAAUUUUCU (SEQ ID NO: 302); and wherein a second HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') UACCAAUUUAUGCCUACAGCC (SEQ ID NO: 162), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') GUGGUGGACUUCUCUCAAUAUU (SEQ ID NO: 307).
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein a first HBV RNAi agent comprises an antisense strand that consists of the nucleobase sequence (5'->3') AGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 171), and a sense strand that consists of the nucleobase sequence (5'^3') GUGGACUUCUCUCAAUUUUCU (SEQ ID NO: 302); and wherein a second HBV RNAi agent comprises an antisense strand that consists of the nucleobase sequence (5'^3') UACCAAUUUAUGCCUACAGCC (SEQ ID NO: 162), and a sense strand that consists of the nucleobase sequence (5'->3*) GUGGUGGACUUCUCUCAAUAUU (SEQ ID NO: 307).
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein ail or substantially ail of the nucléotides in the sense strand are modified and/or ail or substantially ail of the nucléotides in the antisense strand in the first and/or second HBV RNAi agent are modified nucléotides, and wherein the first HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5^31) UAUUGAGAGAAGUCCACCACUU (SEQ ID NO: 175), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') GUGGUGGACUUCUCUCAAUAUU (SEQ ID NO: 307); and wherein the second HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5’->3*) UACCAAUUUAUGCCUACAGUU (SEQ ID NO: 154), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') CUGUAGGCAUAAAUUGGUA (SEQ ID NO: 292).
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein ail or substantially ail of the nucléotides in the sense strand are modified and/or ail or substantially ail of the nucléotides in the antisense strand in the first and/or second HBV RNAi agent are modified nucléotides, and wherein the first HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') AGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 171), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') GUGGACUUCUCUCAAUUUUCU (SEQ ID NO: 302); and wherein the second HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'~>3')
UACCAAUUUAUGCCUACAGCG (SEQ ID NO: 188), and a sense strand that comprises the nucleobase sequence diiTering by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') CGCUGUAGGCAUAAAUUGGUA (SEQ ID NO: 328).
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein ail or substantially ail of the nucléotides in the sense strand are modified and/or ail or substantially ail of the nucléotides in the antisense strand in the first and/or second HBV RNAi agent are modified nucléotides, and wherein the first HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') AGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 171), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') GUGGACUUCUCUCAAUUUUCU (SEQ ID NO: 302); and wherein the second HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3')
UACCAAUUUAUGCCUACAGCG (SEQ ID NO; 162), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') CGCUGUAGGCAUAAAUUGGUA (SEQ ID NO: 294).
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein ail or substantially ail of the nucléotides in the sense strand are modified and/or ail or substantially ail of the nucléotides in the antisense strand in the first and/or second HBV RNAi agent are modified nucléotides, and wherein the first HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'-^3') AGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 171), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') GUGGACUUCUCUCAAUUUUCU (SEQ ID NO: 302); and wherein the second HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'~>3')
UACCAAUUUAUGCCUACAGCG (SEQ ID NO: 162), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') GUGGUGGACUUCUCUCAAUAUU (SEQ ID NO: 307).
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein ail or substantially ail of the nucléotides in the sense strand are modified and/or ail or substantially ail of the nucléotides in the antisense strand in the first and/or second HBV RNAi agent are modified nucléotides, and wherein the first HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') UAUUGAGAGAAGUCCACCACUU (SEQ ID NO: 175), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') GUGGUGGACUUCUCUCAAUAUU (SEQ ID NO: 307); and wherein the second HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3')
UACCAAUUUAUGCCUACAGUU (SEQ ID NO: 154), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') CUGUAGGCAUAAAUUGGUA (SEQ ID NO: 292), and wherein the sense strand of the first HBV RNAi agent and the second HBV RNAi agent are conjugated to a targeting ligand comprising N-acetyl-galactosamine.
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein ail or substantially ail of the nucléotides in the sense strand are modified and/or ail or substantially ail of the nucléotides in the antisense strand in the first and/or second HBV RNAi agent are modified nucléotides, and wherein the first HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') AGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 171), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3*) GUGGACUUCUCUCAAUUUUCU (SEQ ID NO; 302); and wherein the second HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3')
UACCAAUUUAUGCCUACAGCG (SEQ ID NO: 188), and a sense strand that comprises the nucleobase sequence differing by 0, I, 2 or 3 nucleobases from the sequence (5'^3') CGCUGUAGGCAUAAAUUGGUA (SEQ ID NO: 328), and wherein the sense strand of the first HBV RNAi agent and the second HBV RNAi agent are conjugated to a targeting ligand comprising N-acetyl-galactosamine.
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein ail or substantially ail of the nucléotides in the sense strand are modified and/or ail or substantially ail of the nucléotides in the antisense strand in the first and/or second HBV RNAi agent are modified nucléotides, and wherein the first HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') AGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 171), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') GUGGACUUCUCUCAAUUUUCU (SEQ ID NO: 302); and wherein the second HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3')
UACCAAUUUAUGCCUACAGCC (SEQ ID NO: 162), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') GGCUGUAGGCAUAAAUUGGUA (SEQ ID NO: 294), and wherein the sense strand of the first HBV RNAi agent and the second HBV RNAi agent are conjugated to a targeting ligand comprising N-acetyl-galactosamine.
In some embodiments, disclosed herein are compositions for inhibiting expression of an HBV gene in a cell, the composition comprising two HBV RNAi agents, wherein ail or substantially ail of the nucléotides in the sense strand are modified and/or ail or substantially ail of the nucléotides in the antisense strand in the first and/or second HBV RNAi agent are modified nucléotides, and wherein the first HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') AGAAAAUUGAGAGAAGUCCAC (SEQ ID NO: 171), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'^3') GUGGACUUCUCUCAAUUUUCU (SEQ ID NO: 302); and wherein the second HBV RNAi agent comprises an antisense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3')
UACCAAUUUAUGCCUACAGCC (SEQ ID NO: 162), and a sense strand that comprises the nucleobase sequence differing by 0, 1, 2 or 3 nucleobases from the sequence (5'->3') GUGGUGGACUUCUCUCAAUAUU (SEQ ID NO: 307), and wherein the sense strand of the first HBV RNAi agent and the second HBV RNAi agent are conjugated to a targeting ligand comprising N-acetyl-galactosamine.
In some embodiments, disclosed herein are methods of treatment of an HBV infection or prévention of disease or symptoms caused by an HBV infection comprising administering to a subject in need thereof an effective amount of AD04872 and an effective amount of AD05070. In some embodiments, the ratio of AD04872 to AD05070 administered to a subject in need thereof is about 2:1. In some embodiments, the ratio of AD04872 to AD05070 administered to a subject in need thereofis about 3:1. In some embodiments, the ratio of AD04872 to AD05070 administered to a subject in need thereof is about 1:1. In some embodiments, the ratio of AD04872 to AD05070 administered to a subject in need thereof is about 4:1. In some embodiments, the ratio of AD04872 to AD05070 administered to a subject in need thereof is about 5:1. In some embodiments, the ratio of AD04872 to ADO5O7O administered to a subject in need thereof is about 1:2.
In some embodiments, about 1 mg/kg (mpk) of AD04872 and about 1 mg/kg of AD05070 are administered to a subject in need thereof. In some embodiments, about 1.5 mg/kg of AD04872 and about 1.5 mg/kg of AD05070 are administered to a subject in need thereof. In some embodiments, about 2.0 mg/kg of AD04872 and about 1.0 mg/kg of AD05070 are administered to a subject in need thereof. In some embodiments, about 3.0 mg/kg of AD04872 and about 1.0 mg/kg of AD05070 are administered to a subject in need thereof. In some embodiments, about 3.2 mg/kg of AD04872 and about 0.8 mg/kg of AD05070 are administered to a subject in need thereof. In some embodiments, about 2.7 mg/kg of AD04872 and about 1.3 mg/kg of AD05070 are administered to a subject in need thereof. In some embodiments, about 4.0 mg/kg of AD04872 and about 1.0 mg/kg of AD05070 are administered to a subject in need thereof. In some embodiments, about 3.3 mg/kg of AD04872 and about 1.7 mg/kg of AD05070 are administered to a subject in need thereof. In some embodiments, between about 0.05 and about 5 mg/kg of AD04872 and between about 0.05 and about 5 mg/kg of AD05070 are administered to a subject in need thereof. In some embodiments, about AD04872 and about AD05070 are administered separately (e.g., in separate injections). In some embodiments, the respective dose of AD04872 and the respective dose of AD05070 are administered together (e.g., in the same injection). In some embodiments, the respective dose of AD04872 and the respective dose of AD05070 are prepared in a single pharmaceutical composition.
In some embodiments, disclosed herein are methods of treatment of an HBV infection or prévention of diseases or symptoms caused by an HBV infection comprising administering to a subject in need thereof an effective amount of AD04872 and an effective amount of AD04776. In some embodiments, the ratio of AD04872 to AD04776 administered to a subject in need thereof is about 2:1. In some embodiments, the ratio of AD04872 to AD04776 administered to a subject in need thereof is about 3:1. In some embodiments, the ratio of AD04872 to AD04776 administered to a subject in need thereof is about 4:1. In some embodiments, the ratio of AD04872 to AD04776 administered to a subject in need thereof is about 1:1. In some embodiments, the ratio of AD04872 to AD04776 administered to a subject inneed thereofis 5:1. In some embodiments, theratio of AD04872toAD04776 administered to a subject in need thereof is 1:2.
In some embodiments, about 1 mg/kg (mpk) of AD04872 and about 1 mg/kg of AD04776 are administered to a subject in need thereof. In some embodiments, about 1.5 mg/kg of AD04872 and about 1.5 mg/kg of AD04776 are administered to a subject in need thereof. In some embodiments, about 2.0 mg^kg of AD04872 and about 1.0 mg/kg of AD04776 are administered to a subject in need thereof. In some embodiments, about 3.0 mg/kg of AD04872 and about 1.0 mg/kg of AD04776 are administered to a subject in need thereof. In some embodiments, about 3.2 mg/kg of AD04872 and about 0.8 mg/kg of AD04776 are administered to a subject in need thereof. In some embodiments, about 2.7 mg/kg of AD04872 and about 1.3 mg/kg of AD04776 are administered to a subject in need thereof. In some embodiments, about 4.0 mg/kg of AD04872 and about 1.0 mg/kg of AD04776 are administered to a subject in need thereof. In some embodiments, about 3.3 mg/kg of AD04872 and about 1.7 mg/kg of AD04776 are administered to a subject in need thereof. In some embodiments, between about 0.05 and about 5 mg/kg of AD04872 and between about 0.05 and about 5 mg/kg of AD04776 are administered to a subject in need thereof. In some embodiments, the respective doses of AD04872 and AD04776 are administered separately (e.g., in separate injections). In some embodiments, the respective doses of AD04872 and AD04776 are administered together (e.g., in the same injection). In some embodiments, the respective doses of AD04872 and AD04776 are prepared in a single pharmaceutical composition.
In some embodiments, disclosed herein are methods of treatment of an HBV infection or prévention of disease or symptoms caused by an HBV infection comprising administering to a subject in need thereof an effective amount of AD04872 and an effective amount of AD04982.
In some embodiments, the ratio of AD04872 to AD04982 administered to a subject in need thereof is about 2:1. In some embodiments, the ratio of AD04872 to AD04982 administered to a subject in need thereof is about 3:1. In some embodiments, the ratio of AD04872 to AD04982 administered to a subject in need thereof is about 4:1. In some embodiments, the ratio of AD04872 to AD04982 administered to a subject in need thereof is about 1:1. In some embodiments, the ratio of AD04872 to AD04982 administered to a subject in need thereof is about 5:1. In some embodiments, the ratio of AD04872 to AD04982 administered to a subject in need thereof is 1:2.
In some embodiments, about 1 mg/kg (mpk) of AD04872 and about 1 mg/kg of AD04982 are administered to a subject in need thereof. In some embodiments, about 1.5 mg/kg of AD04872 and about 1.5 mg/kg of AD04982 are administered to a subject in need thereof. In some embodiments, about 2.0 mg/kg of AD04872 and about 1.0 mg/kg of AD04982 are administered to a subject in need thereof. In some embodiments, about 3.0 mg/kg of AD04872 and about 1.0 mg/kg of AD04982 are administered to a subject in need thereof. In some embodiments, about 3.2 mg/kg of AD04872 and about 0.8 mg/kg of AD04982 are administered to a subject in need thereof. In some embodiments, about 2.7 mg/kg of AD04872 and about 1.3 mg/kg of AD04982 are administered to a subject in need thereof. In some embodiments, about 4.0 mg/kg of AD04872 and about 1.0 mg/kg of AD04982 are administered to a subject in need thereof. In some embodiments, about 3.3 mg/kg of AD04872 and about 1.7 mg/kg of AD04982 are administered to a subject in need thereof. In some embodiments, between about 0.05 and about 5 mg/kg of AD04872 and between about 0.05 and about 5 mg/kg of AD04982 are administered to a subject in need thereof. In some embodiments, the respective doses of AD04872 and AD04982 are administered separately (e.g., in separate injections). In some embodiments, the respective doses of AD04872 and AD04982 are administered together (e.g., in the same injection). In some embodiments, the respective doses of AD04872 and AD04982 are prepared in a single pharmaceutical composition.
In some embodiments, disclosed herein are methods of treatment of an HBV infection or prévention of disease or symptoms caused by an HBV infection comprising administering to a subject in need thereof an effective amount of AD04580 and an effective amount of AD04585. In some embodiments, the ratio of AD04580 to AD04585 administered to a subject in need thereof is about 2:1. In some embodiments, the ratio of AD04580 to AD04585 administered to a subject in need thereof is about 3:1. In some embodiments, the ratio of AD04580 to AD04585 administered to a subject in need thereof is about 4:1. In some embodiments, the ratio of AD04580 to AD04585 administered to a subject in need thereof is about 5:1. In some embodiments, the ratio of AD04580 to AD04585 administered to a subject in need thereof is about 1:1. In some embodiments, the ratio of AD04580 to AD04585 administered to a subject in need thereof is about 1:2. In some embodiments, about 1 mg/kg (mpk) of AD04580 and about 1 mg/kg of AD04585 are administered to a subject in need thereof. In some embodiments, about 1.5 mg/kg of AD04580 and about 1.5 mg/kg of AD04585 are administered to a subject in need thereof. In some embodiments, between about 0.05 and about 5 mg/kg of AD04580 and between about 0.05 and about 5 mg/kg of AD04585 are administered to a subject in need thereof.
In some embodiments, an HBV RNAi agent disclosed herein consists of or comprises AD05070 linked to (NAG37)s shown as a sodium sait having the structure represented by the following:
In some embodiments, an HBV RNAi agent disclosed herein consists of or comprises AD05070 linked to (NAG25)s shown as a sodium sait having the structure represented by the following:
Ηι rOH ho^-o HO .°H J J O /x z\.NHÂk„, NV ° J OH ° : l ΓΊ -NH /X.,. . — HO NhU° H° A S θ ° . /° Na S—P=O M H--V O—« ° HNH jj + - 1 ,-'Ή Ί Na S—P=O fi N h 1 < A Ο-Ί r. > ° 1 |.o/ । .h ] f o'' o O W .H + . | \ N-Z^N Na O—P—O // Ί\ A °A°V O O^ HjF Na+ O—-P=O f| N 1 Jss °-^o^/N o ox Na+O—P=O ό ^O OH z^oAa | J O=P—s‘ Na+ 4H ^9 y ri / \ O^o n-/n 1 + / fl O=P—O Na /° \ । ^H' ? ? i J——l 1 ^=\ Ao^n -O'xAïj/f^ ? O=P—o’ Na+ 'Άμ o'
In some embodiments, an HBV RNAi agent disclosed herein consists of or comprises AD05070 linked to (NAG37)s shown as a free acid having the structure represented by the following:
In some embodiments, an HBV RNAi agent disclosed herein consists of or comprises AD04580 linked to (NAG31)s shown as a sodium sait having the structure represented by the following:
In some embodiments, an HBV RNAi agent disclosed herein consists of or comprises AD04585 linked to (NAG25)s shown as a sodium sait having the structure represented by the following:
In some embodiments, an HIW RNAi agent disclosed herein consists of or comprises AD04872 linked to (NAG37)s shown as a sodium sait having the structure represented by the following:
In some embodiments, an HIW RNAi agent disclosed herein consists of or comprises AD04872 linked to (NAG25)s shown as a sodium sait having the structure represented by the following:
ι F Ο
Ο Ο,
In some embodiments. an HBV RNAi agent disclosed herein consists of or comprises AD04872 linked to (NAG37)s shown as a free acid having the structure represented by the following:
O Ο. ô
In some embodiments, the described HBV RNAi agent(s) are optionally combined with one or more additional (i.e., second, third, etc.) therapeutics. A second therapeutic can be another HBV RNAi agent (e.g., a HBV RNAi agent which targets a different sequence within an HBV genome). An additional therapeutic can also be a small molécule drug, antibody, antibody fragment, and/or vaccine. The HBV RNAi agents, with or without the one or more additional therapeutics, can be combined with one or more excipients to form pharmaceutical compositions.
In some embodiments, the described HBV RNAi agent(s) are optionally combined with one or more additional therapeutics, tvherein the additional therapeutic is a nucleoside inhibitor or nucléotide inhibitor. In some embodiments, the described HBV RNAi agent(s) are optionally combined with one or more additional therapeutics, wherein the additional therapeutic entecavir, tenofovir, tenofovir alafenamide, tenofovir disoproxil, lamivudine, or another antiviral therapeutic. In some embodiments, the described HBV RNAi agent(s) are optionally combined with one or more additional therapeutics, wherein the additional therapeutic is an interferon. In some embodiments, the described HBV RNAi agent(s) are optionally combined with one or more additional therapeutics, wherein the additional therapeutic is interferon-alpha. In some embodiments, the described HBV RNAi agent(s) are optionally combined with one or more HBV additional therapeutics, wherein the additional therapeutic is an HBV vaccine.
In some embodiments, the described HBV RNAi agent(s) are optionally combined with one or more additional therapeutics in a single dosage form (i.e., a cocktail included in a single injection). In some embodiments, the described HBV RNAi agent(s) may be administered separately from one or more optional additional therapeutics. In some embodiments, the described HBV RNAi agent(s) are administered to a subject in need thereof via subcutaneous injection, and the one or more optional additional therapeutics are administered orally, which together provide for a treatment regimen for diseases and conditions associated with HBV infection. In some embodiments, the described HBV RNAi agent(s) are administered to a subject in need thereof via subcutaneous injection, and the one or more optional additional therapeutics are administered via a separate subcutaneous injection.
In some embodiments, disclosed herein are compositions for delivering an HBV RNAi agent to a liver cell in vivo, the composition including an HBV RNAi agent conjugated or linked to a targeting group. In some embodiments, the targeting group is an asialoglycoprotein receptor ligand. In some embodiments, compositions for delivering an HBV RNAi agent to a liver cell in vivo are described, the composition including an HBV RNAi agent linked to an N-acetylgalactosamine targeting ligand.
In some embodiments, one or more of the described HBV RNAi agents are administered to a mammal in a pharmaceutically acceptable carrier or diluent. In some embodiments. the mammal is a human.
The use of Hepatitis B Virus RNAi agent(s) provides methods for therapeutic and/or prophylactic treatment of diseases/disorders which are associated with HBV infection. The described HBV RNAi agents médiate RNA interférence to inhibit the expression of one or more genes necessaiy for réplication and/or pathogenesis of Hepatitis B Virus. In particular, for example, HBV RNAi agents may inhibit viral polymerase, core protein, surface antigen, eantigen and/or the X protein, in a cell, tissue or mammal. HBV RNAi agents can be used to treat HBV infection. HBV RNAi agents can also be used to treat or prevent chronic liver diseases/disorders, inflammations, fibrotic conditions and proliférative disorders, like cancers, associated with HBV infection. ïn some embodiments, the methods further comprise treatment of Hepatitis D Virus (HDV) in the subject. Such methods comprise administration of HBV RNAi agent to a human being or animal infected with HBV. Further, compositions for deliveiy of HBV RNAi agents to liver cells in vivo are described.
The pharmaceutical compositions comprising one or more HBV RNAi agents can be administered in a number of ways depending upon whether local or systemic treatment is desired. Administration can be, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal, subdermal (e.g., via an implanted device), and intraparenchymal administration. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection.
The described HBV RNAi agents and/or compositions can be used in methods for therapeutic treatment of HBV infection or disease or conditions caused by HBV infection. Such methods include administration of an HBV RNAi agent as described herein to a subject, e.g., a human or animal subject.
As used herein, the terms “oligonucleotide” and “polynucleotide” mean a polymer of linked nucleosides each of which can be independently modified or unmodifïed.
As used herein, an “RNAi agent” or “RNAi trigger” means a composition that contains an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molécule that is capable οΓ degrading or inhibiting translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence spécifie manner. As used herein, RNAi agents may operate through the RNA interférence mechanism (i.e., inducing RNA interférence through interaction with the RNA interférence pathway' machinery (RNA-induced silencing complex or RISC) of mammalian cells), or by any alternative mechanism(s) or pathway(s). While it is believed that RNAi agents, as that term is used herein, operate primarily through the RNA interférence mechanism, the disclosed RNAi agents are not bound by' or limited to any particular pathway or mechanism of action. RNAi agents disclosed herein are comprised of a sense strand and an antisense strand, and include. but are not limited to: short interfering RNAs (siRNAs), double-stranded RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to the mRNA being targeted. RNAi agents may be comprised of modified nucléotides and/or one or more non-phosphodiester linkages.
As used herein, the terms “silence,” “reduce,” “inhibit,” “down-regulate,” or “knockdown” when referring to expression of a given gene, mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein or protein subunit translated from the mRNA in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is treated with oligomeric compounds, such as RNAi agents, described herein as compared to a second cell, group of cells, tissue, organ, or subject that has not or hâve not been so treated.
As used herein, the term “sequence” or “nucléotide sequence” mean a succession or order of nucleobases or nucléotides, described with a succession of letters using standard nomenclature.
As used herein, a “nucléotide base,” or “nucleobase” is a heterocyclic pyrimidine or purine compound, which is a standard constituent of ail nucleic acids, and includes the bases that form the nucléotides adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). A nucleobase may further be modified to include, without limitation, universal bases, hydrophobie bases, promiscuous bases, size-expanded bases, and fluorinated bases.
As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucléotide sequence (e.g., RNAi agent sense strand or targeted mRNA) in relation to a second nucléotide sequence (e.g., RNAi agent antisense strand or a single-stranded antisense oligonucleotide), means the ability of an oligonucleotide or polynucleotide including the first nucléotide sequence to hybridize (form base pair hydrogen bonds under mammalian physiological conditions (or similar conditions in vitro)) and form a duplex or double helical structure under certain conditions with an oligonucleotide or polynucleotide including the second nucléotide sequence. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucléotides or nucléotide mimics, at least to the extent that the above hybridization requirements are fulfîlled. Sequence identity or complementarity is independent of modification. For example, a and Af are complementaiy to U (or T) and identical to A for the purposes of determining identity or complementarity.
As used herein, “perfectly complementary” or “fully complementary” means that ail (100%) of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The contiguous sequence may' comprise ail or a part of a first or second nucléotide sequence.
As used herein, “partially complementary” means that in a hybridized pair of nucleobase sequences, at least 70%, but not ail, of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide.
As used herein, “substantially complementary” means that in a hybridized pair of nucleobase sequences, at least about 85%, but not ail, of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The terms “complementary,” “fully complementary,” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a double-stranded RNAi agent, between the antisense strand of an RNAi agent and a sequence of a target mRNA, or between a singlestranded antisense oligonucleotide and a sequence of a target mRNA.
As used herein. the term “substantially identical” or substantially identity as applied to nucleic acid sequence means that a nucleic acid sequence comprises a sequence that has at least about 85% sequence identity or more, preferably at least 90%, at least 95%, or at least 99%, compared to a reference sequence. Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the resuit by 100 to yield the percentage of sequence identity. The inventions disclosed herein encompasses nucléotide sequences substantially identical to those disclosed herein, e.g., in Tables 2,3, and 4. In some embodiments, the sequences disclosed herein are exactly identical, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent identical to those disclosed herein, e.g., in Tables 1, 2,3 and 4.
As used herein, the terms “treat,” “treatment,” and the like, mean the methods or steps taken to provide relief from or alleviation of the number, severity, and/or frequency of one or more symptoms of a disease or condition in a subject.
As used herein, the phrase “introducing into a cell,” when referring to an oligomeric compound, means functionally delivering the oligomeric compound into a cell. The phrase “functional delivery,” means that delivering the oligomeric compound to the cell in a manner that enables the oligomeric compound to hâve the expected biological activity, e.g., sequence-specific inhibition of gene expression.
Unless stated otherwise, use of the symbol as used herein means that any group or groups may be linked thereto that is in accordance with the scope of the inventions described herein.
As used herein, the term “isomers” refers to compounds that hâve identical molecular formulae, but that differ in the nature or the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereoisomers,” and stereoisomers that are non-superimposable mirror images are termed “enantiomers,” or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is termed a “chiral center.”
As used herein, unless specifically identified in a structure as having a particular conformation, for each structure in which asymmetric centers are présent and thus give rise to enantiomers, diastereomers, or other stereoisomeric configurations, each structure disclosed herein is intended to represent ail such possible isomers, including their optically pure and racemic forms. For example, the structures disclosed herein are intended to cover mixtures of diastereomers as well as single stereoisomers.
As used in a claim herein, the phrase “consisting of ’ excludes any element, step, or ingrédient not specified in the claim When used in a claim herein, the phrase “consisting essentially of ’ limits the scope of a claim to the specified materials or steps and those that do not materiahy affect tire basic and novel characteristic(s) of tire claimed invention.
The person of ordinary skill in the art would readily understand and appreciate that the compounds and compositions disclosed herein may hai e certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated State, depending upon the environment in which the compound or composition is placed. Accordingly, as used herein, the structures disclosed herein envisage that certain functional groups, such as, for example, OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to cover the disclosed compounds and compositions regardless of their state of protonation based on the environment (such as pH), as would be readily understood by the person of ordinary skill in the art.
Unless otherwise defined, ail technical and scientific ternis used herein hâve the same meaning as commonly understood by one of ordinary’ skill in the art. Although methods and materials similar or équivalent to those described herein can be used in the practice or testing of the présent invention, suitable methods and materials are described below. Ail publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety’. In case of conflict, the présent spécification, including définitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
Detailed Description
Described herein are RNAi agents for inhibiting expression of Hepatitis B Virus (HBV) (referred to herein as HBV RNAi agents or HBV RNAi triggers). Each HBV RNAi agent comprises a sense strand and an antisense strand. The sense strand and the antisense strand each can be 16 to 30 nucléotides in length. In some embodiments, the sense and antisense strands each can be 17 to 26 nucléotides in length. The sense and antisense strands can be either the same length or they can be different lengths. In some embodiments, the sense and antisense strands are each independently 17 to 26 nucléotides in length. In some embodiments, the sense and antisense strands are each independently 17-21 nucléotides in length. In some embodiments, both the sense and antisense strands are each 21-26 nucléotides in length. In some embodiments, the sense strand is about 19 nucléotides in length while the antisense strand is about 21 nucléotides in length. In some embodiments, the sense strand is about 21 nucléotides in length while the antisense strand is about 23 nucléotides in length. In some embodiments, both the sense and antisense strands are each 26 nucléotides in length. In some embodiments, the RNAi agent sense and antisense strands are each independently 17,18, 19, 20, 21, 22, 23, 24, 25, or 26 nucléotides in length. In some embodiments, a double-stranded RNAi agent has a duplex length of about 16,17,18,19,20,21, 22, 23 or 24 nucléotides. This région of perfect or substantial complementarity between the sense strand and the antisense strand is typically 15-25 (e.g., 15,16,17,18,19,20,21,22,23,24, or 25) nucléotides in length and occurs at or near the 5' end of the antisense strand (e.g., this région may be separated from the 5’ end of the antisense strand by 0, 1, 2, 3, or 4 nucléotides that are not perfectly or substantially complementary').
The sense strand and antisense strand each contain a core stretch sequence that is 16 to 23 nucleobases in length. An antisense strand core stretch sequence is 100% (perfectly) complementary' or at least about 85% (substantially) complementary’ to a nucléotide sequence (sometimes referred to, e.g., as a target sequence) présent in the HBV mRNA target. A sense strand core stretch sequence is 100% (perfectly) complementary or at least about 85% (substantially) complementary' to a core stretch sequence in the antisense strand, and thus the sense strand core stretch sequence is perfectly identical or at least about 85% identical to a nucléotide sequence (target sequence) présent in the HBV mRNA target. A sense strand core stretch sequence can be the same length as a corresponding antisense core sequence or it can be a different length. In some embodiments, the antisense strand core stretch sequence is 16, 17,18,19, 20.21,22, or 23 nucléotides in length. In some embodiments, the sense strand core stretch sequence is 16,17,18,19,20, 21,22, or 23 nucléotides in length.
Examples of sense and antisense strand nucléotide sequences used in forming HBV RNAi agents are provided in Tables 3 and 4. Examples of RNAi agent duplexes, that include the nucléotide sequences in Tables 3 and 4, are provided in Table 5.
The HBV RNAi agent sense and antisense strands anneal to form a duplex. A sense strand and an antisense strand of an HBV RNAi agent may be partially, substantially, or fully complementary to each other. Within the complementary duplex région, the sense strand core stretch sequence is at least about 85% complementary’ or 100% complementary to the antisense core stretch sequence. In some embodiments, the sense strand core stretch sequence contains a sequence ofat least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucléotides that is at least about 85% or 100% complementary to a corresponding 16,17,18,19, 20, or 21 nucléotide sequence of the antisense strand core stretch sequence (i.e., the sense strand and antisense core stretch sequences of an HBV RNAi agent hâve a région of at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucléotides that is at least 85% base paired or 100% base paired.).
In some embodiments, the antisense strand of an HBV RNAi agent disclosed herein differs by 0,1, 2, or 3 nucléotides from any of the antisense strand sequences in Table 2 or Table 3. In some embodiments, the sense strand of an HBV RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucléotides from any of the sense strand sequences in Table 2 or Table 4.
The length of the HBV RNAi agent sense and antisense strands described herein are independently 16 to 30 nucléotides in length. In some embodiments, the sense and antisense strands are independently 17 to 26 nucléotides in length. In some embodiments, the sense and antisense strands are 19-26 nucléotides in length. In some embodiments, the described RNAi agent sense and antisense strands are independently 17,18,19, 20, 21, 22, 23, 24, 25, or 26 nucléotides in length. The sense and antisense strands can be either the same length or they can be different lengths. In some embodiments, a sense strand and an antisense strand are each 26 nucléotides in length. In some embodiments, a sense strand is 23 nucléotides in length and-an antisense strand is 21 nucléotides in length. In some embodiments, a sense strand is 22 nucléotides in length and an antisense strand is 21 nucléotides in length. In some embodiments, a sense strand is 21 nucléotides in length and an antisense strand is 21 nucléotides in length. In some embodiments, a sense strand is 19 nucléotides in length and an antisense strand is 21 nucléotides in length.
The sense strand and/or the antisense strand may optionally and independently contain an additional 1, 2, 3,4, 5, or 6 nucléotides (extension) al the 3' end, the 5' end, or both the 3' and 5' ends of the core sequences. The antisense strand additional nucléotides, if présent, may or may not be complementaiy to the corresponding sequence in an HBV mRNA. The sense strand additional nucléotides, if présent, may or may not be identical to the corresponding sequence in an HBV mRNA. The antisense strand additional nucléotides, if présent, may or may* not be complementaiy to the corresponding sense strand’s additional nucléotides, if présent.
As used herein, an extension comprises 1, 2, 3, 4, 5, or 6 nucléotides atthe 5' and/or 3' end of the sense strand core stretch sequence and/or antisense strand core stretch sequence. The extension nucléotides on a sense strand may or may not be complementaiy to nucléotides, either core stretch sequence nucléotides or extension nucléotides, in the corresponding antisense strand Conversely, the extension nucléotides on an antisense strand may or may not be complementaiy to nucléotides, either core stretch sequence nucléotides or extension nucléotides, in the corresponding sense strand. In some embodiments, both the sense strand and the antisense strand of an RNAi agent contain 3' and 5' extensions. In some embodiments, one or more of the 3' extension nucléotides of one strand base pairs with one or more 5' extension nucléotides of the other strand. In other embodiments, one or more of 3' extension nucléotides of one strand do not base pair with one or more 5’ extension nucléotides of the other strand. In some embodiments, an HBV RNAi agent has an antisense strand having a 3' extension and a sense strand having a 5' extension.
In some embodiments, an HBV RNAi agent comprises an antisense strand having a 3' extension of 1, 2, 3, 4, 5, or 6 nucléotides in length. In other embodiments, an HBV RNAi agent comprises an antisense strand having a 3' extension of 1,2, or 3 nucléotides in length. In some embodiments, one or more of the antisense strand extension nucléotides comprise uracil or thymidine nucléotides or nucléotides which are complementary to a corresponding HBV niRNA sequence. In some embodiments, a 3' antisense strand extension includes or consists of, but is not limited to: AUA, UGCUU, CUG, UG, UGCC, CUGCC, CGU, CUU, UGCCUA, CUGCCU, UGCCU, UGAUU, GCCUAU, T, TT, U, UU (each listed 5' to 3').
In some embodiments, the 3' end of the antisense strand may include additional abasic nucleosides (Ab). In some embodiments, Ab or AbAb may be added to the 3' end of the antisense strand.
In some embodiments, an HBV RNAi agent comprises an antisense strand having a 5' extension of 1,2,3,4, or 5 nucléotides in length. In other embodiments, an HBV RNAi agent comprises an antisense strand having a 5' extension of 1 or 2 nucléotides in length. In some embodiments, one or more of the antisense strand extension nucléotides comprises uracil or thymidine nucléotides or nucléotides which are complementary to a corresponding HBV mRNA sequence. In some embodiments, the 5' antisense strand extension includes or consists of, but is no limited to, UA, TU, U, T, UU, TT, CUC (each listed 5' to 3'). An antisense strand may hâve any of the 3' extensions described above in combination with any of the 5' antisense strand extensions described, if présent.
In some embodiments, an HBV RNAi agent comprises a sense strand having a 3' extension of 1,2,3, 4, or 5 nucléotides in length. In some embodiments, one or more of the sense strand extension nucléotides comprises adenosine, uracil, or thymidine nucléotides, AT dinucleotide, or nucléotides which correspond to nucléotides in the HBV mRNA sequence. In some embodiments, the 3' sense strand extension includes or consists of, but is not limited to: T, UT, TT, UU, UUT, TTT, or TTTT (each listed 5' to 3'). '
In some embodiments, the 3' end of the sense strand may include additional abasic nucleosides. In some embodiments, UUAb, UAb, or Ab may be added to the 3' end of the sense strand. In some embodiments, the one or more abasic nucleosides added to the 3' end of the sense strand may be inverted (invAb). In some embodiments, one or more inverted abasic nucleosides may be inserted between the targeting ligand and the nucleobase sequence of the sense strand of the RNAi agent. In some embodiments, the inclusion of one or more inverted abasic nucleosides at or near the terminal end or terminal ends of the sense strand of an RNAi agent may allow for enhanced activity or other desired properties of an RNAi agent.
In some embodiments, an HBV RNAi agent comprises a sense strand having a 5' extension of 1,2, 3,4, 5, or 6 nucléotides in length. In some embodiments, one or more of the sense strand extension nucléotides comprise uracil or adenosine nucléotides or nucléotides which correspond to nucléotides in theHBV mRNA sequence. In some embodiments, the sense strand 5' extension can be, but is not limited to: CA, AUAGGC, AUAGG, AU AG, AU A, A, AA, AC, GCA, GGCA, GGC, UAUCA, UAUC, UCA, UAU, U, UU (each listed 5' to 3'). A sense strand may hâve a 3' extension and/or a 5' extension.
In some embodiments, the 5' end of the sense strand may include an additional abasic nucleoside (Ab) or nucleosides (AbAb). In some embodiments, the one or more abasic nucleosides added to the 5' end of the sense strand may be inverted (invAb). In some embodiments, one or more inverted abasic nucleosides may be inserted between the targeting ligand and the nucleobase sequence of the sense strand of the RNAi agent. In some embodiments, the inclusion of one or more inverted abasic nucleosides at or near the terminal end or terminal ends of the sense strand of an RNAi agent may allow for enhanced activity or other desired properties of an RNAi agent.
Examples of nucléotide sequences used in forming HBV RNAi agents are provided in Tables 3 and 4. In some embodiments, an HBV RNAi agent antisense strand includes a nucléotide sequence of any of the sequences in Table 3. In some embodiments, an HBV RNAi agent antisense strand includes the sequence of nucléotides 1-17, 2-15, 2-17,1-18,2-18,1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22,2-22,1-23,2-23,1-24, 2-24,1-25,2-25,1-26, or 2-26 of any of the sequences in Table 3. In some embodiments, an HBV RNAi agent sense strand includes the nucléotide sequence of any of the sequences in Table 4. In some embodiments, an HBV RNAi agent sense strand includes the sequence of nucléotides 1-18,1-19,1-20,1-21,1-22,123, 1-24, 1-25, 1-26, 2-19,2-20,2-21,2-22,2-23, 2-24,2-25, 2-26, 3-20, 3-21,3-22,3-23, 324, 3-25, 3-26,4-21,4-22, 4-23, 4-24, 4-25,4-26, 5-22,5-23, 5-24,5-25, 5-26,6-23, 6-24, 625, 6-26, 7-24, 7-25, 7-25, 8-25, 8-26 of any of the sequences in Table 4.
In some embodiments, the sense and antisense strands of the RNAi agents described herein contain the same number of nucléotides. In some embodiments, the sense and antisense strands of the RNAi agents described herein contain different numbers of nucléotides. In some embodiments, the sense strand 5' end and the antisense strand 3' end of an RNAi agent form a blunt end. In some embodiments, the sense strand 3' end and the antisense strand 5' end of an
RNAi agent form a blunt end. In some embodiments, both ends of an RNAi agent form blunt ends. In some embodiments, neither end of an RNAi agent is blunt-ended. As used herein a blunt end refers to an end of a double stranded RNAi agent in which the terminal nucléotides of the two annealed strands are complementary (form a complementary base-pair). In some embodiments, the sense strand 5' end and the antisense strand 3' end of an RNAi agent form a frayed end. In some embodiments, the sense strand 3' end and the antisense strand 5' end of an RNAi agent form a frayed end. In some embodiments, both ends of an RNAi agent form a frayed end. In some embodiments, neither end of an RNAi agent is a frayed end. As used herein a frayed end refers to an end of a double stranded RNAi agent in which the terminal nucléotides of the two annealed strands from a pair (i.e. do not form an overhang) but are not complementaiy (i.e. form anon-complementaiy pair). As used herein, an overhang is a stretch of one or more unpaired nucléotides at the end of one strand of a double stranded RNAi agent. The unpaired nucléotides may be on the sense strand or the antisense strand, creating either 3' or 5' overhangs. In some embodiments, the RNAi agent contains: a blunt end and a frayed end, a blunt end and 5' overhang end, a blunt end and a 3' overhang end, a frayed end and a 5' overhang end, a frayed end and a 3' overhang end, two 5' overhang ends, two 3' overhang ends, a 5' overhang end and a 3' overhang end, two frayed ends, or two blunt ends.
A nucléotide base (or nucleobase) is a heterocyclic pyrimidine or purine compound which is a constituent of ail nucleic acids and includes adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). As used herein, the term “nucléotide” can include a modified nucléotide (such as, for example, anucleotide mimic, abasic site (Ab), or a surrogate replacement moiety). Modified nucléotides, when used in various polynucleotide or oligonucleotide constructs, may preserve activity of the compound in cells while at the same time increasing the sérum stability of these compounds, and can also minimize the possibility of activating interferon activity in humans upon administering of the polynucleotide or oligonucleotide construct.
In some embodiments, an HBV RNAi agent is prepared or provided as a sait, mixed sait, or a free-acid. In some embodiments, an HBV RNAi agent is prepared as a sodium sait. Such forms are within the scope of the inventions disclosed herein.
Modified Nucléotides
In some embodiments, an HBV RNAi agent contains one or more modified nucléotides. As used herein, a “modified nucléotide” is a nucléotide other than a ribonucleotide (2'-hydroxyl nucléotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucléotides are modified nucléotides. As used herein, modified nucléotides include, but are not limited to, deoxyribonucleotides, nucléotide mimics, abasic nucléotides (represented herein as Ab), 2'modified nucléotides, 3' to 3' linkages (inverted) nucléotides (represented herein as invdN, invN, invn, invAb), non-natural base-comprising nucléotides, bridged nucléotides, peptide nucleic acids (PNAs), 2',3'-seco nucléotide mimics (unlocked nucleobase analogues, represented herein as Nuna or NUNA), locked nucléotides (represented herein as Nlna or NLNA), 3'-O-methoxy (2' intemucleoside linked) nucléotides (represented herein as 3'OMen), 2'-F-Arabino nucléotides (represented herein as NfANA or NFana), 5'-Me, 2'-fluoro nucléotide (represented herein as 5Me-Nf), morpholino nucléotides, vinyl phosphonate deoxyribonucleotides (represented herein as vpdN), vinyl phosphonate containing nucléotides, and cyclopropyl phosphonate containing nucléotides (cPrpN). 2'-modified nucléotides (i.e. a nucléotide with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucléotides (represented herein as a lower case letter ’n1 in a nucléotide sequence), 2'-deoxy-2'-fluoro nucléotides (represented herein as Nf, also represented herein as 2'-iluoro nucléotide), 2'-deoxy nucléotides (represented herein as dN), 2'-methoxyethyl (2'-O-2-methoxylethyl) nucléotides (represented herein as NM or 2'-M0E). 2'-amino nucléotides, and 2'-alkyl nucléotides. It is not necessary for ail positions in a given compound to be uniformly modified. Conversely, more than one modification may be incorporated in a single HBV RNAi agent or even in a single nucléotide thereof. The HBV RNAi agent sense strands and antisense strands may be synthesized and/or modified by methods known in the art. Modification at one nucléotide is independent of modification at another nucléotide.
Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-etliyl, 6-isopropyl, or6-n-butyl) dérivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl dérivatives of adenine and guanine, 2thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8halo, 8-amino, 8-sulfhydiyl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-haIo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.
In some embodiments, ail or substantially ail of thenucléotides of an RNAi agent are modified nucléotides. As used herein, an RNAi agent wherein substantially ail of the nucléotides présent are modified nucléotides is an RNAi agent having four or fewer (i.e., 0,1,2,3, or 4) nucléotides in both the sense strand and the antisense strand being ribonucleotides. As used herein, a sense strand wherein substantially ail of the nucléotides présent are modified nucléotides is a sense strand having two or fewer (i.e., 0,1, or 2) nucléotides in the sense strand being ribonucleotides. As used herein, an antisense sense strand wherein substantially ail of the nucléotides présent are modified nucléotides is an antisense strand having two or fewer (i.e., 0,1, or 2) nucléotides in the sense strand being ribonucleotides. In some embodiments, one or more nucléotides of an RNAi agent is a ribonucleotide.
Modified Intemucleoside Linkages
In some embodiments, one or more nucléotides of an HBV RNAi agent are linked by nonstandard linkages or backbones (i.e., modified intemucleoside linkages or modified backbones). In some embodiments, a modified intemucleoside linkage is a non-phosphatecontaining covalent intemucleoside linkage. Modified intemucleoside linkages or backbones include, but are not limited to, 5’-phosphorothioate groups (represented herein as a lower case “s”), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3'-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkylphosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, a modified intemucleoside linkage or backbone lacks a phosphorus atom. Modified intemucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages. In some embodiments, modified intemucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and Ihiofoimacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH? components.
In some embodiments, a sense strand of an HBV RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, an antisense strand of an HBV RNAi agent can contain 1, 2, 3,4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1,2,3,4,5, or 6 phosphorothioate linkages. In some embodiments, a sense strand of an HBV RNAi agent can contain 1,2,3, or 4 phosphorothioate linkages, an antisense strand of an HBV RNAi agent can contain 1,2,3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, or 4 phosphorothioate linkages.
In some embodiments, an HBV RNAi agent sense strand contains at least two phosphorothioate intemucleoside linkages. In some embodiments, the at least two phosphorothioate intemucleoside linkages are between the nucléotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, the at least two phosphorothioate intemucleoside linkages are between the nucléotides at positions 1-3, 2-4, 3-5,4-6, 4-5, or 6-8 from the 5' end of the sense strand. In some embodiments, an HBV RNAi agent antisense strand contains four phosphorothioate intemucleoside linkages. In some embodiments, the four phosphorothioate intemucleoside linkages are between the nucléotides at positions 1-3 from the 5' end of the sense strand and between the nucléotides at positions 19-21,20-22,21-23,22-24,23-25, or 2426 from the 5' end. In some embodiments, an HBV RNAi agent contains at least two phosphorothioate intemucleoside linkages in the sense strand and three or four phosphorothioate intemucleoside linkages in the antisense strand.
In some embodiments, an HBV RNAi agent contains one or more modified nucléotides and one or more modified intemucleoside linkages. In some embodiments, a 2'-modified nucleoside is combined with modified intemucleoside linkage.
HBV RNAi Agents
In some embodiments, tire HBV RNAi agents disclosed herein target an HBV gene at or near the positions of the HBV genome shown in the following Table 1. In some embodiments, the antisense strand of an HBV RNAi agent disclosed herein includes a core stretch sequence that is fully, substantially. or at least partially complementary to a target ΗΒλ^ 19-mer sequence disclosed in Table 1.
Table 1. Example 19-mer HBV cDNA target sequences for HBV RNAi agents (taken from Hepatitis B virus (subtype ADW2), génotype A, complété genome GenBank AM282986.1 (SEQ IDNO: 1)).
SEQ ID No. HBV 19-mer Target Sequences (5’^3’) Genome Position of SEQ ID NO: 1 Région of HBV Gene Targeted
2 GTGGTGGACTTCTCTCAAT 256-274 S ORF
3 TGGTGGACTTCTCTCAATT 257-275 S ORF
4 GGACTTCTCTCAATTTTCT 261-279 S ORF
5 GCTGTAGGCATAAATTGGT 1780-1798 XORF
6 CTGTAGGCATAAATTGGTC 1781-1799 XORF
In some embodiments, an HBV RNAi agent includes an antisense strand wherein position 19 of the antisense strand (5'->3') is capable of forming a base pair with position 1 of a 19-mer target sequence disclosed in Table 1. In some embodiments, an HBV RNAi agent includes an antisense strand wherein position 1 of the antisense strand (5' -> 3') is capable of forming a base pair with position 19 of the 19-mer target sequence disclosed in Table 1.
In some embodiments, an HBV RNAi agent includes an antisense strand wherein position 2 of the antisense strand (5' -> 3') is capable of forming a base pair with position 18 of the 19-mer target sequence disclosed in Table 1. In some embodiments, an HBV RNAi agent includes an antisense strand wherein positions 2 through 18 of the antisense strand (5' 3') are capable of forming base pairs with each of the respective complementary bases located at positions 18 through 2 of the 19-mer target sequence disclosed in Table 1.
In some embodiments, the HBV RNAi agents include core 19-mer nucléotide sequences shown in the following Table 2.
Table 2. HBV RNAi agent antisense strand and sense strand core stretch sequences (N=any nucléotide).
SEQ ID NO: Antisense Sequence (S’ - 3’) (19-mer) SEQ ID NO: Sense Sequence (5’ - 3’) (19-mer) Genome Position of SEQ ID NO: 1
7 AUUGAGAGAAGUCCACCAC 34 GUGGUGGACUUCUCUCAAU 256-274
8 UUUGAGAGAAGUCCACCAC 35 GUGGUGGACUUCUCUCAAA 256-274
9 AUUGAGAGAAGUCCACCAN 36 NUGGUGGACUUCUCUCAAU 256-274
10 UUUGAGAGAAGUCCACCAN 37 NUGGUGGACUUCUCUCAAA 256-274
11 NUUGAGAGAAGUCCACCAN 38 NUGGUGGACUUCUCUCAAN 256-274
12 AAUUGAGAGAAGUCCACCA 39 UGGUGGACUUCUCUCAAUU 257-275
13 UAUUGAGAGAAGUCCACCA 40 UGGUGGACUUCUCUCAAUA 257-275
14 AAUUGAGAGAAGUCCACCN 41 NGGUGGACUUCUCUCAAUU 257-275
15 UAUUGAGAGAAGUCCACCN 42 NGGUGGACUUCUCUCAAUA 257-275
16 NAUUGAGAGAAGUCCACCN 43 NGGUGGACUUCUCUCAAUN 257-275
17 AGAAAAU UGAGAGAAGUCC 44 GGACUUCUCUCAAUUUUCU 261-279
18 UGAAAAUUGAGAGAAGUCC 45 GGACUUCUCUCAAUUUUCA 261-279
19 AGAAAAUUGAGAGAAGUCN 46 NGACUUCUCUCAAUUUUCU 261-279
20 UGAAAAUUGAGAGAAGUCN 47 NGACUUCUCUCAAUUUUCA 261-279
21 NGAAAAUUGAGAGAAGUCN 48 NGACUUCUCUCAAUUUUCN 261-279
22 ACCAAUUUAUGCCUACAGC 49 GCUGUAGGCAUAAAUUGGU 1780-1798
23 UCCAAUUUAUGCCUACAGC 50 GCUGUAGGCAUAAAUUGGA 1780-1798
24 ACCAAUUUAUGCCUACAGN 51 NCUGUAGGCAUAAAUUGGU 1780-1798
25 UCCAAUUUAUGCCUACAGN 52 NCUGUAGGCAUAAAUUGGA 1780-1798
26 NCCAAUUUAUGCCUACAGN 53 NCUGUAGGCAUAAAUUGGN 1780-1798
27 GACCAAUUUAUGCCUACAG 54 CUGUAGGCAUAAAUUGGUC 1781-1799
28 AACCAAUUUAUGCCUACAG 55 CUGUAGGCAUAAAUUGGUU 1781-1799
29 UACCAAUUUAUGCCUACAG 56 CUGUAGGCAUAAAUUGGUA 1781-1799
SEQ ID NO: Antisense Sequence (5’ - 3’) (19-mer) SEQ ID NO: Sense Sequence (5’ - 3’) (19-mer) Genome Position οΓ SEQ ID NO: 1
30 GACCAAUUUAUGCCUACAN 57 NUGUAGGCAUAAAUUGGUC 1781-1799
31 AACCAAUUUAUGCCUACAN 58 NUGUAGGCAUAAAUUGGUU 1781-1799
32 UACCAAUUUAUGCCUACAN 59 NUGUAGGCAUAAAUUGGUA 1781-1799
33 NACCAAUUUAUGCCUACAN 60 NUGUAGGCAUAAAUUGGUN 1781-1799
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The HBV RNAi agent sense strands and antisense strands that comprise or consist of the nucléotide sequences in Table 2 can be modified nucléotides or unmodified nucléotides. In some embodiments, the HBV RNAi agents having the sense and antisense strand sequences that comprise or consist of the nucléotide sequences in Table 2 are ail or substantially ail modified nucléotides.
In some embodiments, the antisense strand of an HBV RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucléotides from any of the antisense strand sequences in Table 2. In some embodiments, the sense strand of an HBV RNAi agent disclosed herein differs by 0,1,2, or 3 nucléotides from any of the sense strand sequences in Table 2.
Modified HBV RNAi agent antisense strand sequences, as well as their underlying unmodified sequences, are provided in Table 3. Modified HBV RNAi agent sense strands, as well as their underlying unmodified sequences, are provided in Table 4. In forming HBV RNAi agents, each of the nucléotides in each of the unmodified sequences listed in Tables 3 and 4 may be a modified nucléotide.
As used herein (including in Tables 3 and 4), the following notations are used to indicate modified nucléotides, targeting groups, and linking groups. As the person of ordinary skill in the art would readily understand, unless otherwise indicated by the sequence, that when présent in an oligonucleotide, the monomers are mutually linked by 5’-3’-phosphodiester bonds:
A = adenosine-3'-phosphate;
C = cylidine-3'-phosphate;
G = guanosine-3'-phosphate;
U = uridine-3'-phosphate n = any 2'-0Me modified nucléotide a = 2'-O-methyladenosine-3'-phosphate as = 2'-O-methyladenosine-3'-phosphorothioate c = 2'-O-methylcytidine-3'-phosphate es = 2'-O-methylcytidine-3'-phosphorothioate g = 2'-O-methylguanosine-3’-phosphate gs = 2'-O-methylguanosine-3'-phosphorothioate t = 2'-O-methyl-5-methyluridine-3'-phosphate ts = 2'-O-methyl-5-methyluridine-3'-phosphorothioate u = 2'-O-methyluridine-3'-phosphate us = 2'-O-methyluridine-3'-phosphorothioate
101
Nf = any 2'-fluoro modified nucléotide
Af = 2'-fluoroadenosine-3'-phosphate
Afs = 2'-fluoroadenosine-3'-phosporothioate
Cf = 2'-fluorocytidine-3'-phosphate
5 Cfs = 2'-fluorocytidine-3'-phosphorothioate
Gf = 2'-fluoroguanosine-3'-phosphate
Gfs = 2'-fluoroguanosine-3'-phosphorothioate
Tf = 2'-fluoro-5'-methyluridine-3'-phosphate
Tfs = 2'-iluoro-5'-methyluridine-3'-phosphorothioate
10 Uf = 2'-fluorouridine-3'-phosphate
Ufs = 2'-fluorouridine-3'-phosphorothioate
dN = any 2'-deoxyribonucleotide
dT = 2'-deoxythymidine-3'-phosphate
Nuna = 2',3'-seco nucléotide mimics (unlocked nucleobase analogs)
15 Nlna = locked nucléotide
N£ana = 2-F-Arabino nucléotide
NM = 2'-methoxyethyl nucléotide
AM = 2'-methoxyethyladenosine-3'-phosphate
AMs = 2'-methoxyethyladenosine-3'-phosphorothioate
20 TM = 2'-methoxyethylthymidine-3'-phosphate
TMs = 2'-methoxyethylthymidine-3'-phosphorothioate
R = ribitol
(invdN) = any inverted deoxyribonucleotide (3-3' linked nucléotide)
(invAb) = inverted(3-3'linked)abasicdeoxyribonucleotide, seeTable6
25 (invAb)s = inverted (3'-3' linked) abasic deoxyribonucleotide-5'-
phosphorothioate, see Table 6
(invn) = any inverted 2'-0Me nucléotide (3 '-3' linked nucléotide)
s = phosphorothioate linkage
vpdN = vinyl phosphonate deoxyribonucleotide
30 (5Me-Nf) = 5-Me, 2’-fluoro nucléotide
cPrp = cyclopropyl phosphonate, see Table 6
epTcPr = see Table 6
epTM = see Table 6
The person or ordinaiy skill in the art would readily understand that the terminal nucléotide at the 3' end of a given oligonucleotide sequence would typically hâve a hydroxyl (-OH) group at the respective 3' position of the given monomer instead of a phosphate moiety ex vivo. Thus, for example, as shown above in the structure représentation of AD05070, above, the “g” modified nucléotide on the terminal 3' end of the antisense strand of AM06606-AS has a hydroxyl group positioned at its 3' position. Unless expressly indicated otherwise herein, such
102 understandings of the person of ordinary skill in the art are used when describing the HBV RNAi agents and compositions of HBV RNAi agents disclosed herein.
Targeting groups and linking groups include the following. for which their Chemical structures 5 are provided below in Table 6: (PAZ), (NAG13), (NAG13)s, (NAGI 8), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), (NAG39)s. Each sense 10 strand and/or antisense strand can hâve any targeting groups or linking groups listed above, as well as other targeting or linking groups, conjugated to the 5' andzor 3' end of the sequence.
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104
Table 3. HBV RNAi Agent antisense strand sequences.
AS Strand ID Modified sequence (5' —► 3') SEQ ID NO. Unmodified sequence (5' —♦ 3') SEQ ID NO.
AM03508-AS usAfscCfaAfuUfuAfuGfcCfuAfcAfgGfccsusuAu 61 UACCAAUUUAUGCCUACAGGCCUUAU 149
AM04441-AS usAfscCfaAfuUfuAfuGfcCfuAfcAfgGfcscsu 62 UACCAAUUUAUGCCUACAGGCCU 150
AM04442-AS usAfscsCfaAfuUfuAfuGfcCfiiAfcAfgGfccsu 63 UACCAAUUUAUGCCUACAGGCCU 150
AM04443-AS usAfscsCfaAfuUfuAfuGfcCfüAfcAfgGfsc 64 UACCAAUUUAUGCCUACAGGC 151
AM04661-AS usGfsugaAfgCfGfaaguGfcAfcacsusu 65 UGUGAAGCGAAGUGCACACUU 152
AM04768-AS us Afs cCfaAfuU fuAfuGfcCfu Afc AfgC fcsusccgc 66 UACCAAUUUAUGCCUACAGCCUCCGC 153
AM04769-AS vpusAfscCfaAfuUfuAfuGfcCfuAfcAfgCfcsusccgc 67 UACCAAUUUAUGCCUACAGCCUCCGC 153
AM05011-AS usAfscsCfaAfuUfuAfuGfcCfuAfcAfgusu 68 UACCAAUUUAUGCCUACAGUU 154
AM05012-AS usAfscsCfaAfuUfuAfuGfcCfuAfcAfggsc 69 UACCAAUUUAUGCCUACAGGC 151
AM05013-AS vpusAfscsCfaAfuUfuAfuGfcCfuAfcAfgGfsc 70 UACCAAUUUAUGCCUACAGGC 151
AM05014-AS vpusAfscsCfaAfuUfuAfuGfcCfuAfcAfgusu 71 UACCAAUUUAUGCCUACAGUU 154
AM05052-AS as U fsusGfaGfaGfaAfg U fcCfaCfcAfcGfsa 72 AUUGAGAGAAGUCCACCACGA 155
AM05053-AS asUfsusGfaGfaGfaAfgUfcCfaCfcAfcgsa 73 AUUGAGAGAAGUCCACCACGA 155
AM05054-AS asUfsusGfaGfaGfaAfgUfcCfaCfcAfcusu 74 AUUGAGAGAAGUCCACCACUU 156
AM05055-AS vpusUfsusGfaGfaGfaAfgUfcCfaCfcAfcGfsa 75 UUUGAGAGAAGUCCACCACGA 157
AM05056-AS asAfsusUfgAfgAfgAfaGfuCfcAfcCfaCfsg 76 AAUUGAGAGAAGUCCACCACG 158
AM05057-AS asAfsusUfgAfgAfgAfaGfuCfcAfcCfacsg 77 AAU UGAGAGAAGUCC ACC AC G 158
AM05058-AS asAfsusUfgAfgAfgAfaGfuCfcAfcCfausu 78 AAUUGAGAGAAGUCCACCAUU 159
AM05060-AS vpusAfsusUfgAfgAfgAfaGfuCfcAfcCfaCfsg 79 UAUUGAGAGAAGUCCACCACG 160
AM05351-AS usAfscsCfaAfuUfuAfuGfcCfuAfcAfgGfsu 80 UACCAAUUUAUGCCUACAGGU 161
AM05608-AS usAfscCfaAfuUfuAfuGfcCfuAfcAfgsusu 81 UACCAAUUUAUGCCUACAGUU 154
AM05609-AS usAfscsCfaAfuUfuAfuGfcCfuAfcAfgcsc 82 UACCAAUUUAUGCCUACAGGC 162
AM05610-AS usAfscsCfaAfuUfuAfuGfcCfuAfcAfgccusu 83 UACCAAUUUAUGCCUACAGCCUU 163
AM05611-AS usAfscsCfaAfuUfuAfuGfcCfuAfcAfgccusc 84 UACCAAUUUAUGCCUACAGCCUC 164
105
AM05612-AS usAfscscaauUfuAfuGfcCfuacagcsc 85 UACCAAUUUAUGCCUACAGCC 162
AM05613-AS us Afs cscaauUfuAfuGfcCfuacagccusu 86 UACCAAUUUAUGCCUACAGCCUU 163
AM05614-AS usAfscscaauUfuAfuGfcCfuacagccusc 87 UACCAAUUUAUGCCUACAGCCUC 164
AM05618-AS asUfsusgagaGfaAfgUfcCfaccacusu 88 AUUGAGAGAAGUCCACCACUU 156
AM05621-AS usUfsusGfaGfaGfaAfgUfcCfaCfcAfcusu 89 UUUGAGAGAAGUCCACCACUU 165
AM05623-AS asUfsusGfaGfaGraAfgUfcCfaCfcAfcggusu 90 AUUGAGAGAAGUCCACCACGGUU 166
AM05626-AS asUfsusgagaGfaAfgUfcCfaccacggusu 91 AUUGAGAGAAGUCCACCACGGUU 166
AM05628-AS asUfsusGfaGfaGfaAfgUfcCfaCfcAfcgagsu 92 AUUGAGAGAAGUCCACCACGAGU 167
AM05631-AS usAfsusUfgAfgAfgAfaGfuCfcAfcCfaCfsg 93 UAUUGAGAGAAGUCCACCACG 160
AM05632-AS usAfsusugagAfgAfaGfuCfcaccacsg 94 UAUUGAGAGAAGUCCACCACG 160
AM05633-AS usAfsusUfgAfgAfgAfaGfuCfcAfcCfaCfgusu 95 UAUUGAGAGAAGUCCACCACGUU 168
AM05634-AS usAfsusugagAfgAfaGfuCfcaccacgasg 96 UAUUGAGAGAAGUCCACCACGAG 169
AM05635-AS usAfsusüfgAfgAfgAfaGfuCfcAfcCfaCfgasg 97 UAUUGAGAGAAGUCCACCACGAG 169
AM05637-AS usAfsusUfgAfgAfgAfaGfuCfcAfcCfaCfgsa 98 UAUUGAGAGAAGUCCACCACGA 170
AM05638-AS usAfsusugagAfgAfaGfuCfcaccacgsa 99 UAUUGAGAGAAGUCCACCACGA 170
AM05747-AS asGfsasAfaAfuugagAfgAfaGfuCfcAfsc 100 AGAAAAUUGAGAGAAGUCCAC 171
AM05849-AS usAfscsCfaAfuuuauGfcCfuAfcAfgusu 101 UACCAAUUUAUGCCUACAGUU 154
AM05850-AS us Afs csCfaAfuuuauGfcCfu Afc Afgcs c 102 UACCAAUUUAUGCCUACAGCC 162
AM05851-AS usAfscsCfaAfuuuauGfcCfuAfcAfgcusu 103 UACCAAUUUAUGCCUACAGCUU 172
AM05852-AS usAfscsCfaAfuuuauGfcCfuAfcAfgccsu 104 UACCAAUUUAUGCCUACAGCCU 173
AM05853-AS usAfscsCfaAfuuuauGfcCfuAfcAfgccusu 105 UACCAAUUUAUGCCUACAGCCUU 163
AM05854-AS usAfscsCfaAfuuuauGfcCfuAfcAfgccusc 106 UACCAAUUUAUGCCUACAGCCUC 164
AM05855-AS cPrpusAfscsCfaAfuUfuAfuGfcCfuAfcAfgusu 107 UACCAAUUUAUGCCUACAGUU 154
AM05860-AS cPrpusAfsusUfgAfgAfgAfaGfuCfcAfcCfaCfsg 108 UAUUGAGAGAAGUCCACCACG 160
AM05862-AS us AfsusU fg AfgagaaGfuCfcAfcC fau su 109 UAUUGAGAGAAGUCCACCAUU 174
AM05863-AS usAfsusUfgAfgagaaGfuCfcAfcCfacsg 110 UAUUGAGAGAAGUCCACCACG 160
AM05864-AS usAfsusUfgAfgagaaGfuCfcAfcCfacsusu 111 UAUUGAGAGAAGUCCACCACUU 175
AM05865-AS usAfsusUfgAfgagaaGfuCfcAfcCfacsgsa 112 UAUUGAGAGAAGUCCACCACGA 170
106
AM05867-AS vpusAfsusUfgAfgagaaGfuCfcAfcCfaCfsg 113 UAUUGAGAGAAGUCCACCACG 160
AM05873-AS usUfsusGfaGfagaagUfcCfaCfcAfcusu 114 UUUGAGAGAAGUCCACCACUU 165
AM05874-AS usUfsusGfaGfagaagUfcCfaCfcAfcgsa 115 UUUGAGAGAAGUCCACCACGA 157
AM05875-AS usUfsusGfaGfagaagUfcCfaCfcAfcgusu 116 UUUGAGAGAAGUCCACCACGUU 176
AM05876-AS usUfsusGfaGfagaagUfcCfaCfcAfcgasg 117 UUUGAGAGAAGUCCACCACGAG 177
AM05877-AS cPrpusUfsusGraGraGfaAfgUfcCfaCfcAfcusu 118 UUUGAGAGAAGUCCACCACUU 165
AM06074-AS cPrpusAfsusUfgAfgagaaGfuCfcAfcCfacsusu 119 UAUUGAGAGAAGUCCACCACUU 175
AM06142-AS usAfsusUfgAfgagaaGfuCfcAfcCfacusu 120 UAUUGAGAGAAGUCCACCACUU 175
AM06143-AS usAfsusUfgAfgagaaGfuCfcAfcCfacgusu 121 UAUUGAGAGAAGUCCACCACGUU 168
AM06144-AS usAfsusUfgAfgagaaGfuCfcAfcCfacuus(invAb) 122 UAUUGAGAGAAGUCCACCACUU 175
AM06145-AS usAfsusUfgAfgagaaGfuCfcAfcCfacgasg 123 UAUUGAGAGAAGUCCACCACGAG 169
AM06222-AS usAfsusUfgAfgAfgAfaGfuCfcAfcCfacusu 124 UAUUGAGAGAAGUCCACCACUU 175
AM06281-AS asGfsasAfaAfuUfgAfgAfgAfaGfuCfcu.su 125 AGAAAAUUGAGAGAAGUCCUU 178
AM06282-AS asGfsasAfaAfuUfgAfgAfgAfaGfuCfcasc 126 AGAAAAUUGAGAGAAGUCCAC 171
AM06283-AS asGfsasAfaAfuUfgAfgAfgAfaGfuCfcacusu 127 AGAAAAUUGAGAGAAGUCCACUU 179
AM06284-AS asGfsasAfaAfuUfgAfgAfgAfaGfuCfcacsc 128 AGAAAAUUGAGAGAAGUCCACC 180
AM06285-AS usGfsasAfaAfuUfgAfgAfgAfaGfuCfcusu 129 UGAAAAUUGAGAGAAGUCCUU 152
AM06286-AS usGfsasAfaAfuUfgAfgAfgAfaGfuCfcasc 130 UGAAAAUUGAGAGAAGUCCAC 181
AM06299-AS asCfscsAfaUfuUfaUfgCfcUfaCfaGfcusu 131 ACCAAUUUAUGCCUACAGCUU 182
AM06300-AS asCfscsAfaUfuUfaUfgCfcUfaCfaGfccusu 132 ACCAAUUUAUGCCUACAGCCUU 183
AM06301-AS asCfscsAfaUfuUfaUfgCfcUfaCfaGfccusc 133 ACCAAUUUAUGCCUACAGCCUC 184
AM06302-AS usCfscsAfaUfuUfaUfgCfcUfaCfaGfcusu 134 UCCAAUUUAUGCCUACAGCUU 185
AM06303-AS usCfscsAfaUfuUfaUfgCfcUfaCfaGfccusu 135 UCCAAUUUAUGCCUACAGCCUU 186
AM06463-AS cPrpusAfscsCfaAfuUfuAfuGfcCfuAfcAfgcsc 136 UACCAAUUUAUGCCUACAGCC 162
AM06464-AS usAfscsCfaAfuUfuAfuGfcCfuAfcAfgscsc 137 UACCAAUUUAUGCCUACAGCC 162
AM06465-AS cPrpusAfscsCfaAfuUfuAfuGfcCfuAfcAfgscsc 138 UACCAAUUUAUGCCUACAGCC 162
AM06604-AS usAfscsCfaAfuUfuAfuGfcCfuAfcAfgcsu 139 UACCAAUUUAUGCCUACAGCU 187
AM06606-AS usAfscsCfaAfuUfuAfuGfcCfuAfcAfgcsg 140 UACCAAUUUAUGCCUACAGCC 188
AM06608-AS asAfscsCfaAfuUfuAfuGfcCfuAfcAfgcsc 141 AACCAAUUUAUGCCUACAGCC 189
AM06611-AS usAfscsCfaAfuUfUfAfuGfcCfuAfcAfgusu 142 UACCAAUUUAUGCCUACAGUU 154
AM06612-AS us Afs es CfaAfuU fu AfuGfcCfu Afc AfgCfsc 143 UACCAAUUUAUGCCUACAGCG 162
AM066I4-AS asCfscAfaUfuUfaUfgCfcUfaCfaGfcCfsu 144 ACCAAUUUAUGCCUACAGCCU 190
AM06616-AS usCfscAfaUfuUfaUfgCfcUfaCfaGfcCfsu 145 UCCAAUUUAUGCCUACAGCCU 191
AM06618-AS asCfscATaUfuUfaUfgCfcUfaCfaGfccsg 146 ACCAAUUUAUGCCUACAGCCG 192
AM06620-AS usCfscAfaUfuUfaUfgCfcUfaCfaGfccsg 147 UCCAAUUUAUGCCUACAGCCG 193
AM06751-AS us Afs es CfaAfuUfuAfuGfcCfuAfc Afggsg 148 UACCAAUUUAUGCCUACAGCG 194
107
108
Table 4. HBV RNAi agent sense strand sequences.
Strand ID Modified sequence (5'—+3') . SEQ ID NO. Unmodified sequence (5' —» 3') SEQ ID NO.
AM04444-SS (NAG25)uusgsccuguagGfCfAfuaaauugguaus(invdT) 195 UUGCCUGUAGGCAUAAAUUGGUAUT 275
AM04445-SS (NAG25)uauausgsccuguagGÎCfAfuaaauuggu(invdA) 196 UAUAUGCCUGUAGGCAUAAAUUGGUA 276
AM04767-SS (NAG25)gcggagsgcuguagGfCfAfuaaauuggTM(invdA) 197 GCGGAGGCUGUAGGCAUAAAUUGGTA 277
AM05010-SS (NAG25)scsuguagGfCfAfuaaauugguauus(invAb) 198 CUGUAGGCAUAAAUUGGUAUU 278
AM05015-SS (NAG25)sgsccuguagGfCfAfuaaauugguas(invAb) 199 GCCUGUAGGCAUAAAUUGGUA 279
AM05016-SS (NAG25)sgsccuguagGfCfAfuaaauuggus(invdA) 200 GCCUGUAGGCAUAAAUUGGUA 279
AM05017-SS (NAG25)sgsccuguagGfCfAfuaaauugguAMs(invAb) 201 GCCUGUAGGCAUAAAUUGGUA 279
AM05018-SS (NAG25)sgsccuguagGfCfAfuaaauuggTMAMs(invAb) 202 GCCUGUAGGCAUAAAUUGGTA 280
AM05019-SS (NAG25)sasacuguagGfCrAfuaaauugguas(invAb) 203 AACUGUAGGCAUAAAUUGGUA 281
AM05034-SS (NAG25)suscguggugGfAfCfuucucucaaus(invAb) 204 UCGUGGUGGACUUCUCUCAAU 282
AM05046-SS (NAG25)sasaguggugGfAfCfuucucucaaus(invAb) 205 AAGUGGUGGACUUCUCUCAAU 283
AM05047-SS (NAG25)suscguggugGfAfCfuucucucaAMTMs(invAb) 206 UCGUGGUGGACUUCUCUCAAT 284
AM05048-SS (NAG25)scsgugguggAfCfUfucucucaauus(invAb) 207 CGUGGUGGACUUCUCUCAAUU 285
AM05049-SS (NAG25)sasaugguggAfCfUfucucucaauus(invAb) 208 AAUGGUGGACUUCUCUCAAUU 286
AM05050-SS (NAG25)scsgugguggAfCfUfucucucaaTMTMs(invAb) 209 CGUGGUGGACUUCUCUCAATT 287
AM05051-SS (NAG25)sgsgacuucuCfUfCfaauuuucuaas(invAb) 210 GGACUUCUCUCAAUUUUCUAA 288
AM05063-SS (NAG25)scsgugguggAfCfUfucucucaauas(invAb) 211 CGUGGUGGACUUCUCUCAAUA 289
AM05064-SS (NAG25)suscguggugGfAfCfuucucucaaas(invAb) 212 UCGUGGUGGACUUCUCUCAAA 290
AM05346-SS (NAG31 )sasccuguagGfCfAfuaaauugguas(invAb) 213 ACCUGUAGGCAUAAAUUGGUA 291
AM05347-SS (NAG31 )s(invAb)scuguagGfCfAfuaaauugguas(invAb) 214 CUGUAGGCAUAAAUUGGUA 292
AM05606-SS (NAG25)s(invAb)scuguagGfCfAfuaaauugguas(invAb) 215 CUGUAGGCAUAAAUUGGUA 292
AM05607-SS (NAG37)s(invAb)scuguagGfCfAfuaaauugguas(invAb) 216 CUGUAGGCAUAAAUUGGUA 292
AM05615-SS (NAG25)s(invAb)sacuguagGfCfAfuaaauugguas(invAb) 217 ACUGUAGGCAUAAAUUGGUA 293
109
Strand ID Modified sequence (5' —► 3’) SEQID NO. Unmodified sequence (5' —> 3’) SEQ ID NO.
AM05616-SS (NAG25)sgsgcuguagGICfAfuaaauugguas(invAb) 218 GGCUGUAGGCAUAAAUUGGUA 294
AM05617-SS (NAG37)sasaguggugGfAfCfuucucucaaus(invAb) 219 AAGUGGUGGACUUCUCUCAAU 283
AM05620-SS (NAG25)sasaguggugGfAfCfuucucucaaas(invAb) 220 AAGUGGUGGACUUCUCUCAAA 295
AM05622-SS (NAG25)scscguggugGfAICfuucucucaaus(invAb) 221 CCGUGGUGGACUUCUCUCAAU 296
AM05624-SS (NAG25)s(invAb)sccguggugGfAfCfuucucucaaus(invAb) 222 CCGUGGUGGACUUCUCUCAAU 296
AM05627-SS (NAG25)scsucguggugGfAfCfuucucucaaus(invAb) 223 CUCGUGGUGGACUUCUCUCAAU 297
AM05629-SS (NAG25)s(invAb)sguggugGfAfCfuucucucaaus(invAb) 224 GUGGUGGACUUCUCUCAAU 298
AM05630-SS (NAG25)s(invAb)sguggugGfAfCfuucucucaauusu(invAb) 225 GUGGUGGACUUCUCUCAAUUU 299
AM05636-SS (NAG25)suscgugguggAfCfUfucucucaauus(invAb) 226 UCGUGGUGGACUUCUCUCAAUU 300
AM05639-SS (NAG25)s(invAb)sugguggAfCfUfucucucaauus(invAb) 227 UGGUGGACUUCUCUCAAUU 301
AM05640-SS (NAG37)s(invAb)sugguggAfClUfucucucaauus(invAb) 228 UGGUGGACUUCUCUCAAUU 301
AM05746-SS (NAG25)sgsuggacuuCfUICfucaauuuucus(invAb) 229 GUGGACUUCUCUCAAUUUUCU 302
AM05856-SS (NAG25)s(invAb)scuguagGÎCfAfuaaauugguausu(invAb) 230 CUGUAGGCAUAAAUUGGUAUU 278
AM05857-SS (NAG25)s(invAb)sgcuguagGfCfAfuaaauugguausu(invAb) 231 GCUGUAGGCAUAAAUUGGUAUU 303
AM05858-SS (NAG25)s(invAb)sggcuguagGfCfAfuaaauugguausu(invAb) 232 GGCUGUAGGCAUAAAUUGGUAUU 304
AM05859-SS (NAG25)s(invAb)saacuguagGfCfAfuaaauugguausu(invAb) 233 AACUGUAGGCAUAAAUUGGUAUU 305
AM05868-SS (NAG25)s(invAb)ugguggAfCfUfucucucaauausu(invAb) 234 UGGUGGACUUCUCUCAAUAUU 306
AM05869-SS (NAG25)s(invAb)sgugguggAfCfUfucucucaauausu(invAb) 235 GUGGUGGACUUCUCUCAAUAUU 307
AM05870-SS (NAG25)sasaugguggAfCfUfucucucaauausu(invAb) 236 AAUGGUGGACUUCUCUCAAUAUU 308
AM05 871-S S (NAG25)scsgugguggAfCfUfucucucaauausu(invAb) 237 CGUGGUGGACUUCUCUCAAUAUU 309
AM05872-SS (NAG31)scsgugguggAfCfUfucucucaauas(invAb) 238 CGUGGUGGACUUCUCUCAAUA 289
AM05879-SS (NAG25)s(invAb)saaguggugGfAfCfuucucucaaus(invAb) 239 AAGUGGUGGACUUCUCUCAAU 283
AM05880-SS (NAG25)s(invAb)sguggugGfAfCfuucucucaaausu(invAb) 240 GUGGUGGACUUCUCUCAAAUU 310
AM05881-SS (NAG25)s(invAb)scguggugGfAfCfuucucucaaausu(invAb) 241 CGUGGUGGACUUCUCUCAAAUU 311
110
Strand ID Modified sequence (5' —* 3') SEQ ID NO. Unmodified sequence (5' —» 3') SEQ ID NO.
AM05882-SS (NAG25)sasaguggugGfAfCfuucucucaaausu(invAb) 242 AAGUGGUGGACUUCUCUCAAAUU 312
AM05883-SS (NAG25)suscguggugGfAfCfuucucucaaausu(invAb) 243 UCGUGGUGGACUUCUCUCAAAUU 313
AM06146-SS (NAG37)s(invAb)sgugguggAfCfUfucucucaauausu(invAb) 244 GUGGUGGACUUCUCUCAAUAUU 307
AM06147-SS (NAG37)s(invAb)scgugguggAfCfUfucucucaauausu(invAb) 245 CGUGGUGGACUUCUCUCAAUAUU 309
AM06148-SS (NAG37)s(invAb)scucgugguggAfCfUfucucucaauas(invAb) 246 CUCGUGGUGGACUUCUCUCAAUA 314
AM06149-SS (NAG37)s(invAb)scucgugguggAIC,fUfucucucaauausu(invAb) 247 CUCGUGGUGGACUUCUCUCAAUAUU 315
AM06150-SS (NAG37)s(invAb)sggcuguagGICfAfuaaauugguas(invAb) 248 GGCUGUAGGCAUAAAUUGGUA 294
AM06151-SS (NAG37)s(invAb)sgaggcuguagGfCfAfuaaauugguas(invAb) 249 GAGGCUGUAGGCAUAAAUUGGUA 316
AM06152-SS (NAG37)s(invAb)sgaggcuguagGfCfAfuaaauugguausu(invAb) 250 GAGGCUGUAGGCAUAAAUUGGUAUU 317
AM06287-SS (NAG37)s(invAb)sggacuuCfUfCfucaauuuucus(invAb) 251 GGACUUCUCUCAAUUUUCU 318
AM06288-SS (NAG37)s(invAb)sguggacuuCfUfCfucaauuuucus(invAb) 252 GUGGACUUCUCUCAAUUUUCU 302
AM06289-SS (NAG37)s(invAb)sgguggacuuCfUfCfucaauuuucus(invAb) 253 GGUGGACUUCUCUCAAUUUUCU 319
AM06290-SS (NAG37)s(invAb)sggacuuCfUICfucaauuuucas(invAb) 254 GGACUUCUCUCAAUUUUCA 320
AM06291-SS (NAG37)s(invAb)sguggacuuCfUfCfucaauuuucas(invAb) 255 GUGGACUUCUCUCAAUUUUCA 321
AM06304-SS (NAG37)s(invAb)sgcuguaGfGfCfauaaauuggus(invAb) 256 GCUGUAGGCAUAAAUUGGU 322
AM06305-SS (NAG37)s(invAb)sggcuguaGfGfCfauaaauuggus(invAb) 257 GGCUGUAGGCAUAAAUUGGU 323
AM06306-SS (NAG37)s(invAb)sgaggcuguaGfGfCfauaaauuggus(invAb) 258 GAGGCUGUAGGCAUAAAUUGGU 324
AM06307-SS (NAG37)s(invAb)sgcuguaGfGfCfauaaauuggas(invAb) 259 GCUGUAGGCAUAAAUUGGA 325
AM06308-SS (NAG37)s(invAb)sggcuguaGfGfCfauaaauuggas(invAb) 260 GGCUGUAGGCAUAAAUUGGA 326
AM06603-SS (NAG37)s(invAb)sagcuguagGfCfAfuaaauugguas(invAb) 261 AGCUGUAGGCAUAAAUUGGUA 327
AM06605-SS (NAG37)s(invAb)scgcuguagGfCfAfuaaauugguas(invAb) 262 GGCUGUAGGCAUAAAUUGGUA 328
AM06607-SS (NAG37)s(invAb)sggcuguagGfCfAfuaaauugguus(invAb) 263 GGCUGUAGGCAUAAAUUGGUU 329
AM06609-SS (NAG37)s(invAb)scuguagGICfAfuaaauugguasuus(invAb) 264 CUGUAGGCAUAAAUUGGUAUU 278
AM06610-SS (NAG37)s(invAb)scuGfuAfgGfCfAfuAfaAfuUfgGfuasuus(invAb) 265 CUGUAGGCAUAAAUUGGUAUU 278
Strand ID Modified sequence (5' —♦ 3’) SEQ ID NO. Unmodified sequence (5' —> 3') SEQ ID NO.
AM06613-SS (NAG37)s(invAb)saggcuguaGfGfCfauaaauuggus(invAb) 266 AGGCUGUAGGCAUAAAUUGGU 330
AM06615-SS (NAG37)s(invAb)saggcuguaGfGfGfauaaauuggas(invAb) 267 AGGCUGUAGGCAUAAAUUGGA 331
AM06617-SS (NAG37)s(invAb)scggcuguaGfGfCfauaaauuggus(invAb) 268 CGGCUGUAGGCAUAAAUUGGU 332
AM06619-SS (NAG37)s(invAb)scggcuguaGfGfCfauaaauuggas(invAb) 269 CGGCUGUAGGCAUAAAUUGGA 333
AM06750-SS (NAG37)s(invAb)scccuguagGfCfAfuaaauugguas(invAb) 270 CCCUGUAGGCAUAAAUUGGUA 334
AM06752-SS (NAG37)csgcuguagGfCfAfuaaauugguas(invAb) 271 GGCUGUAGGCAUAAAUUGGUA 328
AM06753-SS (NAG37)csccuguagGfCfAfuaaauugguas(invAb) 272 CCCUGUAGGCAUAAAUUGGUA 334
AM06776-SS (NAG25)s(invAb)sguggacuuCfUfCfucaauuuucus(invAb) 273 GUGGACUUCUCUCAAUUUUCU 302
AM06777-SS (NAG25)s(invAb)scgcuguagGfCfAfuaaauugguas(invAb) 274 GGCUGUAGGCAUAAAUUGGUA 328
The HBV RNAi agents described herein are formed by annealing an antisense strand with a sense strand. A sense strand containing a sequence listed in Table 4 can be hybridized to any antisense strand containing a sequence listed in Table 3, provided the two sequences hâve a région of at least about 85% complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucléotide sequence.
In some embodiments, the antisense strand of an HBV RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucléotides from any of the antisense strand sequences in Table 3. In some embodiments, the sense strand of an HBV RNAi agent disclosed herein diiTers by 0,1,2, or 3 nucléotides from any of the sense strand sequences in Table 4.
In some embodiments, an HBV RNAi agent antisense strand comprises a nucléotide sequence of any of the sequences in Table 3. In some embodiments, an HBV RNAi agent antisense strand comprises the sequence of nucléotides (from 5’ end -> 3’ end) 1-17, 2-17, 1-18, 2-18, 1-19,2-19,1-20,2-20,1-21,2-21,1-22,2-22,1-23,2-23,1-24,2-24,1-25,2-25,1-26, or 2-26 of any of the sequences in Table 3.
In some embodiments, an HBV RNAi agent sense strand comprises the nucléotide sequence of any of the sequences in Table 4. In some embodiments, an HBV RNAi agent sense strand comprises the sequence of nucléotides (from5: end -> 3’ end) 1-17,2-17,3-17,4-17,1-18,218, 3-18, 4-18,1-19, 2-19, 3-19, 4-19,1-20,2-20,3-20,4-20, 1-21,2-21, 3-21,4-21, 1-22, 222, 3-22, 4-22,1-23, 2-23, 3-23,4-23, 1-24,2-24, 3-24,4-24, 1-25,2-25, 3-25,4-25, 1-26, 226, 3-26, or 4-26 of any of the sequences in Table 4.
For the HBV RNAi agents disclosed herein, the nucléotide at position 1 of the antisense strand (from 5’ end 3’ end) can be perfectly complementary’ to an HBV gene, or can be noncomplementary to an HBV gene. In some embodiments, the nucléotide at position 1 of the antisense strand (from 5’ end -> 3’ end) is a U, A, or dT. In some embodiments, the nucléotide at position 1 of the antisense strand (from 5’ end 3’ end) forms an A:U or U:A base pair with the sense strand.
In some embodiments, an HBV RNAi agent antisense strand comprises the sequence of nucléotides (from 5’ end 3’ end) 2-18 or 2-19 of any of the antisense strand sequences in
112
Table 3. In some embodiments, an HBV RNAi sense strand comprises the sequence of nucléotides (from 5’ end 3’ end) 1-17 or 1-18 of any of the sense strand sequences in Table 4.
In some embodiments, an HBV RNAi agent includes (i) an antisense strand comprising the sequence of nucléotides (from 5’ end 3’ end) 2-18 or 2-19 of any of the antisense strand sequences in Table 3, and (ii) a sense strand comprising the sequence of nucléotides (from 5’ end -> 3’ end) 1-17 or 1-18 of any of the sense strand sequences in Table 4.
A sense strand containing a sequence listed in Table 4 can be hybridized to any antisense strand containing a sequence listed in Table 3 provided the two sequences hâve a région of at least about 85% complementarity over a contiguous 16, 17, 18,19,20, or 21 nucléotide sequence. Représentative sequence pairings are exemplified by the Duplex ID Nos. shown in Table 5.
In some embodiments, an HBV RNAi agent comprises of any ofthe Duplex ID Nos. presented herein. In some embodiments, an HBV RNAi agent consists of any of the Duplex ID Nos. presented herein. In some embodiments, an HBV RNAi agent comprises the sense strand and/or the antisense strand nucléotide sequences of any of the Duplex ID Nos. presented herein. In some embodiments, an HBV RNAi agent comprises the sense strand and antisense strand nucléotide sequences of any of the Duplex ID Nos. presented herein and a targeting group and/or linking group wherein the targeting group and/or linking group is coval ently linked (i.e. conjugated) to the sense strand or the antisense strand. In some embodiments, an HBV RNAi agent comprises the sense strand and antisense strand modified nucléotide sequences of any of the Duplex ID Nos. presented herein. In some embodiments, an HBV RNAi agent comprises the sense strand and antisense strand modified nucléotide sequences of any of the Duplex ID Nos. presented herein and a targeting group and/or linking group wherein the targeting group and/or linking group is covalently linked to the sense strand or the antisense strand.
In some embodiments, an HBV RNAi agent comprises an antisense strand and a sense strand having the nucléotide sequences of any of the antisense strand/sense strand duplexes of Table 5, and further comprises an asialoglycoprotein receptor ligand targeting group.
113
In some embodiments, an HBV RNAi agent comprises an antisense strand and a sense strand having the nucléotide sequences of any of the antisense strand and/or sense strand nucléotide sequences of any of the duplexes of Table 5, and further comprises a targeting group selected from the group consisting of (PAZ), (NAG13), (NAG13)s, (NAG18), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s.
In some embodiments, an HBV RNAi agent comprises an antisense strand and a sense strand having the modified nucléotide sequences of any of the antisense strand and/or sense strand nucléotide sequences of any of the duplexes of Table 5.
In some embodiments, an HBV RNAi agent comprises an antisense strand and a sense strand having the modified nucléotide sequences of any of the antisense strand and/or sense strand nucléotide sequences of any of the duplexes of Table 5, and further comprises an asialoglycoprotein receptor ligand targeting group.
In some embodiments, an HBV RNAi agent comprises any of the duplexes of Table 5.
In some embodiments, an HBV RNAi agent consists of any of the duplexes of Table 5.
114
Table 5. Examples of HBV RNAi agent duplexes.
Duplex ID Antisense Strand ID Sense Strand ID
AD03498 AM03508-AS AM04445-SS
AD03499 AM04441-AS AM04444-SS
AD03500 AM04442-AS AM04444-SS
AD03501 AM04443-AS AM04444-SS
AD03738 AM04768-AS AM04767-SS
AD03739 AM04769-AS AM04767-SS
AD03967 AM04443-AS AM05010-SS
AD03968 AM05011-AS AM05010-SS
AD03969 AM04443-AS AM05015-SS
AD03970 AM05011-AS AM05019-SS
AD03971 AM05012-AS AM05015-SS
AD03972 AM04443-AS AM05016-SS
AD03973 AM04443-AS AM05017-SS
AD03974 AM04443-AS AMO5O18-SS
AD03975 AM05013-AS AM05015-SS
AD03976 AM05014-AS AM05019-SS
AD03977 AM05013-AS AM05017-SS
AD03978 AM05013-AS AM04444-SS
AD0400I AM05052-AS AM05034-SS
AD04002 AM05053-AS AM05034-SS
AD04003 AM05054-AS AM05046-SS
AD04004 AM05052-AS AM05047-SS
AD04005 AM05055-AS AM05064-SS
AD04006 AM05056-AS AM05048-SS
AD04007 AM05057-AS AM05048-SS
AD04008 AM05058-AS AM05049-SS
AD04009 AM05056-AS AM05050-SS
AD04010 AM05060-AS AM05063-SS
AD04176 AM05351-AS AM05346-SS
AD 04177 AM04443-AS AM05347-SS
AD 04178 AM05011-AS AM05347-SS
AD04412 AM05011-AS AM05606-SS
AD04413 AM05011-AS AM05607-SS
AD04414 AM05608-AS AM05606-SS
AD04415 AM05011-AS AM05615-SS
AD04416 AM05609-AS AM05616-SS
AD04417 AM05610-AS AM05616-SS
AD04418 AM05611-AS AM05616-SS
AD04419 AM05612-AS AM05616-SS
AD04420 AM05613-AS AM05616-SS
AD04421 AM05614-AS AM05616-SS
AD04422 AM05054-AS AM05617-SS
AD04423 AM05618-AS AM05046-SS
AD04425 AM05621-AS AM05620-SS
Duplex ID Antisense Strand ID Sense Strand ID
AD04426 AM05623-AS AM05622-SS
AD04427 AM05623-AS AM05624-SS
AD04428 AM05626-AS AM05622-SS
AD04429 AM05626-AS AM05624-SS
AD04430 AM05628-AS AM05627-SS
AD04431 AM05054-AS AM05629-SS
AD04432 AM05054-AS AM05630-SS
AD04433 AM05631-AS AM05048-SS
AD04434 AM05632-AS AM05048-SS
AD04435 AM05633-AS AM05048-SS
AD04436 AM05635-AS AM05048-SS
AD04437 AM05634-AS AM05048-SS
AD04438 AM05637-AS AM05636-SS
AD04439 AM05638-AS AM05636-SS
AD04440 AM05058-AS AM05639-SS
AD04441 AM05057-AS AM05639-SS
AD04442 AM05057-AS AM05640-SS
AD04511 AM05747-AS AM05746-SS
AD04570 AM05011-AS AM05856-SS
AD04571 AM05849-AS AM05856-SS
AD04572 AM05850-AS AM05856-SS
AD04573 AM05851-AS AM05857-SS
AD04574 AM05852-AS AM05857-SS
AD04575 AM05853-AS AM05858-SS
AD04576 AM05854-AS AM05858-SS
AD04577 AM05011-AS AM05859-SS
AD04578 AM05850-AS AM05858-SS
AD04579 AM05014-AS AM05347-SS
AD04580 AM05855-AS AM05347-SS
AD04581 AM05860-AS AM05063-SS
AD04583 AM05862-AS AM05868-SS
AD04584 AM05863-AS AM05868-SS
AD04585 AM05864-AS AM05869-SS
AD04586 AM05865-AS AM05869-SS
AD04587 AM05862-AS AM05870-SS
AD04588 AM05863-AS AM05871-SS
AD04590 AM05867-AS AM05063-SS
AD04591 AM05860-AS AM05872-SS
AD04592 AM05054-AS AM05879-SS
AD04593 AM05873-AS AM05880-SS
AD04594 AM05874-AS AM05880-SS
AD04595 AM05875-AS AM05881-SS
AD04596 AM05876-AS AM05881-SS
AD04597 AM05873-AS AM05882-SS
115
Duplex ID Antisense Strand ID Sense Strand ID
AD04598 AM05874-AS AM05883-SS
AD04599 AM05877-AS AM05620-SS
AD04734 AM06074-AS AM05869-SS
AD04771 AM06142-AS AM06146-SS
AD04772 AM06143-AS AM06147-SS
AD04773 AM06144-AS AM06146-SS
AD04774 AM06145-AS AM06148-SS
AD04775 AM06145-AS AM06149-SS
AD04776 AM05850-AS AM06150-SS
AD04777 AM05854-AS AM06151-SS
AD04778 AM05854-AS AM06152-SS
AD04822 AM06222-AS AM06146-SS
AD04823 AM05609-AS AM06150-SS
AD04871 AM06281-AS AM06287-SS
AD04872 AM06282-AS AM06288-SS
AD04873 AM06283-AS AM06288-SS
AD04874 AM06284-AS AM06289-SS
ADO4875 AM06285-AS AM06290-SS
AD04876 AM06286-AS AM06291-SS
AD04881 AM06299-AS AM06304-SS
AD04882 AM06300-AS AM06305-SS
AD04883 AM06301-AS AM06306-SS
AD04884 AM06302-AS AM06307-SS
AD04885 AM06303-AS AM06308-SS
AD04962 AM05864-AS AM06146-SS
AD04963 AM05855-AS AM05607-SS
AD04981 AM06463-AS AM06150-SS
AD04982 AM06464-AS AM06150-SS
AD04983 AM06465-AS AM06150-SS
AD05069 AM06604-AS AM06603-SS
AD05070 AM06606-AS AM06605-SS
AD05071 AM06608-AS AM06607-SS
AD05072 AM05011-AS AM06609-SS
AD05073 AM06611-AS AM06610-SS
AD05074 AM06612-AS AM06150-SS
AD05075 AM06614-AS AM06613-SS
AD05076 AM06616-AS AM06615-SS
AD05077 AM06618-AS AM06617-SS
AD05078 AM06620-AS AM06619-SS
AD05147 AM06751-AS AM06750-SS
AD05148 AM06606-AS AM06752-SS
AD05149 AM06751-AS AM06753-SS
AD05164 AM06282-AS AM06776-SS
AD05165 AM06606-AS AM06777-SS
116
In some embodiments, an HBV RNAi agent is prepared or provided as a sait, mixed sait, or a free-acid. The RNAi agents described herein, upon delivery to a cell expressing an ΗΒλζ gene, inhibit or knockdown expression of one or more HBV genes in vivo.
Targeting Groups, Linking Groups, and Delivery Vehicles
In some embodiments, an HBV RNAi agent is conjugated to one or more non-nucleotide groups including, but not limited to a targeting group, linking group, delivery polymer, or a delivery vehicle. The non-nucleotide group can enhance targeting, delivery or attachment of the RNAi agent. Examples of targeting groups and linking groups are provided in Table 6. The non-nucleotide group can be covalently linked to the 3' and/or 5' end of either the sense strand and/or the antisense strand. In some embodiments, an HBV RNAi agent contains a nonnucleotide group linked to the 3' and/or 5' end of the sense strand. In some embodiments, a non-nucleotide group is linked to the 5' end of an HBV RNAi agent sense strand. A nonnucleotide group may be linked directly or indirectly to the RNAi agent via a linker/linking group. In some embodiments. a non-nucleotide group is linked to the RNAi agent via a labile, cleavable, or réversible bond or linker.
In some embodiments, a non-nucleotide group enhances the pharmacokinetic or biodistribution properties of an RNAi agent or conjugale to which it is attached to improve cell- or tissue-specific distribution and cell-specific uptake of the conjugale. In some embodiments, a non-nucleotide group enhances endocytosis of the RNAi agent.
Targeting groups or targeting moieties enhance the pharmacokinetic or biodistribution properties of a conjugate to which they are attached to improve cell-specific distribution and cell-specific uptake of the conjugate. A targeting group can be monovalent, divalent, trivalent, tetravalent, or hâve higher valency. Représentative targeting groups include, without limitation, compounds with afîînity to cell surface molécule, cell receptor ligands, hapten, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with afîînity to cell surface molécules. In some embodiments, a targeting group is linked to an RNAi agent using a linker, such as a PEG linker or one, two, or three abasic and/or ribitol (abasic ribose) groups. In some embodiments, a targeting group comprises a galactose dérivative cluster.
117
The HBV RNAi agents described herein may be synthesized having a reactive group, such as an amine group, at the 5'-terminus. The reactive group may be used to subsequently attach a targeting moiety using methods typical in the art.
In some embodiments, a targeting group comprises an asialoglycoprotein receptor ligand. In some embodiments, an asialoglycoprotein receptor ligand includes or consists of one or more galactose dérivatives. As used herein, the term galactose dérivative includes both galactose and dérivatives of galactose having affinity for the asialoglycoprotein receptor that is equal to or greater than that of galactose. Galactose dérivatives include, but are not limited to: galactose, galactosamine, N-formylgalactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-n-butanoyl-galactosamine, and N-iso-butanoylgalactos-amine (see for example: lobst, S.T. and Drickamer, K. J.B.C. 1996, 271, 6686). Galactose dérivatives, and clusters of galactose dérivatives, that are useful for in vivo targeting of oligonucleotides and other molécules to the liver are known in the art (see, for example, Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257,939-945). Galactose dérivatives hâve been used to target molécules to hépatocytes in vivo through their binding to the asialoglycoprotein receptor (ASGPr) expressed on the surface of hépatocytes. Binding of ASGPr ligands to the ASGPr(s) facilitâtes cell-specific targeting to hépatocytes and endocytosis of the molécule into hépatocytes. ASGPr ligands can be monomeric (e.g., having a single galactose dérivative) or multimeric (e.g., having multiple galactose dérivatives). The galactose dérivative or galactose dérivative cluster may be attached to the 3' or 5' end of the RNAi polynucleotide using methods known in the art. The préparation of targeting groups, such as galactose dérivative clusters, is described in, for example, U.S. Patent Application Serial Nos. 15/452,324 and 15/452,423, the contents of both of which are incorporated herein in their entirety.
As used herein, a galactose dérivative cluster comprises a molécule having two to four terminal galactose dérivatives. A terminal galactose dérivative is attached to a molécule through its ΟΙ carbon. In some embodiments, the galactose dérivative cluster is a galactose dérivative trimer (also referred to as tri-antennary galactose dérivative or tri-valent galactose dérivative). In some embodiments, the galactose dérivative cluster comprises N-acetyl-galactosamines. In some embodiments, the galactose dérivative cluster comprises three N-acetyl-galactosamines. In some embodiments, the galactose dérivative cluster is a galactose dérivative tetramer (also
118 referred to as tetra-antennaiy galactose dérivative or tetra-valent galactose dérivative). In some embodiments, the galactose dérivative cluster comprises fourN-acetyl-galactosamines.
As used herein, a galactose dérivative trimer contains three galactose dérivatives, each linked to a central branch point. As used herein, a galactose dérivative tetramer contains four galactose dérivatives, each linked to a central branch point. The galactose dérivatives can be attached to the central branch point through the C-l carbons of the saccharides. In some embodiments, the galactose dérivatives are linked to the branch point via linkers or spacers. In some embodiments, the linker or spacer is a flexible hydrophilic spacer, such as a PEG group (see, for example, U.S. Patent No. 5,885,968; Biessen et al. J. Med. Chem. 1995 Vol. 39 p. 15381546). In some embodiments, the PEG spacer is a PEG3 spacer. The branch point can be any small molécule which permits attachment of three galactose dérivatives and further permits attachment of the branch point to the RNAi agent. An example of branch point group is a dilysine or di-glutamate. Attachment of the branch point to the RNAi agent can occur through a linker or spacer. In some embodiments, the linker or spacer comprises a flexible hydrophilic spacer, such as, but not limited to, a PEG spacer. In some embodiments, the linker comprises a rigid linker, such as a cyclic group. In some embodiments, a galactose dérivative comprises or consists of N-acetyl-galactosamine. In some embodiments, the galactose dérivative cluster is comprised of a galactose dérivative tetramer, which can be, for example, an N-acetylgalactosamine tetramer.
In some embodiments, pharmaceutical compositions for delivering an HBV RNAi agent to a liver cell in vivo are described. Such pharmaceutical compositions can include, for example, an HBV RNAi agent conjugated to a galactose dérivative cluster. In some embodiments, the galactose dérivative cluster is comprised of a galactose dérivative trimer, which can be, for example, an N-acetyl-galactosamine trimer, or galactose dérivative tetramer, which can be, for example, an N-acetyl-galactosamine tetramer.
Targeting groups include, but are not limited to, (PAZ), (NAG13), (NAG13)s, (NAG18), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27).
(NAG27)s, (NAG28). (NAG28)s, (NAG29). (NAG29)s, (NAG30). (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), and
119 (NAG39)s. Other targeting groups, including galactose cluster targeting ligands, are known in the art.
In some embodiments, a linking group is conjugated to the RNAi agent. The linking group facilitâtes covalent linkage of the agent to a targeting group or delivery polymer or delivery7 vehicle. The linking group can be linked to the 3' or the 5' end of the RNAi agent sense strand or antisense strand. In some embodiments, the linking group is linked to the RNAi agent sense strand. In some embodiments, the linking group is conjugated to the 5' or 3' end of an RNAi agent sense strand. In some embodiments, a linking group is conjugated to the 5' end of an RNAi agent sense strand. Examples of linking groups, include, but are not limited to: reactive groups such a primary amines and alkynes, alkyl groups, abasic nucleosides, ribitol (abasic ribose), and/or PEG groups.
A linker or linking group is a connection between two atoms that links one Chemical group (such as an RNAi agent) or segment of interest to another Chemical group (such as a targeting group or delivery' polymer) or segment of interest via one or more covalent bonds. A labile linkage contains a labile bond. A linkage may optionally include a spacer that increases the distance between the two joined atoms. A spacer may further add flexibility and/or length to the linkage. Spacers may include, but are not be limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups; each of which can contain one or more heteroatoms, heterocy’cles, amino acids, nucléotides, and saccharides. Spacer groups are well known in the art and the preceding list is not meant to limit the scope of the description.
Any' of the HBV RNAi agent nucléotide sequences listed in Tables 3 and 4, whether modified or unmodifïed, may contain 3' or 5' targeting group and/or linking group. Any of the HBV RNAi agent sequences listed in Table 3 and 4 which contain a 3' or 5' targeting group and/or linking group, may alternatively contain no 3' or 5' targeting group and/or linking group, or may contain a different 3' or 5' targeting group and/or linking group including, but not limited to, those depicted in Table 3. Any of the HBV RNAi agent duplexes listed in Table 5, whether modified or unmodifïed, may further comprise a targeting group and/or linking group, including, but not limited to, those depicted in Table 3, and the targeting group or linking group
120 may be attached to the 3' or 5' terminus of either the sense strand or the antisense strand of the HBV RNAi agent duplex.
Examples of targeting groups and linking groups are provided in Table 6. Table 4 provides 5 several embodiments of HBV RNAi agent sense strands having a targeting group or linking group linked to the 5' or 3' end.
Table 6. Structures representing various modified nucléotides, targeting groups, and linking groups.
o, °τ~γ=ο B \\ p H O. \___/ Z---n. o=\ y-- Z--Λ X O ΛΛΛΛΛΛ O—D—O g P 2 S ” V/z O
0 ° o > 1 V'o cPrpTM cPr c / y— o / / \ / —2 /=o
121 ο
Ο—Ρ ο
ο
Ο—Ρ ο
epTM epTcPr
When positioned intemally on oligonucleotide:
linkage towards 5' end of oligonucleotide
o ïr° 0 linkage towards 3' end of oligonucleotide (invAb)
When positioned intemally on oligonucleotide:
linkage towards 5' end of oligonucleotide
S /7^° linkage towards 3' end of oligonucleotide (invAb)s
122
When positioned at the 3' terminal end of oligonucleotide:
linkage towards 5' end of oligonucleotide
123
124
125
126
127
(NAG28)s
Oy O ^NH
(NAG29)
128
(NAG29)s
(NAG30)
129
(NAG31)
130
H
(NAG32)s
(NAG33)
(NAG33)s
131
H
(NAG34)
(NAG34)s H
(NAG35)
H
(NAG35)s
132
133
134
(NAG39)s
In each of the above structures in Table 6, NAG comprises an N-acetyl-galactosamine or another ASGPr ligand, as would be understood by a person of ordinary skill in the art to be attached in view of the structures above and description provided herein. For example, in some embodiments, NAG in the structures provided in Table 6 is represented by the following structure:
(N-acetyl-galactosamine)
135
Each (NAGx) may be attached to an HBV RNAi agent via a phosphate group (as in (NAG25), (NAG30), and (NAG31)), or a phosphorothioate group, (as is (NAG25)s, (NAG29)s, (NAG30)s, (NAG31)s, or (NAG37)s), or another linking group.
O O
HH |_0_β4
O s
Phosphate group Phosphorothioate group Other linking groups known in the art may be used.
Delivery' Vehicles
In some embodiments, a delivery vehicle may be used to deliver an RNAi agent to a cell or tissue. A delivery' vehicle is a compound that improves delivery of the RNAi agent to a cell or tissue. A delivery' vehicle can include, or consist of, but is not limited to: a polymer, such as an amphipathic polymer, a membrane active polymer, a peptide, a melittin peptide, a melittinlike peptide (MLP), a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane active polyamine.
In some embodiments, the RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs or other delivery' Systems available in the art. The RNAi agents can also be chemically conjugated to targeting groups, lipids (including, but not limited to cholestérol and cholesteryl dérivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, for example WO 2000/053722, WO 2008/0022309, WO 2011/104169, and WO 2012/083185, WO 2013/032829, WO 2013/158141, each of which is incorporated herein by reference), or other delivery Systems available in the art.
Pharmaceutical Compositions and Formulations
The HBV RNAi agents disclosed herein may be prepared as pharmaceutical compositions or formulations. In some embodiments, pharmaceutical compositions include at least one HBV RNAi agent. These pharmaceutical compositions are particularly useful in the inhibition of the expression of the target mRNA in a target cell, a group of cells, a tissue, or an organism. The pharmaceutical compositions can be used to treat a subject having a disease or disorder tirât
136 would benefit from réduction in the level of the target mRNA, or inhibition in expression of the target gene. The pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from réduction of the level ofthe target mRNA or an inhibition in expression the target gene. In one embodiment, the method includes administering an HBV RNAi agent linked to atargeting ligand as described herein. to a subject to be treated. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and/or delivery polymers) are added to the pharmaceutical compositions including an HBV RNAi agent, thereby forming a pharmaceutical formulation suitable for in vivo delivery' to a human.
The pharmaceutical compositions that include an HBV RNAi agent and methods disclosed herein may decrease the level of the target mRNA in a cell, group of cells, group of cells, tissue, or subject, including: administering to the subject a therapeutically effective amount of a herein described HBV RNAi agent, thereby inhibiting the expression of a target mRNA in the subject.
In some embodiments, the described pharmaceutical compositions including an HBV RNAi agent are used for treating or managing clinical présentations associated with HBV infection. In some embodiments, a therapeutically or prophylactically effective amount of one or more of pharmaceutical compositions is administered to a subject in need of such treatment. prévention or management. In some embodiments, administration of any ofthe disclosed HBV RNAi agents can be used to decrease the number, severity, and/or frequency of symptoms of a disease in a subject.
The described pharmaceutical compositions including an HBV RNAi agent can be used to treat at least one symptom in a subject having a disease or disorder that would benefit from réduction or inhibition in expression of HBV mRNA. In some embodiments, the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions including an HBV RNAi agent thereby treating the symptom. In other embodiments, the subject is administered a prophylactically effective amount of one or more HBV RNAi agents, thereby preventing the at least one symptom.
137
The route of administration is the path by which an HBV RNAi agent is brought into contact with the body. In general, methods of administering drugs and nucleic acids for treatment of a mammal are well known in the art and can be applied to administration of the compositions described herein. The HBV RNAi agents disclosed herein can be administered via any suitable route in a préparation appropriately tailored to the particular route. Thus, herein described pharmaceutical compositions can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, intraarticularly, or intraperitoneally. In some embodiments, there herein described pharmaceutical compositions via subcutaneous injection.
The pharmaceutical compositions including an HBV RNAi agent described herein can be delivered to a cell, group of cells, tumor, tissue, or subject using oligonucleotide delivery’ technologies known in the art. In general, any suitable method recognized in the art for delivering a nucleic acid molécule (in vitro or in vivo) can be adapted for use with a herein described compositions. For example, delivery can be by local administration, (e.g., direct injection, implantation, or topical administering), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parentéral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aérosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration. In certain embodiments, tire compositions are administered by subcutaneous or intravenous infusion or injection.
Accordingly, in some embodiments, the herein described pharmaceutical compositions may comprise one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions described herein can be formulated for administration to a subject.
As used herein, a pharmaceutical composition or médicament includes a pharmacologically effective amount of at least one of the described therapeutic compounds and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical ingrédient (API, therapeutic product, e.g., HBV RNAi agent) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients may act to a) aid in processing of the drug delivery system during manufacture, b) protect,
138 support or enhance stability, bioavailability or patient acceptabilité' of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery’ of the API during storage or use. A pharmaceutically acceptable excipient may’ or may not be an inert substance.
Excipients include, but are not limited to: absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery’ enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricanls, oils, polymers, preservatives, saline, salis, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity' agents, vehicles, water-repelling agents, and wetting agents.
Pharmaceutical compositions suitable for injectable use include stérile aqueous solutions (where water soluble) or dispersions and stérile powders for the extemporaneous préparation of stérile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline. Il should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, éthanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by' the use of surfactants. In màny cases, it will be préférable to include isotonie agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Stérile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingrédients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a stérile vehicle which contains a basic dispersion medium and the required other ingrédients from those enumerated above. In the case of stérile
139 powders for the préparation of stérile injectable solutions, methods of préparation include vacuum drying and freeze-drying which yields a powder of the active ingrédient plus any additional desired ingrédient from a previously sterile-filtered solution thereof.
Formulations suitable for intra-articular administration can be in the form of a stérile aqueous préparation of the drug that can be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodégradable polymer Systems can also be used to présent the drug for both intra-articular and ophthalmic administration.
The active compounds can be prepared with carriers that will protect the compound against rapid élimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery Systems. Biodégradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for préparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
The HBV RNAi agents can be formuiated in compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrète units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The spécification for the dosage unit forms of the disclosure are dictated by and directly dépendent on the unique characteristics of the active compound and the therapeutic effect to be achieved, and the limitations inhérent in the art of compounding such an active compound for the treatment of individuals.
A pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to: antipruritics, astringents, local anesthetics, or anti-inflammatoiy agents (e.g., antihistamine, diphenhydramine, etc.). It is also envisioned that cells, tissues or isolated organs that express or comprise the herein defined RNAi agents may be used as “pharmaceutical compositions.”
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As used herein, “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to that amount of an RNAi agent to produce a pharmacological, therapeutic or préventive resuit.
Generally, an effective amount of an active compound will be in the range of from about 0.1 to about 100 mg/kg of body weight/day, e.g., from about 1.0 to about 50 mg/kg of body weight/day. In some embodiments, an effective amount of an active compound will be in the range of from about 0.25 to about 5 mg/kg of body weight per dose. In some embodiments, an effective amount of an active ingrédient will be in the range of from about 0.5 to about 3 mg/kg of body weight per dose. The amount administered will also likely dépend on such variables as the overall health status of the patienk the relative biological efficacy of the compound delivered, the formulation of the drug, the presence and types of excipients in the formulation, and the route of administration. Also, it is to be understood that the initial dosage administered can be increased beyond the above upper level in order to rapidly achieve the desired blood-level or tissue level, or the initial dosage can be smaller than the optimum.
For treatment of disease or for formation of a médicament or composition for treatment of a disease, the pharmaceutical compositions described herein including an HBV RNAi agent can be combined with an excipient or with a second therapeutic agent or treatment including, but not limited to: a second or other RNAi agent, a small molécule drug, an antibody, an antibody fragment, and/or a vaccine.
The described HBV RNAi agents, when added to pharmaceutically acceptable excipients or adjuvants, can be packaged into kits, containers, packs, or dispensera. The pharmaceutical compositions described herein may be packaged in pre-filled syringes or vials.
Methods of Treatment and Inhibition of Expression
The HBV RNAi agents disclosed herein can be used to treat a subject (e.g., a human or mammal) having a disease or disorder that would benefit from administration of the compound. In some embodiments, the RNAi agents disclosed herein can be used to treat a subject (e.g., a human) having a disease or disorder that would benefit from réduction or inhibition in expression of HBV mRNA. The subject is administered a therapeutically effective amount of any one or more RNAi agents. The subject can be a human, patient, or human patient. The
141 subject may be an adult, adolescent, child, or infant. The described pharmaceutical compositions including an HBV RNAi agent can be used to provide methods for the therapeutic treatment of diseases. Such methods include administration of a pharmaceutical composition described herein to a human being or animal.
In some embodiments, the HBV RNAi agents described herein are used to treat a subject infected with HBV. In some embodiments, the described HBV RNAi agents are used to treat at least one symptom in a subject having a HBV infection. The subject is administered a therapeutically effective amount of any one or more of the described RNAi agents.
In some embodiments, the subject has both a HBV infection and a HDV infection. In some embodiments, the HBV RNAi agents described herein are used to treat a subject infected with both HBV and HDV. In some embodiments, the described HBV RNAi agents are used to treat at least one symptom in a subject having a HBV or a HDV infection. The subject is administered a therapeutically effective amount of any one or more of the described RNAi agents.
In some embodiments, the HBV RNAi agents are used to treat or manage a clinical présentation wherein a subject infected with HBV. The subject is administered atherapeutically or effective amount of one or more of the HBV RNAi agents or HBV RNAi agent-containing compositions described herein. In some embodiments, the method comprises administering a composition comprising an HBV RNAi agent described herein to a subject to be treated.
In some embodiments, the gene expression level and'or mRNA level of an HBV gene in a subject to whom a described HBV RNAi agent is administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subject prior to being administered the HBV RNAi agent or to a subject not receiving the HBV RNAi agent. The gene expression level and/or mRNA level in the subject may be reduced in a cell, group of cells, and/or tissue of the subject. In some embodiments, the expressed protein level of an HBV gene in a subject to whom a described HBV RNAi agent has been administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subject
142 prior to being administered the HBV RNAi agent or to a subject not receiving the HBV RNAi agent. The protein level in the subject may be reduced in a cell, group of cells, tissue, blood, and/or other fluid of the subject. For example, in some embodiments, the amount or level of Hepatitis B surface antigen (HBsAg) in a subject to whom a described HBV RNAi agent has been administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%. 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subject prior to being administered the HBV RNAi agent or to a subject not receiving the HBV RNAi agent. In some embodiments, the amount or level of Hepatitis B e-antigen (HBeAg) in a subject to whom a described HBV RNAi agent has been administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subject prior to being administered the HBV RNAi agent or to a subject not receiving the HBV RNAi agent. In some embodiments, the amount or level ofsérum HBV DNA in a subject to whom a described HBV RNAi agent has been administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subject prior to being administered the HBV RNAi agent or to asubject not receiving the HBV RNAi agent. A réduction in the presence of sérum HBV DNA, HBV gene expression, HBV mRNA, or HBV protein amounts or levels may be assessed by methods known in the art. Réduction or decrease in HBV mRNA amount or level, expressed protein amount or level, and/or sérum HBV DNA amount or level, are collectively referred to herein as a réduction or decrease in HBV or inhibiting or reducing the expression of HBV.
Cells and Tissues and non-Human organisms
Cells, tissues, and non-human organisms that include at least one of the HBV RNAi agents described herein is contemplated. The cell, tissue, or non-human organism is made by delivering the RNAi agent to the cell, tissue, or non-human organism.
The above provided embodiments and items are now illustrated with the following, nonlimiting examples.
Examples
Exantple 1. Synthesis of HBVRNAi agents.
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HBV RNAi agent duplexes shown in Table 5 were synthesized in accordance with the following:
A. Synthesis. The sense and antisense strands of the HBV RNAi agents were synthesized according to phosphoramidite technology on solid phase used in oligonucleotide synthesis. Depending on the scale, either a MerMade96E© (Bioautomation), a MerMadel2® (Bioautomation), or an OP Pilot 100 (GE Healthcare) was used. Synthèses were performed on a solid support made of controlled pore glass (CPG, 500 À or 600Â, obtained from Prime Synthesis, Aston, PA, USA). Ail RNA and 2'-modified phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the following 2'-O-methyl phosphoramidites were used: (5'-O-dimethoxytrityl-N6-(benzoyl)-2'-O-methyl-adenosine-3'-O(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, 5'-O-dimethoxy-trityl-N4-(acetyl)-2'-Omethyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino) phosphoramidite, (5'-Odimethoxytrityl-N2-(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,Ndiisopropylamino) phosphoramidite, and 5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2cyanoethyl-N,N-diisopropylamino) phosphoramidite. The 2'-deoxy-2'-fluoro-phosphoramidites carried the same protecting groups as the 2'-O-methyl amidites. The abasic (3'-Odimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropyIamino) phosphoramidites were purchased from ChemGenes (Wilmington, MA, USA). Targeting ligand containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while ail other amidites were dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3Â) were added. 5-Benzylthio-lH-tetrazoIe (BTT, 250 mM in acetonitrile) or 5Ethylthio-lH-tetrazole (ETT, 250 mM in acetonitrile) was used as activator solution. Coupling times were 12 min (RNA), 15 min (targeting ligand), 90 sec (2'OMe), and 60 sec (2'F). In order to introduce phosphorothioate linkages, a 100 mM solution of 3-phenyl l,2,4-dithiazoline-5one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous Acetonitrile was employed.
B. Cleavage and deprotection of support bound oligomer. After finalization of the solid phase synthesis, the dried solid support was treated with a 1:1 volume solution of 40 wt % methylamine in water and 28% ammonium hydroxide solution (Aldrich) for 1.5 hours at 30°C. The solution was evaporated and the solid residue was reconstituted in w'ater (see below).
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C. Purification. Crude oligomers were purified by anionic exchange HPLC using a TSKgel SuperQ-5PW 13pm column and Shimadzu LC-8 System. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0 and contained 20% Acetonitrile and buffer B was the same as buffer A with tire addition of 1.5 M sodium chloride. UV traces at 260 nm were recorded. Appropriate fractions were pooled then run on size exclusion HPLC using a GE Healthcare XK 26/40 column packed with Sephadex G-25 fine with a running buffer of filtered DI w'ater or lOOmM ammonium bicarbonate, pH 6.7 and 20% Acetonitrile.
D. Annealing. Complementary' strands were mixed by combining equimolar RNA solutions (sense and antisense) in 1 xPhosphate-Buffered Saline (Corning, Cellgro) to form the RNAi agents. Some RNAi agents wære lyophilized and stored at -15 to -25°C. Duplex concentration w'as determined by measuring the solution absorbance on a UV-Vis spectrometer in 1 x Phosphate-Buffered Saline. The solution absorbance at 260 nm was then multiplied by a conversion factor and the dilution factor to détermine the duplex concentration. Unless otherwise stated, ail conversion factor was 0.037 mg/(mL-cm). For some experiments, a conversion factor was calculated from an experimentally determined extinction coefficient.
Exemple 2. pHBV model mice.
Six to eight-week-old female NOD.CB17-Prkdscid/NcrCrl (NOD-SCID) mice were transiently transfected in vivo with MC-HBV1.3 by hydrodynamic tail vein injection (Yang PL et al. “Hydrodynamic injection of viral DNA: a mouse model of acute hepatitis B virus infection,” PNAS USA 2002 Vol. 99: p. 13825-13830), administered 30 to 45 days prior to administration of anHBV RNAi agent or control. MC-HBV1.3 is a plasmid-derived minicircle that contains the same terminally redundant human hepatitis B virus sequence HBV1.3 as in plasmid pHBV1.3 and in the HBVI.3.32 transgenic mice (GenBank accession #V01460) (Guidotti LG et al., “High-level hepatitis B virus réplication in transgenic mice,” J Virol 1995 Vol. 69, p6158-6169.). 5 or 10 pg MC-HBVL3 in Ringer’s Solution in a total volume of 10% of the animal’s body w'eight was injected into mice via tail vein to create pHBV model of chronic HBV infection. The solution was injected through a 27-gauge needle in 5-7 seconds as previously described (Zhang G et al., “High levels of foreign gene expression in hépatocytes after tail vein injection of naked plasmid DNA.” Human Gene Therapy 1999 Vol. 10, pl7351737.). At pre-dose (either day 1 pre-dose, day -1, or day -2), Hepatitis B surface antigen
145 (HBsAg) HBsAg expression levels in sérum were measured by ELISA and the mice were grouped according to average HBsAg expression levels.
Analyses: At various times, before and after administration of HBV RNAi agents, sérum HBsAg, sérum HBeAg, sérum HBV DNA, or liver HBV RNA may be measured. HBV expression levels were normalized to pre-administration expression levels and to control mice injected with phosphate buffered saline (“PBS”).
i) Sérum collection: Mice were anesthetized with 2-3% isoflurane and blood samples were collected from the submandibular area into sérum séparation tubes (Sarstedt AG & Co., Nümbrecht, Germany). Blood was allow'ed to coagulate at ambient température for 20 min. The tubes were centrifuged at 8,000 xg for 3 min to separate the sérum and stored at 4°C.
ii) Sérum Hepatitis B surface antigen (HBsAg) levels: Sérum was collected and diluted 10 to 8000-fold in PBS containing 5% nonfat dry milk. Secondary HBsAg standards diluted in the nonfat milk solution w'ere prepared from sérum of ICR mice (Harlan Sprague Dawley) that had been transfected with 10 pg HBsAg-expressing plasmid pRc/CMV-HBs (Aldevron, Fargo, ND). HBsAg levels w'ere determined with a GS HBsAg EIA 3.0 kit (Bio-Rad Laboratories, Inc., Redmond, WA) as described by the manufacturer. Recombinant HBsAg protein, aywr subtype, also diluted in nonfat milk in PBS, was used as a primary standard (Aldevron).
HBsAg expression for each animal was normalized to the control group of mice injected with PBS in order to account for the non-treatment related décliné in expression of MC-HBV1.3. First, the HBsAg level for each animal at a time point was divided by the pretreatment level of expression in that animal in order to détermine the ratio of expression “normalized to pre-treatmenf’. Expression at a spécifie time point was then normalized to the control group by dividing the “normalized to pre-treatment” ratio for an individual animal by the average “normalized to pre-treatmenf’ ratio of ail mice in the normal PBS control group.
iii) Sérum Hepatitis B e-antigen (HBeAg) levels: HBeAg analysis was performed with the HBeAg enzyme linked immunosorbent assay (ELISA) as described by the manufacturer (DiaSorin) using sérum diluted 4- to 20-fold in 5% nonfat dry milk. The amount of antigen
146 was determined in the linear range of the assay and quantitated against HBeAg protein standards (Fitzgerald Industries International, catalog # 30-AH18, Acton, MA).
HBeAg expression for each animal was normalized to the control group of mice injected with PBS in order to accounl for the non-treatment related décliné in expression of MC-HBV1.3. For évaluation of HBeAg in sérum, HBeAg is analyzed from pooled group or subgroup sérum samples. First, the HBeAg level for each pooled group or subgroup was divided by the pre-treatment level of expression in the same group or subgroup in order to détermine the ratio of expression “normalized to pre-treatment”. Expression at a spécifie time point was then normalized to the control group by dividing the “normalized to pre-treatment” ratio for a group or subgroup by the average “normalized to pre-treatment” ratio of ail samples from the normal PBS control group.
iv) Sérum HBVDNA levels: Equal volumes of sérum from mice in a group or subgroup were pooled to a final volume of 100 pL. DNA was isolated from sérum samples using the QIAamp MinElute Virus Spin Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. Stérile 0.9% saline was added to each sample to a final volume of 200 pL. Sérum samples were added to tubes containing buffer and protease. Carrier RNA was added to aid in the isolation of small amounts of DNA. 1 ng of pHCR/UbC-SEAP plasmid DNA (Wooddell CI, et al. Long-term RNA interférence from optimized siRNA expression constructs in adult mice. Biochem Biophys Res Commun (2005) 334,117-127) was added as a recovery control. After incubating 15 min at 56°C, nucleic acids were precipitated from the lysâtes wdth éthanol and the entire solution applied to a column. After w'ashing, the samples were eluted into a volume of 50 pL Buffer AVE.
The number of copies of HBV genomes in DNA isolated from the pHBV mouse model sérum was determined by qPCR. Plasmid pSEAP-HBV353-777, encoding a short segment of the HBV genome within the S gene (bases 353-777 of GenBank accession #V01460), wras used to create a six log standard curve. Samples wdth recovery of DNA below 2 standard déviations from the average, based on détection of pHCR/UbC-SEAP were omitted. TaqMan chemistrybased primers and probes with fluor/ZEN/IBFQ are utilized.
147 qPCR assays were performed on a 7500 Fast or StepOne Plus Real-Time PCR system (Life Technologies). For évaluation of HBV DNA in sérum, DNA was isolated from singlet or duplicate purification steps from pooled group sérum samples. Quantitations of HBV DNA and recovery control plasmid were determined by qPCR reactions performed in triplicate. The probes to quantitate HBV and pHCR/UbC-SEAP were included in each reaction.
Example 3. HBVRNAi agents in pHBVmode! mice.
The pHBV mouse model described in Example 2, above, was used. At day 1, each mouse was administered a single subcutaneous injection of 200 μΐ containing 2 mg/kg (mpk) of an HBV RNAi agent formulated in phosphate buffered saline (“PBS”), or 200 μΐ of phosphate buffered saline without an HBV RNAi agent, to be used as a control. Each of the HBV RNAi agents included N-acetyl-galactosamine targeting ligands conjugated to the 5'-terminal end of the sense strand, as shown in Tables 4 and 5. The HBV RNAi agents tested included those having the duplex numbers shown in Table 7, below7. The injections w;ere performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Three (3) mice in each group w'ere tested (n=3).
Sérum was collected on day 8, day 15, day 22, and day 29, and sérum Hepatitis B surface antigen (HBsAg) levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment is shown in the following Table:
Table 7. Average HBsAg levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 3 (standard déviation reflected as (+/-)).
Group Day 8 Day 15 Day 22 Day 29
PBS 1.000 ±0.185 1.000 ±0.288 1.000 ±0.540 1.000 ±0.326
AD04178 0.164 ±0.043 0.206 ±0.044 0.293 ± 0.050 0.348 ±0.099
AD04579 0.083 ± 0.028 0.099 ±0.022 0.112 ±0.022 0.138 ±0.056
AD04580 0.048 ±0.007 0.073 ±0.012 0.085 ±0.012 0.126 ±0.014
AD04570 0.241 ± 0.076 0.294 ±0.071 0.276 ±0.068 0.474 ± 0.092
AD04572 0.190 ±0.040 0.279 ±0.011 0.323 ± 0.049 0.441 ±0.046
AD04573 0.333 ±0.143 0.505 ±0.106 0.361 ±0.060 0.444 ±0.068
AD04574 0.291 ± 0.032 0.650 ±0.056 0.388 ±0.048 0.485 ± 0.070
AD04575 0.397 ±0.189 0.514 ±0.234 0.574 ±0.204 0.689 ±0.207
AD04419 0.262 ±0.038 0.174 ±0.042 0.258 ±0.064 0.311 ±0.089
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AD04578 0.210 + 0.056 0.235 ± 0.033 0.298 ± 0.035 0.336 ± 0.049
RNAi agents AD0417S, AD04579, AD04580, AD04570, AD04572, AD04573, AD04574, AD04575, AD04419, and AD04578 were each designed to hâve antisense strand sequences at least partially complementary to the X open reading frame at positions 1781-1789 of the HBV genome shown in Tables 1 and 2, above. Each of the HBV RNAi agents showed substantial réduction in HBsAg as compared to the PBS control across ail measured time points. For example, AD04580 showed greater than 95% réduction in s-antigen levels at day 8 (0.048 ± 0.007 HBsAg level) when normalized to pre-treatment and PBS control.
Additionally, sérum HBV DNA levels were determined for the PBS, AD04579, and AD04580 groups from sérum samples collected on days 8, 15, 22, 29, 36, 43 and 50, pursuant to the procedure set forth in Example 2, above. Sérum from each group w'as pooled and then DNA w'as isolated from the sérum in duplicate isolations. Data are presented in the following Table:
Table 8. Average Sérum HBV DNA levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 3 (standard déviation reflected as (+/-)).
Group Day 8 Day 15 Day 22 Day 29
PBS 1.0000+0.1185 1.0000 ±0.0591 1.0000 ±0.0322 1.0000 ±0.0597
AD04579 0.1541 ±0.0070 0.1776 ±0.0027 0.1810 ±0.0450 0.3738 ± 0.0302
AD04580 0.0921 ±0.0253 0.0869 ±0.0117 0.1444 ±0.0755 0.0950 ±0.0026
Group Day 36 Day 43 Day 50
PBS 1.0000 ±0.1625 1.0000 ±0.0055 1.0000 ±0.1484
AD04579 0.9670 ±0.1247 0.7643 ±0.1334 0.6299 ±0.1319
AD04580 0.4949 ±0.0096 0.4350 ± 0.0344 0.6819 ±0.0266
The data in Table 8 indicate that both RNAi agents examined provided a substantial réduction in HBV DNA levels compared to the PBS group, with AD04580 achieving slightly greater than 1 log knockdown at nadir (e.g., 0.0869 ± 0.0117 average sérum DNA level at day 15).
Example 4. HBVRNAi agents in pHBVmodel mice.
The pHBV mouse model described in Example 2, above, was used. At day 1, each mouse wras given a single subcutaneous administration of 200 μΐ containing 2 mg/kg (mpk) of an HBV
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RNAi agent formulated in phosphate buffered saline, or 200 μΐ of phosphate buffered saline without an HBV RNAi agent to be used as a control. Each of the HBV RNAi agents included N-acetyl-galactosamine targeting ligands conjugated to the 5'-terminal end of the sense strand, as shown in Tables 4 and 5. The HBV RNAi agents administered included those listed in Table 9, below. The injections w'ere performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area Three (3) mice in each group were tested (n=3).
Sérum w'as collected on day 8, day 15, day 22, and day 29, and sérum Hepatitis B surface antigen (HBsAg) levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment is shown in the following Table:
Table 9. Average HBsAg levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 4 (standard déviation reflected as (+/-)).
Group Day 8 Day 15 Day 22 Day 29
PBS 1.000 + 0.085 1.000 + 0.235 1.000+0.171 1.000 + 0.099
AD04010 0.229 + 0.141 0.165 + 0.091 0.142+0.085 0.116 + 0.076
AD04581 0.379+0.042 0.221 ± 0.066 0.135 + 0.040 0.112 + 0.050
AD04591 0.285 + 0.101 0.145 + 0.064 0.086+0.024 0.081 ± 0.026
AD04434 0.295 ± 0.041 0.191 + 0.008 0.147 + 0.016 0.187 + 0.049
AD04583 0.488 + 0.018 0.545 ± 0.037 0.511 + 0.086 0.663 + 0.112
AD04584 0.392 + 0.136 0.337 + 0.073 0.364 + 0.075 0.515 + 0.155
AD04585 0.099 + 0.016 0.042 + 0.014 0.030 + 0.009 0.044 + 0.014
AD04586 0.222 + 0.056 0.107 + 0.034 0.074+0.016 0.106 + 0.039
AD04588 0.255 + 0.065 0.205 ± 0.021 0.185 + 0.021 0.207 + 0.024
AD04438 0.265 + 0.106 0.113 + 0.045 0.091 ± 0.031 0.130 + 0.038
RNAi agents AD04010, AD04581, ADÛ4591, AD04434, AD04583, AD04584, AD04585, AD04586, AD04588, and AD04438 w7ere designed to hâve antisense strand sequences that are at least partially complementary' to the S open reading frame at positions 257-275 of the HBV genome, as shown in Tables 1 and 2. The HBV RNAi agents shown in Table 9, directly above, each showed substantial réduction in HBsAg as compared to the PBS control across ail measured time points. For example, AD04585 exhibited approximately a 90% réduction of HBsAg at day 8, a 95% réduction at day 15, a 97% réduction at day 22, and a 95% réduction at day 29.
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Additionally, sérum HBV DNA levels were determined for the PBS, AD04585 groups from sérum samples collected on days 8, 15, 22, 29, 36, 43 and 50, pursuant to the procedure set forth in Example 2, above. Sérum from each group was pooled and then DNA was isolated from the sérum in duplicate isolations. Data are presented in the following Table:
Table 10. Average Sérum HBV DNA levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 4 (standard déviation reilected as (+/-)).
Group Day 8 Day 15 Day 22 Day 29
PBS 1.000 ±0.248 1.000 ±0.089 1.000 ±0.195 1.000 ±0.180
AD04585 0.901 ±0.183 0.225 ± 0.003 0.187 ±0.023 0.191 ±0.004
Group Day 36 Day 43 Day 50
PBS 1.000 ±0.018 1.000 ±0.033 1.000 ±0.778
AD04585 0.209 ±0.017 0.171 ±0.019 0.305 ±0.010
The data in Table 10 indicate that HBV RNAi agent AD04585 provided a réduction in HBV DNA levels compared to the PBS group.
Example 5. Dose response and combinations of HBVRNAi Agents in pHBV model mice.
The pHBV mouse model described in Example 2, above, was used. The mice were divided into various groups including those set forth in Table 11, below, and the mice were given 200 μΐ subcutaneous injections pursuant to the dosing regimen set forth in Table 11:
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Table 11. Dosing groups of pHBV mice for Example 5.
Group RNAi Agent and Dose Dosing Regimen
A PBS (no RNAi agent) Single injection on day 1
B 3.0 mg/kg AD04585 Single injection on day 1
C 3.0 mg/kg AD04585 Injection on day 1, day 8, and day 15 (i.e., three Aveekly injections)
D 3.0 mg/kg AD04580 Single injection on day 1
E 3.0 mg/kg AD04580 Injection on day 1, day 8, and day 15 (i.e., three w'eekly injections)
F 1.0 mg/kg AD4585 + 1.0 mg/kg AD04580 Injection on day 1, and another injection on day 22
G 1.0 mg/kg AD4585 + 1.0 mg/kg AD04580 Injection on day 1, day 8, day 15, and day 43
H 1.5 mg/kg AD4585 + 1.5 mg/kg AD04580 Injection on day 1, day 22, and day 43
I 1.5 mg/kg AD4585 + 1.5 mg/kg AD04580 Injection on day 1, day 8, day 15, and day 43
Each mouse was given a subcutaneous administration of200 μΐ containing the amount of HBV RNAi agent(s) formuiated in phosphate buffered saline, or 200 pl of phosphate buffered saline 5 without an HBV RNAi agent, as set forth in Table 11. Each of the HBV RNAi agents included
N-acetyl-galactosamine targeting ligands conjugated to the 5'-terminal end ofthe sense strand, as shown in Tables 4 and 5. The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Three (3) mice in each group were tested (n=3).
Sérum was collected on day 8, day 15, day 22, day 29, day 36, day 43, day 50, and day 57, and sérum Hepatitis B surface antigen (HBsAg) levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment is shown in the following Table:
Table 12. Average HBsAg levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 5 (standard déviation reflected as (+/-)).
Group Day 8 Day 15 Day 22 Day 29
A 1.000 ±0.162 1.000 ±0.138 1.000 ±0.083 1.000 ±0.204
B 0.060 ±0.015 0.010 ±0.003 0.006 ±0.002 0.007 ± 0.002
C 0.087 ±0.014 0.004 ±0.001 0.001 ± 0.0003 0.0002 ± 0.0001
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D 0.026 + 0.009 0.035 + 0.013 0.037 + 0.014 0.046 + 0.006
E 0.023 ± 0.005 0.002 + 0.001 0.001 ± 0.0003 0.001 ± 0.0004
F 0.063 ± 0.046 0.083 + 0.051 0.086 + 0.016 0.027 ± 0.006
G 0.062 + 0.011 0.022 ± 0.008 0.009 + 0.003 0.008 ± 0.002
H 0.055 ± 0.015 0.062 + 0.002 0.072 + 0.013 0.011 + 0.001
I 0.031 ± 0.006 0.008 ± 0.001 0.003 ± 0.0004 0.003 ± 0.0003
Group Day 36 Day 43 Day 50 Day 57
A 1.000 + 0.211 1.000 + 0.189 1.000 + 0.179 1.000 + 0.062
B 0.013 ± 0.005 0.027 + 0.004 0.026 + 0.004 0.057 + 0.012
C 0.001 ± 0.0002 0.002 + 0.001 0.008 ± 0.004 0.020 + 0.015
D 0.116 + 0.019 0.214 + 0.056 0.263 ± 0.046 0.404 ± 0.030
E 0.003 ± 0.0001 0.007 ± 0.001 0.012 + 0.002 0.033 ± 0.011
F 0.029 + 0.003 0.065 ± 0.005 0.064 + 0.004 0.161 + 0.033
G 0.014+0.008 0.039 + 0.011 0.018 + 0.008 0.046 ± 0.008
H 0.017 + 0.005 0.039 + 0.008 0.007 + 0.001 0.013 ± 0.003
I 0.007 ± 0.001 0.020 + 0.002 0.005 + 0.001 0.011 + 0.002
HBV RNAi agents AD04580 and AD04585 each individually showed a réduction in HBsAg as compared to the PBS control across ail measured time points. Furthermore, combination treatment of ADO4585 and AD04580, which as noted in the Examples above target different 5 régions of the HBV genome, also showed réduction in HBsAg as compared to the PBS control across ail measured time points.
Additionally, sérum HBV DNA levels were determined for each of the groups in Table 11 from sérum samples collected on days 8,15,22, 29, and 36, pursuant to the procedure set forth 10 in Example 2, above. Sérum from each group was pooled and then DNA was isolated from the sérum in duplicate reactions. Data are presented in the following Table:
Table 13. Average Sérum HBV DNA levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 5 (standard 15 déviation reflected as (+/-)).
Group Day 8 Day 15 Day 22 Day 29
A 1.000 + 0.063 1.000 + 0.059 1.000 + 0.372 1.000 + 0.237
B 0.267 + 0.003 0.043 + 0.016 0.038 + 0.008 0.044 ± 0.004
C 0.236 + 0.016 0.023 + 0.001 0.004 + 0.001 0.002 ± 0.000
D 0.058 + 0.016 0.085 + 0.017 0.252 + 0.071 0.217 + 0.009
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E 0.056 ±0.002 0.0009 ± 0.0004 0.0005 ± 0.0002 0.003 ± 0.002
F 0.298 ±0.013 0.351 ±0.032 0.823 ±0.127 0.217 ±0.007
G 0.276 ±0.035 0.112 ±0.020 0.061 ±0.002 0.073 ± 0.002
H 0.232 ±0.012 0.213 ± 0.028 0.403 ± 0.047 0.079 ± 0.005
I 0.092 ±0.026 0.055 ± 0.000 0.002 ±0.003 0.010 ±0.004
Group Day 36
A 1.000 ±0.024
B 0.046 ±0.007
C 0.003 ± 0.000
D 0.319 ±0.034
E 0.002 ±0.000
F 0.122 ±0.004
G 0.047 ±0.006
H 0.056 ±0.003
I 0.021 ± 0.007
The data in Table 13 indicate that the RNAi agents examined, both individually and in combination, provided a réduction in HBV DNA levels compared to the PBS group. Re-dosing or increasing the dose amount yielded additional HBV DNA réductions.
Example 6. HBVRNAi agents in pHBV mice: dose response and combination studies.
The pHBV mouse model described in Example 2, above, was used. Mice were divided into various groups as set forth in Table 14, below, and each mouse was administered a single 200 μΐ subcutaneous injection pursuant to the dosing regimen set forth in Table 14:
Table 14. Dosing groups of pHBV mice for Example 6.
Group RNAi Agent and Dose Dosing Regimen
A PBS (no RNAi agent) Single injection on day 1
B 4.0 mg/kg AD04981 Single injection on day 1
C 1.0 mg/kg AD04981 Single injection on day 1
D 2.0 mg/kg AD04981 Single injection on day 1
E 1.0 mg/kg AD04963 Single injection on day 1
F 2.0 mg/kg AD04963 Single injection on day 1
G 3.0 mg/kg AD04872 Single injection on day 1
H 3.0 mg/kg AD04872 + 1.0 mg/kg AD04981 Single injection on day 1
I 3.0 mg/kg AD04872 + 1.0 mg/kg AD04963 Single injection on day 1
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J 3.0 mg/kg AD04872 + 2.0 mg/kg AD04981 Single injection on day 1
Each mouse was given a subcutaneous administration of200 μΐ containing the amount of HBV RNAi agent(s) formulated in phosphate buffered saline, or 200 μΐ of phosphate buffered saline without an HBV RNAi agent, as set forth in Table 14. Each ofthe HBV RNAi agents included 5 N-acetyl-galactosamine targeting ligands conjugated to the 5 '-terminal end of the sense strand, as shown in Tables 4 and 5. The injections w;ere performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Three (3) mice in each group were tested (n=3).
Sérum was collected on day -1 prior to administration, and then on day 8, day 15, day 22, day 29, and day 36, and sérum HBsAg levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment is shown in the following Table 15, with Average HBsAg reflecting the normalized average value of HBsAg:
Table 15. Average HBsAg levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 6 (standard déviation reflected as (+/-)).
Group Day 8 Day 15 Day 22
A 1.000 ±0.068 1.000 ±0.183 1.000 ±0.181
B 0.085 ± 0.020 0.068 ±0.005 0.089 ±0.014
C 0.283 ± 0.039 0.343 ±0.055 0.436 ±0.004
D 0.161 ±0.052 0.137 ±0.036 0.190 ±0.068
E 0.182 ±0.040 0.233 ±0.023 0.436 ±0.029
F 0.078 ± 0.024 0.093 ±0.015 0.167 ±0.028
G 0.066 ±0.030 0.013 ±0.002 0.010 ±0.002
H 0.033 ±0.012 0.016 ±0.005 0.020 ± 0.005
I 0.040 ±0.011 0.028 ±0.003 0.032 ±0.007
J 0.035 ±0.010 0.019 ±0.002 0.021 ±0.001
Group Day 29 Day 36
A 1.000 ±0.032 1.000 ±0.141
B 0.148 ±0.016 0.194 ±0.047
C 0.622 ± 0.041 0.741 ±0.132
D 0.234 ± 0.055 0.280 ±0.071
E 0.623 ±0.116 0.782 ±0.114
F 0.259 ±0.014 0.368 ±0.068
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G 0.010 ±0.003 0.009 ±0.004
H 0.022 ± 0.005 0.024 ± 0.009
I 0.065 ±0.014 0.087 ±0.015
J 0.031 ±0.0001 0.044 ±0.002
The HBV RNAi agents tested showed a réduction in HBsAg as compared to the PBS control across ail measured time points. Furthermore, combination treatment of AD04872 (which includes an antisense strand sequence that is at least partially complementary to the S ORF at positions 261-279 of the HBV genome, as shown in Tables 1 and 2) and either AD04981 or AD04963 (both of which include antisense strand sequences that are at least partially complementary’ to the X ORF at positions 1781-1799 of the HBV genome, as shown in Tables 1 and 2), w’hich are shown in Groups H, I, and J of Example 6, illustrate that combination treatment of two RNAi agents targeting, one which targets in the S ORF, and the other which targets in the X ORF of the HBV genome, similarly show'ed réduction in HBsAg compared to the PBS control across ail measured time points.
Additionally, Sérum Hepatitis B e-antigen (HBeAg) levels were also assessed. Samples from the mice in each respective group were first pooled, and the resulting sérum samples were assayed in singlet. Data from the experiment is shown in the following Table:
Table 16. Average HBeAg levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 6.
Group Day 8 Day 15 Day 22 Day 29 Day 36
A 1.000 1.000 1.000 0.183 1.000
B 0.138 0.180 0.274 0.005 0.089
C 0.316 0.376 0.588 0.055 0.436
D 0.167 0.250 0.262 0.036 0.190
E 0.301 0.327 0.447 0.023 0.436
F 0.167 0.172 0.305 0.015 0.167
G 0.275 0.135 0.158 0.002 0.010
H 0.080 0.053 0.094 0.005 0.020
I 0.165 0.124 0.185 0.003 0.032
J 0.120 0.057 0.101 0.002 0.021
As shown in Table 16, the combination AD04872 (which targets the S ORF of the HBV genome) with either AD04981 or AD04963 (both of which target the X ORF of the HBV
156 genome), showed a further réduction in HBeAg levels relative to administering AD04872 alone.
Example 7. HBVRNAi Agents inpHBVmice: additional dose response and combination studies.
The pHBV mouse model described in Example 2, above, was used. Mice were divided into various groups as set forth in Table 17, below, and each mouse was administered a single 200 μΐ subcutaneous injection pursuant to the dosing regimen set forth in Table 17:
Table 17. Dosing groups of pHBV mice for Example 7.
Group RNAi Agent and Dose Dosing Regimen
A PBS (no RNAi agent) Single injection on day 1
B 4.0 mg/kg AD04776 Single injection on day 1
C 1.0 mg/kg AD04982 Single injection on day 1
D 2.0 mg/kg AD04982 Single injection on day 1
E 1.0 mg/kg AD04776 Single injection on day 1
F 2.0 mg/kg AD04776 Single injection on day 1
G 3.0 mg/kg AD04872 Single injection on day 1
H 3.0 mg/kg AD04872 + 1.0 mg/kg AD04982 Single injection on day 1
I 3.0 mg/kg AD04872 + 2.0 mg/kg AD04982 Single injection on day 1
Each mouse was given a subcutaneous administration of200 pl containing the amount of HBV RNAi agent(s) formulated in phosphate buffered saline, or 200 pl of phosphate buffered saline without an HBV RNAi agent, as set forth in Table 17. Each of the HBV RNAi agents included N-acetyl-galactosamine targeting ligands conjugated to the 5'-terminal end of the sense strand, as shown in Tables 4 and 5. The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Four (4) mice in each group were tested on day -1 and day 8 (n=4), and then one mouse per group was euthanized for histological évaluation. Three (3) mice in each group were tested at day 22 and day 29 (n=3).
157
Sérum was collected on day -1 prior to administration, and then on day 8, day 15, day 22, and day 29, and sérum Hepatitis B surface antigen (HBsAg) levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment is shown in the following Table 18:
Table 18. Average HBsAg levels normalized to pre-treatment (day -1) and PBS control in pHBV mice following administration of HBV RNAi agents from Example 7 (standard déviation reflected as (+/-)).
Group Day 8 Day 15 Day 22 Day 29
A 1.000 ±0.347 1.000 ±0.278 1.000 ±0.194 1.000±0.318
B 0.117 ±0.069 0.085 ±0.039 0.148 ±0.045 0.198 ±0.049
C 0.519 ±0.058 0.375 ±0.012 0.422 ± 0.046 0.525 ± 0.037
D 0.342 ±0.062 0.255 ±0.046 0.272 ±0.122 0.314 ±0.068
E 0.279 ±0.057 0.245 ±0.032 0.374 ±0.121 0.304 ±0.035
F 0.224 ±0.018 0.161 ±0.009 0.310 ±0.016 0.482 ±0.053
G 0.029 ±0.010 0.005 ±0.001 0.004 ±0.001 0.006 ±0.001
H 0.016 ±0.005 0.004 ±0.001 0.010 ±0.006 0.015 ±0.008
I 0.026 ±0.012 0.008 ±0.001 0.010 ± 0.002 0.015 ±0.005
The HBV RNAi agents tested show’ed a réduction in HBsAg as compared to the PBS control across ail measured time points.
Additionally, Sérum Hepatitis B e-antigen (HBeAg) levels were also assessed. Samples from the mice in each respective group w’ere first pooled, and the resulting sérum samples were 15 assayed in singlet. Data from the experiment is shown in the following Table:
Table 19. Average HBeAg levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 7.
Group Day 8 Day 15 Day 22 Day 29 Day 36
A 1.000 1.000 1.000 1.000 1.000
B 0.193 0.213 0.260 0.307 0.464
C 0.471 0.424 0.562 0.513 0.705
D 0.335 0.310 0.411 0.442 0.500
E 0.381 0.368 0.355 0.564 0.483
F 0.275 0.255 0.370 0.495 0.449
G 0.323 0.218 0.205 0.250 0.190
H 0.124 0.102 0.099 0.156 0.156
I 0.081 0.059 0.045 0.063 0.086
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Table 19-1. Average HBeAg fold knockdown normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 7.
Group Day 8 (Fold KD) Day 15 (Fold KD) Day 22 (Fold KD) Day 29 (Fold KD) Day 36 (Fold KD)
A 1.0 1.0 1.0 1.0 1.0
B 5.2 4.7 3.8 3.3 2.2
C 2.1 2.4 1.8 2.0 1.4
D 3.0 3.2 2.4 2.3 2.0
E 2.6 2.7 2.8 1.8 2.1
F 3.6 3.9 2.7 2.0 2.2
G 3.1 4.6 4.9 4.0 5.3
H 8.1 9.8 10.1 6.4 6.4
I 12.3 17.0 22.3 15.7 11.6
Table 19-1 reflects the fold knockdown ratio of HBeAg compared to control, which is calculated as normalized HBeAg level ofthe control (PBS) group/normalized HBeAg level of the respected RNAi agent(s) group (i.e., 1.000/HBeAg level). The data in Table 19-1 indicate that the combination of AD04872 (which, as noted above, includes an antisense strand sequence that is at least partially complementary' to the S ORF at positions 261-279 ofthe HBV genome) with AD04982 (which includes an antisense strand sequence that is at least partially complementary’ to the X ORF at positions 1781-1799 of the HBV genome), show’ed a further réduction in HBeAg levels relative to administering the individual RNAi agents alone (See, e.g., Tables 19 and 19-1 for Groups H and I). Further, the data from this Example also show' that the combination of AD04872 with AD04982 resulted in fold decrease of HBeAg greater than the sum of the fold decrease of HBeAg in AD04872 and AD04982 administered individually. For example, Group I (which is the administration of 3.0 mg/kg AD04872 + 2.0 mg/kg AD04982) resulted in a fold decrease of HBeAg at day 15 of 17.0, w'hich is greater than the sum of the fold decrease for Group G (3.0 mg/kg AD04872) of 4.6 plus the fold decrease for Group D (2.0 mg/kg AD04982) of 3.2.
Further, sérum HBV DNA levels w'ere determined for each of the groups in Table 17 from sérum samples collected on days -1, 8, 15,22, 29, and 36, pursuant to the procedure set forth in Example 2, above. Sérum HBV DNA w’as isolated from each animal at each time point. Data are presented in the following Table:
159
Table 20. Average Sérum HBV DNA levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 7 (standard déviation reflected as (+/-)).
Group Day 8 Day 15 Day 22 Day 29
A 1.000 ±0.493 1.000 ±0.358 1.000 ±0.424 1.000 ±0.387
B 0.224 ±0.150 0.263 ±0.185 0.335 ± 0.204 0.449 ±0.108
C 0.358 ±0.207 0.428 ±0.073 0.433 ± 0.220 0.474 ±0.090
D 0.516± 0.163 0.523 ±0.264 0.244 ±0.123 0.241 ± 0.085
E 0.601 ± 0.388 0.319 ±0.125 0.279 ±0.138 0.506 ±0.525
F 0.363 ±0.128 0.374 ±0.197 0.275 ±0.146 0.385 ±0.141
G 0.071 ± 0.032 0.022 ±0.009 0.015 ±0.015 0.025 ± 0.005
H 0.069 ±0.070 0.018 ±0.014 0.019 ±0.020 0.022 ±0.001
I 0.044 ±0.024 0.033 ±0.016 0.017 ±0.012 0.022 ± 0.014
Group Day 36
A 1.000 ±0.326
B 0.603 ± 0.068
C 0.509 ±0.163
D 0.543 ± 0.079
E 0.444 ±0.407
F 0.721 ± 0.043
G 0.058 ±0.030
H 0.047 ±0.021
I 0.058 ±0.051
The data in Table 20 indicate that the RNAi agents examined, both individually and in combination, provided a réduction in HBV DNA levels compared to the PBS group, and further show that the combination of AD04872 (which targets the S ORF) and AD04982 (which targets the X ORF) reduces sérum HBV DNA to a similar degree as an equal amount 10 of AD04872 alone.
Example 8. HBV RNAi Agents in pHBV mice: further dose response and combination studies.
The pHBV mouse model described in Example 2, above, wras used. Mice were divided into 15 various groups as set forth in Table 21, below, and each mouse was administered a single 200 μΐ subcutaneous injection pursuant to the dosing regimen set forth in Table 21 :
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Table 21. Dosing groups of pHBV mice for Example 8.
Group RNAi Agent and Dose Dosing Regimen Number of Animais (n)
1 PBS (no RNAi agent) Single injection on day 1 4
2A 4.0 mg/kg AD04872 + 1.0 mg/kg AD05070 Single injection on day 1 4
2B 4.0 mg/kg AD04872 + 1.0 mg/kg AD05070 Single injection on day 1 4
3A 3.3 mg/kg AD04872 + 1.7 mg/kg AD05070 Single injection on day 1 4
3B 3.3 mg/kg AD04872 + 1.7 mg/kg AD05070 Single injection on day 1 4
4A 3.2 mg/kg AD04872 + 0.8 mg/kg AD05070 Single injection on day 1 4
4B 3.2 mg/kg AD04872 + 0.8 mg/kg AD05070 Single injection on day 1 4
5A 2.7 mg/kg AD04872 + 1.3 mg/kg AD05070 Single injection on day 1 4
5B 2.7 mg/kg AD04872 + 1.3 mg/kg AD05070 Single injection on day 1 4
6A 4.0 mg/kg AD05070 Single injection on day 1 4
6B 4.0 mg/kg AD05070 Single injection on day 1 4
7A 1.7 mg/kg AD05070 Single injection on day 1 4
7B 1.7 mg/kg AD05070 Single injection on day 1 4
8A 0.8 mg/kg AD05070 Single injection on day 1 4
8B 0.8 mg/kg ADOS 070 Single injection on day 1 4
9 1.7 mg/kg AD05148 Single injection on day 1 4
10 2.7 mg/kg AD04872 Single injection on day 1 3
11 1.7 mg/kg AD05147 Single injection on day 1 3
12 4.0 mg/kg AD04872 Single injection on day 1 3
13 1.7 mg/kg AD05149 Single injection on day 1 3
Additionally, the mice are scheduled to be euthanized pursuant to the following schedule:
• Day 11: Euthanize 2 mice from groups 2A, 3A, 4A, 5A, 6A, 7A and 8A, and 5 euthanize one mouse from group 9.
• Day 14: Euthanize 2 mice from groups 2A, 3A, 4A, 5A, 6A, 7A, and 8A.
• Day 21 : Euthanize 2 mice from groups 2B, 3B, 4B, 5B, 6B, 7B, and 8B.
161 • Day 28: Euthanize 2 mice from groups 1,2B, 3B, 4B, 5B, 6B, 7B, and 8B, and ail mice (4) from groups 10 and 12.
Each mouse was given a subcutaneous administration of200 μΐ containing the amount of HBV 5 RNAi agent(s) formuiated in phosphate buffered saline, or 200 pl of phosphate buffered saline without an HBV RNAi agent, as set forth in Table 21. Each of the HBV RNAi agents included N-acetyl-galactosamine targeting ligands conjugated to the 5'-terminal end ofthe sense strand, as shown in Tables 4 and 5. The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. As shown in
Table 14 above, four (4) mice in each group w'ere tested (n=4), except for groups 10, 11,12 and 13, in which three mice were tested (n=3).
Sérum was collected on day -1 prior to administration, and on days 8,14,21 and 28, and sérum
Hepatitis B surface antigen (HBsAg) levels w’ere determined pursuant to the procedure set 15 forth in Example 2, above. Data from the experiment is shown in the following Table:
Table 22. Average HBsAg levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 8 (standard déviation reflected as (+/-)).
Group Number Day 8 Day 14 Day 21 Day 28
1 1.000 ±0.089 1.000 ±0.087 1.000 ±0.132 1.000± 0.138
2A 0.009 ±0.003 0.005 ± 0.001
2B 0.006 ±0.003 0.002 ±0.001 0.004 ±0.001 0.005 ±0.001
3A 0.032 ±0.021 0.009 ±0.004
3B 0.028 ±0.027 0.008 ± 0.006 0.012 ±0.005 0.015 ±0.005
4A 0.036 ±0.020 0.012 ±0.006
4B 0.029 ±0.025 0.010 ±0.008 0.015 ±0.005 0.022 ±0.004
5A 0.027 ±0.014 0.008 ± 0.002
5B 0.027 ±0.013 0.007 ± 0.003 0.019 ±0.004 0.031 ±0.005
6A 0.058 ±0.035 0.069 ±0.039
6B 0.117 ±0.058 0.079 ±0.047 0.145 ±0.082 0.135 ±0.061
7A 0.189 ±0.100 0.084 ±0.029
7B 0.099 ±0.010 0.147 ±0.025 0.267 ±0.048 0.345 ±0.063
8A 0.355 ±0.099 0.366 ±0.069
8B 0.271 ± 0.058 0.334 ±0.060 0.464 ±0.055 0.624 ±0.053
9 0.239 ±0.148 0.179 ±0.127 0.309 ±0.213 0.345 ± 0.225
10 0.018 ±0.009 0.005 ± 0.003 0.005 ± 0.002 0.007 ± 0.003
11 0.129 ±0.068 0.138 ±0.060 0.239 ±0.092 0.315 ±0.119
162
12 0.033 ± 0.022 0.002 ±0.001 0.002 ±0.001 0.002 ± 0.0004
13 0.200 ±0.093 0.239 ±0.114 0.367 ±0.123 0.477 ±0.125
The HBV RNAi agents tested, both alone and in combination, showed a substantial réduction in HBsAg as compared to the PBS control across ail measured time points.
Example 9. RNAi agent delivery.
The pHBV mouse model described in Example 2, above, was used. At day 1, each mouse was administered a single subcutaneous injection of 200 μΐ containing 10 mg/kg (mpk) of an HBV RNAi agent formulated in phosphate buffered saline, or 200 μΐ of phosphate buffered saline without an HBV RNAi agent, to be used as a control. The HBV RNAi agents tested included those having the duplex numbers shown in Table 23, below7, which each included N-acetylgalactosamine targeting ligands conjugated to the 5'-terminal end of the sense strand, as shown in Tables 4 and 5. The injections w7ere performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Three (3) mice in each group were tested (n=3).
Sérum w7as collected prior to administration, and then on day 8, day 15, day 22, and day 29, and sérum Hepatitis B surface antigen (HBsAg) levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment is shown in the following Table:
Table 23. Average HBsAg levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 9 (standard déviation reflected as (+/-)).
RNAi agent HBsAg in sérum at nadir (norm. fraction) %KD at nadir Day of nadir
PBS 1.000 N/A N/A
AD03498 0.087 ± 0.016 91.3% 8
AD03499 0.069 ±0.011 93.1% 15
AD03500 0.095 ±0.031 90.5% 8
AD03501 0.046 ±0.020 95.4% 15
163
Each of the HBV RNAi agents shown in Table 23, aboi’e, included an antisense strand sequence that is at least partially complementaiy to the X ORF at positions 1781-1799 of the HBV genome. Each of the RNAi agents showed a significant knockdown compared to PBS control.
Example 10. HBVRNAiAgents in pHBVmice:further combination studies.
The pHBV mouse model described in Example 2, above, was used. Mice were divided into various groups as set forth in Table 24, below, and each mouse was administered a single 200 μΐ subcutaneous injection pursuant to the dosing regimen set forth in Table 24:
Table 24. Dosing groups of pHBV mice for Example 10.
Group RNAi Agent and Dose Dosing Regimen
A PBS Group I (no RNAi agent) Single injection on day 1 and day 22
B PBS Group II (no RNAi agent) Single injection on day 1 and day 22
C 3.0 mg/kg AD045 85 Single injection on day 1, day 22, day 50, and day 64
D 3.0 mg/kg AD04771 Single injection on day 1 and day 22
E 3.0 mg/kg AD04580 Single injection on day 1, day 22, day 50, and day 64
F 3.0 mg/kg AD04776 Single injection on day 1 and day 22
G 1.5 mg/kg AD04585 + 1.5 mg/kg AD045 80 Single injection on day 1, day 22, day 50, and day 64 ' '
H 1.5 mg/kg AD04771 + 1.5 mg/kg AD04776 Single injection on day 1 and day 22
I 2.0 mg/kg AD04771 + 1.0 mg/kg AD04776 Single injection on day 1 and day 22
J 2.25 mg/kg AD04771 + 0.75 mg/kg AD04776 Single injection on day 1 and day 22
Each mouse was given a subcutaneous administration of200 pl containing the amount of HBV RNAi agent(s) formulated in phosphate buffered saline, or 200 pl of phosphate buffered saline 15 without an HBV RNAi agent, as set forth in Table 24. Each of the HBV RNAi agents included
N-acetyl-galactosamine targeting ligands conjugated to the 5'-terminal end of the sense strand, as shown in Tables 4 and 5. The injections w'ere performed betw'een the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Three (3) mice in each group w'ere tested (n=3).
164
Sérum was collected prior to administration, and then on day -1, day 8, day 15, day 22, day 29, day 36, day' 43, day 50, day 57, and day 64. Sérum Hepatitis B surface antigen (HBsAg) levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment is shown in the following:
Table 25. Average HBsAg levels normalized to pre-treatment and PBS control (Group A used as control) in pHBV mice following administration of HBV RNAi agents from Example 10 (standard déviation reilected as (+/-)).
Group Day 8 Day 15 Day 22
A 1.000 ±0.146 1.000 ±0.095 1.000 ±0.202
B 0.931 ±0.161 1.091 ±0.156 1.132 ±0.259
C 0.071 ± 0.050 0.031 ±0.022 0.024 ±0.013
D 0.134 ±0.035 0.130 ±0.024 0.119 ±0.028
E 0.015 ± 0.001 0.041 ± 0.012 0.087 ±0.015
F 0.197 ±0.081 0.308 ±0.138 0.476 ±0.156
G 0.029 ±0.015 0.069 ±0.029 0.094 ±0.016
H 0.191 ±0.057 0.315 ±0.094 0.420 ±0.126
I 0.153 ±0.050 0.194 ±0.076 0.233 ±0.116
J 0.155 ±0.059 0.177 ±0.067 0.316 ± 0.117
Group Day 29 Day 36 Day 43
A 1.000 ±0.182 1.000 ±0.287 1.000 ±0.298
B 1.417 ±0.414 1.166 ±0.248
C 0.007 ± 0.005 0.004 ±0.003 0.006 ± 0.001
D 0.048 ± 0.023 0.036 ±0.020 0.052 ±0.027
E 0.014 ±0.006 0.021 ±0.011 0.026 ±0.011
F 0.246 ±0.081 0.244 ±0.097 0.179 ±0.061
G 0.023 ± 0.009 0.027 ±0.009 0.037 ±0.013
H 0.200 ±0.080 0.185 ±0.081 0.194 ±0.055
I 0.141 ±0.082 0.133 ±0.051 0.151 ±0.082
J 0.133 ±0.064 0.102 ±0.039 0.129 ±0.050
Group Day 50 Day 57 Day 64
A 1.000 ±0.296 1.000 ±0.394 1.000 ±0.395
B
C 0.015 ± 0.0001 0.002 ±0.001 0.004 ± 0.001
D
E 0.052 ±0.015 0.009 ± 0.002 0.018 ± 0.007
F
G 0.076 ±0.020 0.012 ±0.003 0.020 ±0.007
H
I
165
J
HBV RNAi agents AD04585 and AD04771 were designed to hâve antisense strand sequences that are at least partially complementary to the S open reading frame at positions 257-275 of the HBV genome, as shown in Tables 1 and 2. HBV RNAi agents AD04580 and AD04776 5 were designed to hâve antisense strand sequences that are at least partially complementary to the X open reading frame at positions 1781-1799 of the HBV genome, as shown in Tables 1 and 2 The HBV RNAi agents tested, both alone and in combination, showed a réduction in HBsAg as compared to the PBS control across ail measured time points. Each subséquent dose further reduced the nadir of HBsAg réduction.
Additionally, sérum HBV DNA levels were determined for Group C (3.0 mg/kg AD04585), Group E (3.0 mg/kg AD04580), and Group G (1.5 mg/kg AD04585 + 1.5 mg/kg AD04580) in Table 24, from sérum samples collected on days -1, 8,15,22,29, and 36,43 and 50 pursuant to the procedure set forth in Example 2, above. Sérum HBV DNA was isolated for each animal 15 at each of these time points. Data are presented in the following Table:
Table 26. Average Sérum HBV DNA levels normalized to pre-treatment and PBS Controls (both PBS groups A and B) in pHBV mice following administration of HBV RNAi agents from Example 10 (standard déviation reflected as (+/-)).
Group Day 8 Day 15 Day 22 Day 29
A/B (PBS) 1.000+0.316 1.000 + 0.427 1.000 + 0.428 1.000 + 0.475
C 0.172 + 0.151 0.142 + 0.079 0.252 + 0.132 0.072 + 0.086
E 0.024 + 0.015 0.042 + 0.037 0.449 + 0.184 0.053 + 0.048
G 0.093 ± 0.053 0.083 + 0.037 0.370 + 0.153 0.211 + 0.060
Group Day 36 Day 43 Day 50
A/B (PBS) 1.000 + 0.623 1.000 + 0.532 1.000 + 0.532
C 0.044 + 0.020 0.104 + 0.033 0.156 + 0.016
E 0.012+0.004 0.061 + 0.031 0.161 + 0.019
G 0.048 + 0.022 0.147 + 0.010 0.295 ± 0.041
The data in Table 26 indicate that the HBV RNAi agents examined, both individually and in combination, provided a réduction in HBV DNA levels compared to the PBS group.
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Example 11. HBVRNAiAgents inpHBV mice: combination studies.
The pHBV mouse model described in Example 2, above, was used. Mice were divided into various groups as set forth in Table 27, below, and each mouse was administered a single 200 pl subcutaneous injection pursuant to the dosing regimen set forth in Table 27:
Table 27. Dosing groups of pHBV mice for Example 11.
Group RNAi Agent and Dose Dosing Regimen
A PBS (no RNAi agent) Single injection on day 1
B 3.0 mg/kg AD04962 Single injection on day 1
C 3.0 mg/kg AD04963 Single injection on day 1
D 1.5 mg/kg AD04962 + 1.5 mg/kg AD04963 Single injection on day 1
E 2.0 mg/kg AD04962 + 1.0 mg/kg AD04963 Single injection on day 1
F 2.25 mg/kg AD04962 + 0.75 mg/kg AD04963 Single injection on day 1
G 1.5 mg/kg AD04962 + 1.5 mg/kg AD04963 Single injection on day 1
H .3.0 mg/kg AD04962 + 3.0 mg/kg AD04963 Single injection on day 1
I 1.5 mg/kg AD04962 + 1.5 mg/kg AD04963 Single injection on day 1
J 4.5 mg/kg AD04962 + 4.5 mg/kg AD04963 Single injection on day 1
K 3.0 mg/kg AD04872 Single injection on day 1
L 3.0 mg/kg AD04882 Single injection on day 1
M 3.0 mg/kg AD04885 Single injection on day 1
Each mouse w'as given a subcutaneous administration of200 μΐ containing the amount of HBV RNAi agent(s) formulated in phosphate buffered saline, or 200 pl of phosphate buffered saline 10 without an HBV RNAi agent, as set forth in Table 24. Each of the HBV RNAi agents included
N-acetyl-galactosamine targeting ligands conjugated to the 5'-terminal end of the sense strand, as shown in Tables 4 and 5. The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area Three (3) mice in each group w'ere tested (n=3).
167
Sérum w as collected on day -1 prior to administration, and then on day 8, day 15, day 22, day 29, and day 36 (except for Group L (AD04882) and Group M (AD04885), and sérum Hepatitis B surface antigen (HBsAg) levels w'ere determined pursuant to the procedure set forth in Example 2, above. Data from the experiment is shown in the following Table:
Table 28. Average HBsAg normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 11 (standard déviation reflected as (+/-)).
Group Day 8 Day 15 Day 22
A 1.000 ±0.048 1.000 ±0.144 1.000 ±0.083
B 0.125 ±0.025 0.083 ± 0.014 0.063 ±0.016
C 0.019 ±0.005 0.035 ± 0.008 0.052 ±0.009
D 0.054 ±0.013 0.079 ± 0.009 0.108 ±0.021
E 0.099 ±0.025 0.098 ± 0.053 0.142 ±0.050
F 0.070 ±0.015 0.103 ±0.036 0.140 ±0.020
G 0.041 ±0.021 0.012 ± 0.008 0.021 ±0.013
H 0.020 ±0.006 0.044 ± 0.010 0.062 ±0.019
I 0.077 ±0.017 0.019 ± 0.004 0.004 ±0.001
J 0.012 ±0.002 0.021 ± 0.001 0.032 ±0.002
K 0.045 ±0.014 0.013 ± 0.005 0.008 ±0.005
L 0.106 ±0.020 0.176 ±0.044 0.215 ± 0.082
M 0.275 ± 0.029 0.378 ± 0.080 0.572 ±0.043
Group Day 29 Day 36
A 1.000 ±0.209 1.000 ±0.270
B 0.079 ±0.020 0.096 ±0.007
C 0.087 ±0.014 0.164 ±0.026
D 0.176 ±0.014 0.292 ± 0.030
E 0.223 ±0.082 0.373 ±0.150
F 0.213 ±0.020 0.328 ± 0.034
G 0.031 ±0.013 0.078 ± 0.064
H 0.97 ±0.028 0.160 ±0.060
I 0.008 ±0.001 0.002 ±0.0003
J 0.044 ±0.008 0.069 ± 0.009
K 0.011 ±0.007 0.011 ±0.009
L 0.299 ±0.009
M 0.792 ±0.057
RNAi agent AD04962 was designed to hâve an antisense strand sequence that is at least partially complementary to the S open reading frame at positions 257-275 of the HBV genome, as shown in Tables 1 and 2. RNAi agent AD04872 w'as designed to hâve an antisense strand
168 sequence that is at least partially complementary to the S open reading frame at positions 261279 of the HBV genome, as shown in Tables 1 and 2. RNAi agent AD04963 was designed to hâve an antisense strand sequence that is at least partially complementary to the X open reading frame at positions 1781-1799 of the HBV genome, as shown in Tables 1 and 2. RNAi agents AD04882 and AD04885 were designed to hâve antisense strand sequences that are at least partially complementary' to the X open reading frame at positions 1780-1798 of the HBV genome, as shown in Tables 1 and 2. The HBV RNAi agents shown in Table 9, directly above, each showed a réduction in HBsAg as compared to the PBS control across ail measured timepoints, both individually and in combination. Re-dosing yielded additional HBsAg réduction.
Additionally, Sérum Hepatitis B e-antigen (HBeAg) levels were also assessed for ail groups except Groups L and M. Samples from the mice in each respective group w^re first pooled, and the resulting sérum samples were assayed in singlet. Data from the experiment is shown in the following Table:
Table 29. Average HBeAg levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 11.
Group Day 8 Day 22 Day 29 Day 36
A 1.000 1.000 1.000 1.000
B 0.425 0.291 0.371 0.365
C 0.152 0.170 0.328 0.356
D 0.266 0.249 0.456 0.440
E 0.278 0.295 0.589 0.561
F 0.306 0.291 0.718 0.522
G 0.183 0.138 0.291 0.249
H 0.091 0.131 0.315 0.238
I 0.183 0.052 0.069 0.036
J 0.089 0.114 0.190 0.236
K 0.458 0.172 0.322 0.207
Further, sérum HBV DNA levels w'ere determined for each of the groups in Table 27 from sérum samples collected on days 8, 15, 22, and 29, pursüant to the procedure set forth in Example 2, above. Sérum HBV DNA w'as isolated from each animal at each time point. Data are presented in the following Table:
169
Table 30. Average Sérum HBV DNA levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 7 (standard déviation reflected as (+/-)).
Group Day 8 Day 15 Day 22 Day 29
A 1.000 + 0.232 1.000 + 0.463 1.000 + 0.272 1.000 + 0.205
B 0.577 + 0.219 0.222 + 0.064 0.196 + 0.055 0.261 + 0.117
C 0.165 + 0.051 0.070 + 0.042 0.142 + 0.105 0.228 + 0.174
D 0.343 + 0.125 0.307 + 0.091 0.300 + 0.092 0.356 ± 0.032
E 0.262 + 0.033 0.216 + 0.018 0.227 + 0.028 0.279 + 0.090
F 0.320 + 0.134 0.332 + 0.208 0.344 + 0.209 0.338 + 0.211
G 0.231 + 0.036 0.034 + 0.024 0.069 + 0.039 0.077 ± 0.020
H 0.229 + 0.101 0.155 + 0.121 0.148 + 0.079 0.215 ± 0.035
I 0.281 + 0.129 0.109 + 0.071 0.023 + 0.019 0.011 + 0.009
J 0.078 + 0.050 0.061+0.020 0.074 + 0.029 0.056 ± 0.030
K 0.314 + 0.064 0.119 + 0.043 0.076 + 0.067 0.078 ± 0.095
L 0.295 + 0.077 0.305 + 0.101 0.213 ± 0.088 0.186 + 0.084
M 0.515 + 0.247 0.505 + 0.293 0.488 + 0.318 0.478 + 0.267
The data in Table 30 indicate that the RNAi agents examined, both individually and in combination, provided a réduction in HBV DNA levels compared to the PBS group. Re-dosing yielded addition réduction of HBV DNA.
Example 12. HBVRNAi Agents inpHBV mice.
The pHBV mouse model described in Example 2, above, was used. Mice were divided into various groups as set forth in Table 31, below, and each mouse was administered a single 200 μΐ subcutaneous injection pursuant to the dosing regimen set forth in Table 31:
Table 31. Dosing groups of pHBV mice for Example 12.
Group RNAi Agent and Dose Dosing Regimen
A PBS (no RNAi agent) Single injection on day 1
B 2.0 mg/kg AD04871 Single injection on day 1
C 2.0 mg/kg AD04872 Single injection on day 1
D 2.0 mg/kg AD04874 Single injection on day 1
E 2.0 mg/kg AD04875 Single injection on day 1
F 2.0 mg/kg AD04876 Single injection on day 1
G 2.0 mg/kg AD04881 Single injection on day 1
H 2.0 mg/kg AD04883 Single injection on day 1
170
I 2.0 mg/kg AD04884 Single injection on day 1
Each mouse was given a subcutaneous administration of200 μΐ containing the amount of HBV RNAi agent formulated in phosphate buffered saline, or 200 μΐ of phosphate buffered saline without an HBV RNAi agent, as set forth in Table 24. Each ofthe HBV RNAi agents included 5 N-acetyl-galactosamine targeting ligands conjugated to the 5 '-terminal end of the sense strand, as shown in Tables 4 and 5. The injections w’ere performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Three (3) mice in each group were tested (n=3).
Sérum was collected prior to administration, and then on day 8, day 15, and day 22. Group A (PBS), Group B (2.0 mg/kg AD04871), Group C (2.0 mg/kg AD04872), Group D (2.0 mg/kg AD04874), Group E (2.0 mg/kg AD04875), and Group F (2.0 mg/kg AD04876) also had sérum collected on day 29, day 36, day 43, and day 50. Sérum Hepatitis B surface antigen (HBsAg) levels W'ere determined pursuant to the procedure set forth in Example 2, above. Data from the experiment is showm in the following Table:
Table 32. Average HBsAg normalized to pre-treatment and PBS control in pHBV mice following administration ofHBV RNAi agents from Example 12 (standard déviation reflected as (+/-)).
Group Day 8 Day 15 Day 22 Day 29
A 1.000 ±0.132 1.000 ±0.089 1.000 ±0.080 1.000 ±0.098
B 0.102 ±0.034 0.041 ± 0.021 0.049 ±0.033 0.048 ±0.031
C 0.153 ±0.064 0.064 ±0.032 0.063 ± 0.034 0.042 ±0.017
D 0.123 ±0.022 0.049 ±0.017 0.039 ±0.010 0.023 ± 0.001
E 0.190 ±0.075 0.094 ±0.038 0.107 ±0.061 0.081 ±0.051
F 0.190 ±0.031 0.076 ±0.035 0.084 ±0.038 0.049 ±0.024
G 0.159 ±0.047 0.216 ±0.057 0.235 ±0.151
H 0.508 ±0.078 0.666 ±0.131 0.543 ±0.048
I 0.279 ±0.087 0.357 ± 0.078 0.614 ±0.156
Group Day 36 Day 43 Day 50
A 1.000 ±0.065 1.000 ±0.242 1.000 ±0.224
B 0.054 ±0.038 0.064 ± 0.030 0.092 ±0.025
C 0.049 ±0.017 0.054 ±0.015 0.085 ±0.010
D 0.037 ±0.004 0.037 ±0.010 0.065 ±0.012
E 0.126 ±0.077 0.125 ±0.063 0.170 ±0.079
171
F 0.089 ±0.044 0.082 ± 0.034 0.115 ±0.028
G
H
I
HBV RNAi agents AD04871, AD04872, AD04874, AD04875, and AD04876 were each designed to hâve antisense strand sequences that are at least partially complementary to the S open reading frame at positions 261-279 of the HBV genome, as shown in Tables 1 and 2. Each of these HBV RNAi agents should a substantial réduction in HBsAg compared to PBS control. For example, a single 2 mg/kg dose of each of AD04871 (Group B). AD04872 (Group C) and AD04874 (Group D), and AD04876 (Group F), exhibited a greater than 90% réduction in HBsAg for each of the timepoints measured from day 15 through day 43 compared to control. HBV RNAi agents AD04881, AD04883, AD04884 w'ere each designed to hâve antisense strand sequences that are at least partially complementary to the X open reading frame at positions 1780-1798 of the HBV genome, as shown in Tables 1 and 2.
Example 13. Dose response and combinations of HBVRNAi Agents in XRégion Knockout model mice.
As an alternative means in assessing the effects of the combination of an RNAi agent that includes an antisense strand sequence that is at least partially complementary to a région located in the S ORF of an HBV mRNA, and a second RNAi agent that includes an antisense strand sequence that is at least partially complementary to a région located in the X ORF of an HBV mRNA, a plasmid was generated that included the HBV genome with a knockout of the binding site for HBV RNAi agents that target positions 1780 and 1781, as shown in Tables 1 and 2 (hereinafter referred to as X Région Knockout mice). This model was generated by mutating ten (10) bases in the pHBVl.3 plasmid within the binding site of these RNAi agents. The remainder of the HBV mRNA, including the S-region, remained functional. Thus, in this HBV mouse model, inclusion of an HBV RNAi agent having an antisense strand that targets positions 1780 and 1781 of the HBV genome disclosed herein is expected to be ineffective in silencing expression.
The mice were divided into various groups including those set forth in Table 33, below, and the mice wrere given 200 μΐ subcutaneous injections pursuant to the dosing regimen set forth in the following Table:
172
Table 33. Dosing groups of X Région Knockout mice for Example 13.
Group RNAi Agent and Dose Dosing Regimen Number of Animais (n)
1 PBS (no RNAi agent) Single injection on day 1 4
2 2.0 mg/kg AD04585 + 1.0 mg/kg AD04963 Single injection on day 1 4
3 2.0 mg/kg AD04872 + 1.0 mg/kg AD04963 Single injection on day 1 4
4 2.5 mg/kg AD04585 + 0.5 mg/kg AD04963 Single injection on day 1 4
5 2.5 mg/kg AD04872 + 0.5 mg/kg AD04963 Single injection on day 1 4
6 3.0 mg/kg AD04963 Single injection on day 15 1
Each mouse was given a subcutaneous administration of200 μΐ containing the amount of HBV 5 RNAi agent(s) formulated in phosphate buffered saline, or 200 pl of phosphate buffered saline without an HBV RNAi agent, as set forth in Table 33. Each of the HBV RNAi agents included N-acetyl-galactosamine targeting ligands conjugated to the 5 '-terminal end of the sense strand, as shown in Tables 4 and 5. Tire injections w'ere performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Three (3) mice 10 in each group w'ere tested (n=3).
Sérum w'as collected on day 5, day 8, day 15, day 22, and day 29 and sérum Hepatitis B surface antigen (HBsAg) levels were determined pursuant to the procedure set forth in Example 2, above. Sérum wras also collected for Groups 1 through 5 on days 36 and 43. Data from the experiment is shown in the following Table 34:
Table 34. Average HBsAg normalized to pre-treatment and PBS control in X Région Knockout mice following administration of HBV RNAi agents from Example 13 (standard déviation rellected as (+/-)).
Group Day 8 Day 15 Day 22
1 1.000 + 0.186 1.000 + 0.165 1.000 + 0.132
2 0.061+0.034 0.041 ± 0.035 0.030 + 0.015
3 0.020 + 0.011 0.007 + 0.003 0.003 ± 0.002
4 0.063 ± 0.039 0.022 + 0.011 0.029 + 0.013
173
5 0.027 ± 0.014 0.003 ± 0.003 0.001 ± 0.001
6 0.948 1.360 1.652
Day 29 Day 36 Day 43
1 1.000 ±0.059 1.000 ±0.044 1.000 ±0.045
2 0.051 ±0.029 0.062 ±0.029
3 0.004 ± 0.003 0.008 ± 0.003 0.018 ±0.007
4 0.040 ± 0.022 0.061 ±0.030
5 0.002 ±0.001 0.003 ±0.002 0.014 ±0.006
6 1.831
As expected. Group 6, which was a single dose of 3.0 mgkg of HBV RNAi agent AD04963 and includes an antisense strand that is at least partially complementary to the X open reading frame at positions 1781-1799 of the HBV genome, was unable to provide knockdown of 5 HBsAg. Additionally, each of Groups 2 through 5 provided substantial knockdown of HBsAg compared to PBS control, with both Group 3 and Group 5 exhibiting a greater than 2 log réduction in HBsAg at nadir (day 22).
Example 14. Dose response and combinations of HBVRNAi Agents in XRégion Knockout 10 model mice.
The X Région Knockout mouse model described in Example 13, above, w7as used. Mice were divided into various groups including those set forth in Table 31, below7, and each mouse w7as administered a single 200 pl subcutaneous injection pursuant to the dosing regimen set forth in Table 35:
Table 35. Dosing groups of X Région Knockout mice for Example 14.
Group RNAi Agent and Dose Dosing Regimen
1 PBS (no RNAi agent) Single injection on day 1
2 2.0 mg/kg AD04872 Single injection on day 1
3 2.0 mg/kg AD04872 + 0.7 mg/kg AD05070 Single injection on day 1
4 2.0 mg/kg AD04872 + 1.0 mg/kg AD05070 Single injection on day 1
5 2.0 mg/kg AD04872 + 2.0 mg/kg AD05070 Single injection on day 1
Each mouse w7as given a subcutaneous administration of200 gl containing the amount of HBV RNAi agent(s) formuiated in phosphate buffered saline, or 200 pl of phosphate buffered saline
174 without an HBV RNAi agent, as set forth in Table 35. Each of the HBV RNAi agents included N-acetyl-galactosamine targeting ligands conjugated to the 5 '-terminal end of the sense strand, as shown in Tables 4 and 5. The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Three (3) mice in each group shown in Table 35 were tested (n=3).
Sérum w'as collected on day 1 (pre-dose), day 8, day 15, day 22, and day 29, and sérum Hepatitis B surface antigen (HBsAg) levels w’ere determined pursuant to the procedure set forth in Example 2, above. Data from the experiment is shown in the following Table:
Table 36. Average HBsAg levels normalized to pre-treatment and PBS control in X Région Knockout mice from Example 14.
Group Day 8 Day 15 Day 22 Day 29
1 1.000 ±0.120 1.000 ±0.255 1.000 ±0.224 1.000 ±0.143
2 0.104 ±0.104 0.009 ±0.009 0.005 ±0.004 0.005 ± 0.003
3 0.076 ±0.041 0.010 ±0.009 0.006 ±0.005 0.005 ± 0.005
4 0.036 ±0.008 0.002 ±0.001 0.001 ±0.001 0.002 ±0.001
5 0.019± 0.017 ’ 0.003 ±0.002 0.003 ±0.001 0.004 ± 0.000
Table 36 shows that HBV RNAi agent AD04872 administered alone, and the combination of AD04872 (w'hich includes an antisense strand that is at least partially complementary to the S open reading from at positions 261-279 of the HBV genome) and AD05070 (which includes an antisense strand that is at least partially complementary' to the X open reading frame at positions 1781-1799 of the HBV genome), provided significant knockdown of HBsAg compared to PBS control across each of the time points measured.. Addition of 0.7 mg/kg to 2 mg/kg HBV RNAi agent AD05070 for w'hich there was a mutated target site in this X Région Knockout model did not diminish the activity of the 2 mg/kg HBV RNAi agent AD04872.
Additionally, sérum HBV DNA levels w'ere determined from sérum samples collected on days 8,15, and 22 pursuant to the procedure set forth in Example 2, above. Sérum from each group was pooled and then DNA w'as isolated from the sérum in singlet. Data are presented in the following Table:
175
Table 37. Average Sérum HBV DNA levels normalized to pre-treatment and PBS Controls in X Région Knockout mice following administration of HBV RNAi agents from Example 14 (standard déviation reflected as (-/-)).
Group Day 8 Day 15 Day 22
1 1.000 ±0.007 1.000 ±0.011 1.000 ±0.066
2 0.225 ±0.019 0.022 ±0.001 0.036 ±0.001
3 0.151 ±0.002 0.029 ±0.001 0.042 ±0.003
4 0.140 ±0.006 0.016 ±0.000 0.018 ±0.000
5 0.069 ±0.002 0.018 ±0.003 0.043 ± 0.002
Addition of 0.7 mg/kg to 2 mg/kg HBV RNAi agent AD05070 for which there was a mutated target site in this X Région Knockout model did not diminish the activity' of the 2 mg/kg HBV RNAi agent AD04872.
Example 15. HBVRNAi agents inpHBVmice.
The pHBV mouse model described in Example 2, above, was used. Mice were divided into various groups including those set forth in Table 38, below, and each mouse was administered a single 200 pl subcutaneous injection pursuant to the dosing regimen set forth in Table 38:
Table 38. Dosing groups of pHBV mice for Example 15.
Group RNAi Agent and Dose Dosing Regimen
1 PBS (no RNAi agent) Single injection on day 1
2 2.0 mg/kg AD04776 Single injection on day 1
3 2.0 mg/kg AD05069 Single injection on day 1
4 2.0 mg/kg AD05070 Single injection on day 1
5 2.0 mg/kg AD05071 Single injection on day 1
6 2.0 mg/kg AD05073 Single injection on day 1
7 2.0 mg/kg AD05074 Single injection on day 1
8 2.0 mg/kg AD05075 Single injection on day 1
9 2.0 mg/kg AD05076 Single injection on day 1
10 2.0 mg/kg AD05077 Single injection on day 1
11 2.0 mg/kg AD05078 Single injection on day 1
12 3.0 mg/kg AD04872 + 1.0 mg/kg AD04776 Single injection on day 1
176
13 3.0 mg/kg AD04872 + 1.0 mg/kg AD05069 Single injection on day 1
14 3.0 mg/kg AD04872 + 1.0 mg/kg AD05070 Single injection on day 1
15 3.0 mg/kg AD04872 + 1.0 mg/kg AD05071 Single injection on day 1
16 3.0 mg/kg AD04872 + 1.0 mg/kg AD05073 Single injection on day 1
17 3.0 mg/kg AD04872 + 1.0 mg/kg AD05074 Single injection on day 1
18 3.0 mg/kg AD04872 + 1.0 mg/kg AD05075 Single injection on day 1
19 3.0 mg/kg AD04872 + 1.0 mg/kg AD05076 Single injection on day 1
20 3.0 mg/kg AD04872 + 1.0 mg/kg AD05077 Single injection on day 1
21 3.0 mg/kg AD04872 + 1.0 mg/kg AD05078 Single injection on day 1
Each mouse was given a subcutaneous administration of200 μΐ containing the amount of HBV RNAi agent(s) formulated in phosphate buffered saline, or 200 μΐ of phosphate buffered saline without an HBV RNAi agent, as set forth in Table 38. Each of the HBV RNAi agents included 5 N-acetyl-galactosamine targeling ligands conjugated to the 5'-terminal end ofthe sense strand, as shown in Tables 4 and 5. The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Three (3) mice in each group w'ere tested (n=3).
Sérum was collected on day -1 prior to administration, and then on day 8, day 15, day 22, day 29, day 36, day 43, and day 50. Sérum Hepatitis B surface antigen (HBsAg) levels wære determined pursuant to the procedure set forth in Example 2, above. Data from the experiment is shown in the following Table 39, with Average HBsAg reflecting the normalized average value of HBsAg:
Table 39. Average HBsAg normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 15.
Group Day 8 Day 15 Day 22 Day 29
1 1.000 + 0.119 1.000 + 0.047 1.000 + 0.080 1.000 + 0.027
2 0.339 ± 0.076 0.414 + 0.126 0.385 ± 0.067 0.450 + 0.075
3 0.240 ± 0.096 0.361 ± 0.078 0.446 + 0.073 0.508 + 0.114
177
4 0.081 ±0.026 0.127 ±0.031 0.223 ± 0.057 0.330 ±0.112
5 0.452 ± 0.020 0.431 ±0.126 0.373 ± 0.079 0.383 ± 0.080
6 0.375 ±0.181 0.632 ±0.192 0.463 ±0.117 0.567 ±0.159
7 0.325 ± 0.032 0.438 ±0.125 0.393 ±0.056 0.443 ± 0.096
8 0.155 ±0.031 0.322 ±0.019 0.333 ±0.077 0.463 ± 0.043
9 0.245 ± 0.063 0.467 ± 0.090 0.477 ±0.045 0.562 ±0.049
10 0.120 ±0.062 0.173 ±0.029 0.289 ±0.019 0.331 ±0.042
11 0.128 ±0.042 0.172 ±0.046 0.179 ±0.015 0.215 ± 0.049
12 0.040 ±0.015 0.014 ±0.004 0.014 ±0.006 0.015 ± 0.004
13 0.050 ±0.020 0.015 ±0.011 0.017 ±0.008 0.022 ± 0.009
14 0.020 ±0.011 0.011 ±0.006 0.015 ± 0.006 0.023 ± 0.004
15 0.043 ± 0.005 0.013 ±0.005 0.010 ±0.002 0.011 ±0.004
16 0.021 ± 0.017 0.008 ± 0.004 0.012 ±0.003 0.011 ±0.001
17 0.032 ±0.011 0.009 ±0.003 0.007 ±0.002 0.008 ±0.0003
18 0.023 ± 0.014 0.010 ±0.006 0.009 ±0.006 0.009 ± 0.004
19 0.025 ± 0.006 0.010 ±0.004 0.009 ±0.002 0.010 ± 0.003
20 0.061 ± 0.013 0.027 ± 0.006 0.020 ±0.003 0.029 ± 0.006
21 0.061 ± 0.050 0.013 ±0.010 0.012 ±0.005 0.018 ±0.006
Group Day 36 Day 43 Day 50
1 1.000 ±0.031 1.000± 0.114 1.000 ±0.112
2 0.617 ±0.116 0.643 ±0.154 0.665 ±0.199
3 0.638 ± 0.067 0.743 ±0.015 0.792 ±0.115
4 0.472 ±0.121 0.515 ±0.126 0.689 ±0.167
5 0.591 ±0.159 0.604 ±0.086 0.709 ±0.115
6 0.717 ±0.136 0.686 ±0.194 0.781 ±0.301
7 0.586 ± 0.069 0.775 ±0.143 0.747 ±0.095
8 0.666 ±0.066 0.803 ± 0.096 0.856 ± 0.180
9 0.801 ± 0.047 0.667 ±0.055 0.765 ± 0.208
10 0.640 ±0.059 0.667 ±0.034 0.742 ±0.133
11 0.429 ±0.063 0.383 ± 0.005 0.497 ±0.060
12 0.037 ± 0.013 0.044 ±0.012 0.056 ±0.014
13 0.046 ±0.011 0.055 ±0.010 0.070 ±0.010
14 0.054 ± 0.016 0.070 ±0.018 0.096 ±0.012
15 0.029 ±0.011 0.032 ±0.015 0.051 ±0.020
16 0.033 ± 0.005 0.038 ±0.007 0.062 ±0.004
17 0.021 ± 0.002 0.031 ±0.004 0.061 ± 0.005
18 0.034 ±0.014 0.047 ±0.016 0.079 ±0.017
19 0.028 ± 0.005 0.037 ± 0.006 0.060 ±0.011
20 0.070 ±0.009 0.063 ±0.018 0.097 ±0.018
21 0.040 ±0.012 0.066 ±0.007 0.120 ±0.036
RNAi agents AD04776, AD05069, AD05070, AD05071, AD05073, and AD05074 were each designed to hâve an antisense strand sequence that is at least partially complementary to the X open reading frame at positions 1781-1799 of the HBV genome, as shown in Tables 1 and 2.
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RNAi agents AD05075, AD05076, AD05077, and AD05078 were each designed to hâve antisense strand sequences that are at least partially complementary to the X open reading frame at positions 1780-1798 of the HBV genome, as shown in Tables 1 and 2.
Table 39 shows that HBV RNAi agents AD04776, AD05069, AD05070, AD05071, AD05073, and AD05074 administered alone or their combination with AD04872 (which includes an antisense strand that is at least partially complementary to the S open reading from at positions 261-279 of the HBV genome) provided signifîcant knockdown of HBsAg compared to PBS control across each of the time points measured.
Example 16. HBVRNAi Agents in pHBV mice: dose response and combination studies. The pHBV mouse model described in Example 2, above, w'as used. Mice were divided into various groups as set forth in Table 40, below, and each mouse wzas administered a single 200 μΐ subcutaneous injection pursuant to the dosing regimen set forth in Table 40:
Table 40. Dosing groups of pHBV mice for Example 16.
Group RNAi Agent and Dose Dosing Regimen
1 PBS (no RNAi agent) Single injection on day 1
2 3.2 mg/kg AD04872 Single injection on day 1
3 3.2 mg/kg AD04872 Single injection on day' 1 and day 22
4 3.0 mg/kg AD04872 + 0.8 mg/kg AD05070 Single injection on day 1
5 3.0 mg/kg AD04872 + 0.8 mg/kg AD05070 Single injection on day 1 and day 22
6 3.0 mg/kg AD04872 + 1.0 mg/kg AD05070 Single injection on day 1
7 3.0 mg/kg AD04872 + 1.0 mg/kg AD05070 Single injection on day 1 and day 22
8 2.7 mg/kg AD04872 + 1.3 mg/kg AD05070 Single injection on day 1
9 2.7 mg/kg AD04872 + 1.3 mg/kg AD05070 Single injection on day 1 and day 22
10 2.0 mg/kg AD04872 + 2.0 mg/kg AD04776 Single injection on day 1 and day 22
11 0.8 mg/kg AD05070 Single injection on day 1 and day 22
12 1.3 mg/kg AD05070 Single injection on day 1 and day 22
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Each mouse was given a subcutaneous administration of200 μΐ containing the amount of HBV RNAi agent(s) formulated in phosphate buffered saline, or 200 μΐ of phosphate buffered saline without an HBV RNAi agent, as set forth in Table 40. Each of the HBV RNAi agents included N-acetyl-galactosamine targeting ligands conjugated to the 5'-terminal end of the sense strand, as shown in Tables 4 and 5. The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Six (6) mice in each group were tested (n=6).
Sérum w'as collected prior to administration, and then on day 8, day 15, day 22, and day 29, and sérum Hepatitis B surface antigen (HBsAg) levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment is shown in the following Table 41:
Table 41. Average HBsAg levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 16 (standard déviation reflected as (+/-)).
Group Day 8 Day 15 Day 22 Day 29
1 1.000 ±0.117 1.000 ±0.213 1.000 ±0.169 1.000 ±0.130
2 0.050 ±0.018 0.015 ±0.007 0.011 ±0.005 0.009 ±0.006
3 0.051 ±0.037 0.014 ±0.011 0.010 ± 0.006 0.002 ±0.001
4 0.029 ±0.018 0.010 ±0.006 0.011 ±0.006 0.010 ±0.005
5 0.022 ±0.003 0.007 ±0.001 0.009 ± 0.003 0.001 ±0.001
6 0.027 ±0.012 0.007 ±0.004 0.008 ± 0.005 0.011 ±0.005
7 0.028 ±0.012 0.010 ±0.005 0.009 ± 0.005 0.001 ± 0.000
8 0.033 ±0.016 0.016 ±0.008 0.020 ± 0.009 0.021 ±0.011
9 0.034 ±0.025 0.015 ±0.011 0.018 ±0.013 0.003 ±0.002
10 0.038 ±0.021 0.015 ±0.005 0.019 ± 0.004 0.003 ±0.001
11 0.446 ±0.143 0.376 ±0.120 0.474 ±0.149 0.338 ±0.123
12 0.307 ±0.111 0.257 ±0.122 0.236 ± 0.057 0.138 ±0.031
The HBV RNAi agents tested, both individually and in combination, showed a réduction in HBsAg as compared to the PBS control across ail measured time points. HBsAg expression w'as further reduced in ail groups that w'ere re-dosed on day 22.
Additionally, Sérum Hepatitis B e-antigen (HBeAg) levels w'ere also assessed. For the day 8 measurement, the sérum samples for ail six mice in each group w'ere pooled, and the resulting samples w'ere assayed in singlet. For the day -1, day 15, day 22, and day 29 measurements,
180 the six mice from each group were paired within each group and their respective sérum samples w'ere pooled, forming three subgroups for each group. The sérum samples for each of the three subgroups for each group were then assayed. Data from the experiment is shown in the following Table 42:
Table 42. Average HBeAg levels normalized to pre-treatment and PBS control in pHBV mice following administration ofHBV RNAi agents from Example 16 (standard déviation for days 15, 22, and 29 reflected as (+/-)).
Group Day 8 Day 15 Day 22 Day 29
1 1.000 1.000+0.011 1.000+0.170 1.000+0.173
2 0.510 0.308 + 0.031 0.217 + 0.021 0.226 + 0.035
3 0.488 0.301 ± 0.065 0.283 + 0.081 0.147 + 0.030
4 0.213 0.216 + 0.067 0.192 + 0.029 0.141 + 0.048
5 0.192 0.211 + 0.053 0.216 + 0.088 0.047 + 0.016
6 0.176 0.163 + 0.022 0.238 ± 0.069 0.117 + 0.011
7 0.165 0.175 + 0.046 0.215 + 0.061 0.028 + 0.012
8 0.128 0.166 + 0.065 0.386 ± 0.284 0.167 + 0.118
9 0.172 0.171 + 0.037 0.244 + 0.052 0.032 + 0.010
10 0.180 0.211+0.012 0.283 ± 0.034 0.034 ± 0.001
11 0.634 0.594 + 0.082 0.840 + 0.152 0.271 + 0.029
12 0.486 0.441 ± 0.066 0.804 + 0.096 0.214 + 0.039
The HBV RNAi agents tested, both individually and in combination, showed a réduction in HBeAg as compared to the saline control across ail measured time points. HBeAg expression w'as further reduced in ail groups that were re-dosed on day 22.
Further, sérum HBV DNA levels w'ere determined for each of the groups in Table 40 from 15 sérum samples collected on days -1, 8, 15, and 22, pursuant to the procedure set forth in
Example 2, above. Sérum from each pair of mice was pooled and then DNA w'as isolated from each sérum pool in a single isolation. Data are presented in the following Table:
Table 43. Average Sérum HBV DNA levels normalized to pre-treatment and PBS control in 20 pHBV mice following administration of HBV RNAi agents from Example 16 (standard déviation reflected as (+/-)).
Group Day 8 Day 15 Day 22
1 1.000 + 0.122 1.000 + 0.299 1.000 + 0.241
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2 0.312 ±0.016 0.126 ±0.008 0.087 ±0.018
3 0.264 ± 0.065 0.081 ± 0.023 0.073 ± 0.028
4 0.321 ± 0.254 0.120 ±0.066 0.134 ±0.101
5 0.319 ±0.081 0.108 ±0.038 0.098 ±0.051
6 0.260 ±0.095 0.068 ± 0.010 0.076 ±0.031
7 0.170 ±0.028 0.082 ±0.013 0.062 ±0.018
8 0.188 ±0.020 0.192 ±0.160 0.307 ± 0.309
9 0.242 ±0.003 0.100 ±0.042 0.075 ± 0.028
10 0.322 ±0.028 0.159 ±0.025 0.086 ±0.016
11 1.124 ±0.142 0.742 ±0.127 0.807 ±0.192
12 1.004 ±0.144 0.541 ± 0.340 0.569 ±0.060
The HBV RNAi agents tested, both individually and in combination, showed a réduction in sérum HBV DNA as compared to the saline control across ail measured time points except in groups 11 and 12 that had no réduction in sérum HBV DNA at Day 8.
Example 17. HBVRNAiAgents in inpHBVmice.
The pHBV mouse model described in Example 2, above, was used. Mice were divided into various groups as set forth in Table 44, below, and each mouse was administered a single 200 μΐ subcutaneous injection pursuant to the dosing regimen set forth in Table 44:
Table 44. Dosing groups of pHBV mice for Example 17.
Group RNAi Agent and Dose Dosing Regimen
1 PBS (no RNAi agent) Single injection on day 1
2 5 mg/kg AD04585 + 1 mg/kg AD04963 Single injection on day 1
3 5 mg/kg AD04872 + 1 mg/kg AD04963 Single injection on day 1
4 5 mg/kg AD04585 + 1 mgkg AD04963 Single injection on day 1 and day 8
5 5 mgkg AD04872 + 1 mg/kg AD04963 Single injection on day 1 and day 8
6 2.5 mgkg AD04585 + 0.5 mg/kg AD04963 Single injection on day 1
7 2.0 mg/kg AD04585 + 1.0 mg/kg AD04963 Single injection on day 1
8 2.5 mgkg AD04872 + 0.5 mg/kg AD04963 Single injection on day 1
9 2.0 mg/kg AD04872 + 1.0 mg/kg AD04963 Single injection on day 1
10 5 mgkg AD04872 + 1 mgkg AD04981 Single injection on day 1
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11 2.5 mg/kg AD04872 + 0.5 mg/kg AD04981 Single injection on day 1 and day 8
12 2.5 mg/kg AD04872 + 0.5 mg/kg AD04981 Single injection on day 1
13 2 mg/kg AD04872 + 1 mg/kg AD04981 Single injection on day 1
14 2.5 mg/kg AD04585 + 0.5 mg/kg AD04981 Single injection on day 1
15 2 mg/kg AD04585 + 1 mg/kg AD04981 Single injection on day 1
16 0.5 mg/kg AD04981 Single injection on day 1
Each mouse was given a subcutaneous administration of 200 μΐ containing the amount of HBV RNAi agent(s) formulated in phosphate buffered saline, or 200 μΐ of phosphate buffered saline without an HBV RNAi agent, as set forth in Table 44. Each ofthe HBV RNAi agents included 5 N-acetyl-galactosamine targeting ligands conjugated to the 5 '-terminal end of the sense strand, as shown in Tables 4 and 5. The injections were performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area. Three (3) mice in each group were tested (n=3).
Sérum was collected prior to administration, and then on day 8, day 14, day 21, and day 29 and day 36, and sérum Hepatitis B surface antigen (HBsAg) levels were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment is shown in the following Table 45:
Table 45. Average HBsAg levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 17 (standard déviation reflected as (+/-)).
Group Day 8 Day 14 Day 21 Day 29
1 1.000 ±0.068 1.000 ±0.125 1.000 ±0.152 1.000 ±0.110
2 0.058 ±0.033 0.059 ±0.022 0.085 ±0.023 0.158 ±0.021
3 0.025 ± 0.009 0.014 ± 0.006 0.015 ± 0.008 0.026 ±0.015
4 0.032 ±0.007 0.005 ± 0.001 0.006 ±0.002 0.014 ±0.002
5 0.024 ±0.009 0.003 ± 0.001 0.001 ±0.0004 0.001 ± 0.0005
6 0.063 ± 0.020 0.077 ± 0.013 0.131 ±0.011 0.214 ±0.026
7 0.041 ±0.018 0.059 ±0.017 0.091 ±0.016 0.140 ±0.045
8 0.070 ±0.008 0.046 ±0.016 0.043 ±0.009 0.055 ±0.012
9 0.043 ± 0.006 0.027 ± 0.003 0.064 ±0.017 0.064 ±0.014
10 0.015 ± 0.008 0.005 ± 0.003 0.005 ±0.003 0.005 ± 0.003
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11 0.047 ± 0.014 0.005 ± 0.003 0.003 ± 0.002 0.003 + 0.003
12 0.062 + 0.006 0.025 ± 0.007 0.027 ± 0.005 0.033 ± 0.005
13 0.092 + 0.029 0.050 + 0.021 0.050 + 0.022 0.054 + 0.0019
14 0.310 + 0.180 0.056 + 0.010 0.081 + 0.010 0.112 + 0.0018
15 0.304 + 0.044 0.083 ± 0.021 0.115 + 0.013 0.165 + 0.025
16 1.667 + 0.217 0.416 + 0.163 0.341 + 0.179 0.511 + 0.0011
Group Day 36
1 1.000 + 0.225
2
3 0.049 + 0.019
4
5 0.004 ± 0.0004
6
7
8 0.081 + 0.010
9 0.108 + 0.026
10 0.009 + 0.004
11 0.005 ± 0.003
12 0.060 + 0.014
13 0.094 + 0.027
14
15
16 0.634 + 0.005
The HBV RNAi agent combinations tested showed a réduction in HBsAg as compared to the saline control across ail measured time points. Combinations containing AD04872 showed greater réductions than the équivalent combinations with AD04585 in place of AD04872.
Additionally, sérum HBV DNA levels were determined for sérum samples collected on days 8,14,21, and 29 pursuant to the procedure set forth in Example 2, above. Sérum HBV DNA was isolated from each animal at each time point. Data are presented in the following Table 46:
Table 46. Average Sérum HBV DNA levels normalized to pre-treatment and PBS control in pHBV mice following administration of HBV RNAi agents from Example 17 (standard déviation reflected as (+/-)).
Group Day 8 Day 14 Day 21 Day 29
1 1.000 + 0.280 1.000 + 0.269 1.000 + 0.418 1.000 + 0.383
2 0.136 + 0.068 0.192 + 0.071 0.173 + 0.032 0.292 ± 0.039
3 0.097 + 0.034 0.068 + 0.016 0.076 + 0.034 0.131+0.061
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4 0.061 ± 0.039 0.002 ± 0.001 0.003 ±0.001 0.019 ±0.013
5 0.068 ± 0.025 0.003 ± 0.002 0.0009 ± 0.0003 0.0009 ± 0.0003
6 0.354 ±0.299 0.345 ±0.187 0.522 ± 0.234 0.509 ±0.106
7 0.103 ±0.064 0.291 ± 0.025 0.203 ± 0.043 0.203 ±0.015
8 0.336 ±0.142 0.185 ±0.071 0.183 ±0.065 0.162 ±0.064
9 0.198 ±0.055 0.093 ± 0.023 0.118 ±0.054 0.143 ±0.032
10 0.122 ±0.071 0.024 ± 0.026 0.023 ± 0.020 0.014 ±0.017
11 0.160 ±0.069 0.016 ± 0.023 0.003 ± 0.001 0.005 ±0.004
12 0.158 ±0.039 0.120 ±0.044 0.100 ±0.049 0.091 ±0.034
13 0.190 ±0.038 0.169 ±0.025 0.066 ±0.015 0.081 ±0.015
14 0.434 ±0.136 0.318 ±0.104 0.144 ±0.094 0.240 ±0.029
15 0.358 ±0.185 0.287 ±0.108 0.279 ± 0.080 0.303 ±0.038
16 0.713 ±0.085 0.674 ±0.140 0.496 ±0.128 0.590 ±0.093
The HBV RNAi agent combinations tested showed a réduction in sérum HBV DNA as compared to the saline control across ail measured time points. Combinations containing AD04872 showed greater réductions than the équivalent combinations with AD04585 in place of AD04872. These greater réductions w'ere observed at Day 22 and Day 29.
Example 18. HBV RNAi Agents in a HBV-infected Humanized Mouse Model.
For this study, Male FRG® (génotype Fah -/-/ Rag2 -/-/ I12rg -/- triple knockout mice on a C57BL/6 background (Y ecuris) were transplanted with human hépatocytes when they w’ere 1-2 months old. The human hépatocytes w'ere allowed to repopulate the liver for approximately 6 months with periodicNTBC treatment to discourage growlh of mouse hépatocytes. At 9 months of âge the mice were given an intravenous inoculation of 4 x 108 genomes/kg HBV génotype C, which infected the human hépatocytes. After 2-3 months, sérum HBV DNA levels reached a plateau indicating the human hépatocytes were maximally infected (mouse hépatocytes cannot be infected by HBV). Mice w'ere one year old at the start of treatment with HBV RNAi agents, thus nearing the end of their life span.
Pre-treatment sérum samples w'ere taken on day -10 and day -3. Beginning on day 1, each mouse was administered an oral daily gavage with 0.01 mg/kg Entecavir dissolved in water to inhibit HBV réplication. Daily dosing of Entecavir continued until the day mice were euthanized. Entecavir administration w'as expected to reduce sérum HBV DNA in chronically infected human patients, but not reduce HBsAg.
Mice W'ere divided into various groups including those set forth in Table 47, below:
185
Table 47. Dosing groups of HBV-infected FRG humanized model mice for Example 18.
Group RNAi Agent and Dose Dosing Regimen Terminal Day
A- mouse 1 PBS (no RNAi agent) Single injection on day 1 Euthanized day 21 (unhealthy animal)
A- mouse 2 PBS (no RNAi agent) Single injection on day 1 and day 29 Euthanized day 36
B- mouse 1 4.0 mg/kg AD04872 + 2.0 mg/kg AD05070 Single injection on day 1 and day 29 Euthanized day 36
B- mouse 2 4.0 mg/kg AD04872 + 2.0 mg/kg AD05070 Single injection on day 1 and day 29 Euthanized day 40
C- mouse 1 4.5 mg/kg AD04872 + 1.5 mg/kg AD05070 Single injection on day 1 Euthanized day 15
C- mouse 2 4.5 mg/kg AD04872 + 1.5 mg/kg AD05070 Single injection on day 1 and day 29 Euthanized day 36
C- mouse 3 4.5 mg/kg AD04872 + 1.5 mg/kg AD05070 Single injection on day 1 and day 29 Euthanized day 40
Each mouse was also given a subcutaneous administration of 100 μΐ per 20 grams body weight 5 containing the amount of HBV RNAi agent(s) formulated in phosphate buffered saline, or an equal volume of phosphate buffered saline without an HBV RNAi agent, on day 1 and on day 29 (if still alive on day 29), pursuant to the schedule as set forth in Table 47, directly above. Each of the HBV RNAi agents included N-acetyl-galactosamine targeting ligands conjugated to the 5'-terminal end of the sense strand, as shown in Tables 4 and 5. The injections were 10 performed between the skin and muscle (i.e. subcutaneous injections) into the loose skin over the neck and shoulder area.
Sérum was collected on day 8, day 15, day 22, day 29, day 36, and day 40 and sérum Hepatitis
B surface antigen (HBsAg) levels wrere determined pursuant to the procedure set forth in 15 Example 2, above. Data from the experiment is shown in the following Table:
Table 48. Average HBsAg levels normalized to pre-treatment (day -3) for each individual
HBV-infected humanized FRG model mouse from Example 18.
Group Day 8 Day 15 Day 22 Day 29 Day 36 Day 40
A-l 0.830 0.828 0.932 0.858 1.107
A-2 1.303 1.328
B-l 0.548 0.314 0.272 0.207 0.138
B-2 0.592 0.337 0.243 0.215 0.160 0.175
186
C-l 0.643 0.460 0.415 0.251 0.164
C-2 0.353 0.228 0.182 0.172 0.224 0.216
C-3 0.814 0.674
Additionally, sérum HBV DNA levels were determined from sérum samples collected on days -10,-3, 8,15,22,29,36, and 40, pursuantto the procedure set forth in Example 2, above. Data are presented in the following Table 49:
Table 49. Sérum HBV DNA levels normalized to the average of pre-treatment day -10 and day -3 for each HBV-infected FRG humanized mouse following administration of HBV RNAi agents from Example 14.
Group Day -10 Day-3 Day 8 Day 15 Day 22 Day 29 Day 36 Day 40
A-l 0.883 1.117 0.072 0.038 0.015 0.027 0.060
A-2 1.070 0.930 0.130 0.075
B-l 1.538 0.462 0.032 0.017 0.011 0.006 0.010
B-2 1.350 0.650 0.042 0.018 0.012 0.007 0.008 0.007
C-l 1.348 0.652 0.041 0.020 0.016 0.005 0.004
C-2 1.030 0.970 0.031 0.015 0.006 0.011 0.008 0.008
As expected, administration of Entecavir reduced viral réplication in both the absence and presence of HBV RNAi agents.
Other Embodiments
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims (61)

1. An RNAi agent comprising a sense strand comprising a nucleobase sequence according to any one of SEQ ID NO: 282-284, 286-290, 296, 297, 300, 302, 306-315, and 318-321, and an antisense strand at least partially complementary to the sense strand.
2. The RNAi agent of claim 1, wherein the antisense strand comprises a nucleobase sequence accordingto any one of SEQ IDNO: 152, 155-160, 165-169, 170-171, and 174-181.
3. The RNAi agent of claim 1 or claim 2, wherein at least one nucléotide of the sense strand and/or at least one nucléotide of the antisense strand of the RNAi agent is a modified nucléotide and/or has a modified intemucleoside linkage.
4. The RNAi agent of any one of claims 1-3, wherein the sense strand comprises a sequence according to any one of SEQ ID NO: 204-206,208-212,219-223,226,229,234-247, 251-255, and 273.
5. The RNAi agent of any one of claims 1-4, wherein the antisense strand comprises a sequence according to any one of SEQ ID NO: 72-76, 78, 79, 88-92, 98-100, and 108-130.
6. The RNAi agent of any one of claims 1-5, wherein the sense strand and the antisense strand are each 17 to 30 nucléotides in length.
7. The RNAi agent of any one of claims 1-6, wherein the RNAi agent is conjugated to a targeting ligand.
8. The RNAi agent of claim 7, wherein the targeting ligand comprises N-acetylgalactosamine.
9. The RNAi agent of claim 8, wherein the targeting ligand is (NAG13), (NAG13)s, (NAG18), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), or (NAG39)s.
10. The RNAi agent of any one of claims 7-9, wherein the targeting ligand is conjugated to the sense strand of the RNAi agent.
11. The RNAi agent of any one of claims 1-10, wherein the RNAi agent comprises an antisense strand and a sense strand having modified nucléotide sequences, the RNAi agent having the duplex structure of AD04001 (SEQ ID.NO: 72 and SEQ ID NO: 204); AD04002 (SEQ ID NO: 73 and SEQ ID NO: 204); AD04003 (SEQ ID NO: 74 and SEQ ID NO: 205);
189
AD04004 (SEQ ID NO: 72 and SEQ ID NO: 206); AD04005 (SEQ ID NO: 75 and SEQ ID NO: 212);AD04008 (SEQ ID NO: 78 and SEQ IDD NO: 208); AD04009 (SEQ ID NO: 76 and SEQ ID NO: 209); AD04010 (SEQ ID NO: 79 and SEQ ID NO: 211); AD04422 (SEQ ID NO: 74 and SEQ ID NO: 219); AD04423 (SEQ ID NO: 88 and SEQ ID NO: 205); AD04425 (SEQ ID NO: 89 and SEQ ID NO: 20); AD04426 (SEQ ID NO: 90 and SEQ ID NO: 221); AD04427 (SEQ ID NO: 90 and SEQ ID NO: 222); AD04428 (SEQ ID NO: 91 and SEQ ID NO: 221); AD04429 (SEQ ID NO: 91 and SEQ ID NO: 222); AD04430 (SEQ ID NO: 92 and SEQ ID NO: 223); AD04438 (SEQ ID NO: 98 and SEQ ID NO: 226); AD04439 (SEQ ID NO: 99 and SEQ ID NO: 226); AD04511 (SEQ ID NO: 100 and SEQ ID NO: 229); AD04581 (SEQ ID NO: 108 and SEQ ID NO: 211); AD04583 (SEQ IDNO: 109 and SEQ ID NO: 234); AD04584 (SEQ ID NO: 110 and SEQ ID NO: 234); AD04585 (SEQ ID NO: 111 and SEQ ID NO: 235); AD04586 (SEQ ID NO: 112 and SEQ ID NO: 235); AD04587 (SEQ ID NO: 109 and SEQ ID NO: 236); AD04588 (SEQ ID NO: 110 and SEQ ID NO: 237); AD04590 (SEQ ID NO: 113 and SEQ ID NO: 211); AD04591 (SEQ ID NO: 108 and SEQ ID NO: 238); AD04592 (SEQ ID NO: 74 and SEQ ID NO: 239); AD04593 (SEQ ID NO: 114 and SEQ ID NO: 240); AD04594 (SEQ ID NO: 115 and SEQ ID NO: 240); AD04595 (SEQ ID NO: 116 and SEQ ID NO: 241); AD04596 (SEQ ID NO: 117 and SEQ ID NO: 241); AD04597 (SEQ ID NO: 114 and SEQ ID NO: 242); AD04598 (SEQ ID NO: 115 and SEQ ID NO: 243); AD04599 (SEQ ID NO: 118 and SEQ ID NO: 220); AD04734 (SEQ ID NO: 119 and SEQ JD NO: 235); AD04771 (SEQ ID NO: 120 and SEQ ID NO: 244); AD04772 (SEQ ID NO: 121 and SEQ ID NO: 245); AD04773 (SEQ ID NO: 122 and SEQ ID NO: 244); AD04774 (SEQ ID NO: 123 and SEQ ID NO: 246); AD04775 (SEQ ID NO: 123 and SEQ ID NO: 247); AD04822 (SEQ ID NO: 124 and SEQ ID NO: 244); AD04871 (SEQ ID NO: 125 and SEQ ID NO: 251); AD04872 (SEQ ID NO: 126 and SEQ ID NO: 252); AD04873 (SEQ ID NO: 127 and SEQ ID NO: 252); AD04874 (SEQ ID NO: 128 and SEQ ID NO: 253); AD04875 (SEQ ID NO: 129 and SEQ ID NO: 254); AD04876 (SEQ ID NO: 130 and SEQ ID NO: 255); AD04962 (SEQ ID NO: 111 and SEQ ID NO: 244); or AD05164 (SEQ ID NO: 126 and SEQ ID NO: 273).
12. The RNAi agent of claim 11, wherein the RNAi agent has the duplex structure of AD04580 (SEQ ID NO: 107 and SEQ ID NO: 214).
13. The RNAi agent of claim 11, wherein the RNAi agent has the duplex structure of AD04585 (SEQ ID NO: 111 and SEQ ID NO: 235).
14. The RNAi agent of claim 11, wherein the RNAi agent has the duplex structure of AD04776 (SEQ ID NO: 102 and SEQ ID NO: 248).
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15. The RNAi agent of claim 11, wherein the RNAi agent has the duplex structure of AD04872 (SEQ ID NO: 126 and SEQ ID NO: 252).
16. The RNAi agent of claim 11, wherein the RNAi agent has the duplex structure of AD04962 (SEQ ID NO: 111 and SEQ ID NO: 244).
17. The RNAi agent of claim 11, wherein the RNAi agent has the duplex structure of AD04963 (SEQ ID NO: 107 and SEQ ID NO: 216).
18. The RNAi agent of claim 11, wherein the RNAi agent has the duplex structure of AD04982 (SEQ ID NO: 137 and SEQ ID NO: 248).
19. A combination, comprising a first RNAi agent according to of any one of claims 1-18, and a second RNAi agent comprising a sense strand and an antisense strand for inhibiting expression of a Hepatitis B Virus gene.
20. The combination of claim 19, wherein the first RNAi agent targets the S open reading frame (ORF) of an HBV gene, and the second RNAi agent targets the X open reading frame (ORF) of an HBV gene.
21. A combination comprising a first RNAi agent that targets the S open reading frame (ORF) of an HBV gene and a second RNAi agent that targets the X open reading frame (ORF) of an HBV gene, wherein the first RNAi agent or the second RNAi agent is conjugated to a targeting ligand.
22. The combination of claim 21, wherein the first RNAi agent comprises a sense strand comprising a nucleobase sequence according to any one of SEQ ID NO: 34-48, 282-290, 295-302, 306-315, and 318-321.
23. The combination of claim 21 or claim 22, wherein the first RNAi agent comprises an antisense strand comprising a nucleobase sequence according to any one of SEQ ID NO: 7-21, 152, 155-160, 165-171, 174-181.
24. The combination of any one of claims 21-23, wherein the second RNAi agent comprises a sense strand comprising a nucleobase sequence according to any one of SEQ ID NO: 49-60, 275-281,291-294, 303-305, 316, 317, and 322-334.
25. The combination of any one of claims 21-24, wherein the second RNAi agent comprises an antisense strand comprising a nucleobase sequence according to any one of SEQ ID NO: 22-33, 149-151, 153, 154, 161-164, 172, 173, and 182-194.
26. The combination of any one of claims 21-25, wherein the first RNAi agent and the second RNAi agent are each conjugated to a targeting ligand.
27. The combination of any one of claims 21-26, wherein the targeting ligand comprises N-acetyl-galactosamine.
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28. The combination of claim 27, wherein the targeting ligand is (NAG13), (NAG13)s, (NAG18), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), or (NAG39)s.
29. The combination of any one of claims 21-28, wherein the targeting ligand is conjugated to the sense strand of the first RNAi agent or the sense strand of the second RNAi agent.
30. The combination of any one of claim 21-25, wherein the first RNAi agent comprises a sense strand comprising a sequence according to any one of SEQ ID NO: 204-212, 219-229, 234-247, 251-255, and 273.
31. The combination of any one of claims 21-25 and 30, wherein the first RNAi agent comprises an antisense strand comprising a sequence according to any one of SEQ ID NO: 65, 72-79, 88-100, and 108-130.
32. The combination of any one of claims 21-25, 30, and 31, wherein the second RNAi agent comprises a sense strand comprising a sequence according to any one of SEQ ID NO: 195-203, 213-218, 230-233, 248-250, 256-272, and 274.
33. The combination of any one of claims 21-25 and 30-32, wherein the second RNAi agent comprises an antisense strand comprising a sequence according to any one of SEQ ID NO: 6164, 66-71, 80-87, 101-107, and 131-148.
34. The combination of any one of claims 21-33, wherein the first RNAi agent comprises an antisense strand and a sense strand having modified nucléotide sequences, the first RNAi agent having the duplex structure of AD04001 (SEQ ID NO: 72 and SEQ ID NO: 204); AD04002 (SEQ ID NO: 73 and SEQ ID NO: 204); AD04003 (SEQ ID NO: 74 and SEQ ID NO: 205); AD04004 (SEQ ID NO: 72 and SEQ ID NO: 206); AD04005 (SEQ ID NO: 75 and SEQ ID NO: 212); AD04006 (SEQ ID NO: 76 and SEQ ID NO: 207); AD04007 (SEQ ID NO: 77 and SEQ ID NO: 207); AD04008 (SEQ ID NO: 78 and SEQ IDD NO: 208); AD04009 (SEQ ID NO: 76 and SEQ ID NO: 209); AD04010 (SEQ ID NO: 79 and SEQ ID NO: 211); AD04422 (SEQ ID NO: 74 and SEQ ID NO: 219); AD04423 (SEQ ID NO: 88 and SEQ ID NO: 205); AD04425 (SEQ ID NO: 89 and SEQ ID NO: 20); AD04426 (SEQ ID NO: 90 and SEQ ID NO: 221); AD04427 (SEQ ID NO: 90 and SEQ ID NO: 222); AD04428 (SEQ ID NO: 91 and SEQ ID NO: 221); AD04429 (SEQ ID NO: 91 and SEQ ID NO: 222); AD04430 (SEQ ID NO: 92 and SEQ ID NO: 223); AD04431 (SEQ ID NO: 74 and SEQ ID NO: 224); AD04432 (SEQ ID NO: 74 and SEQ ID NO: 225); AD04433 (SEQ ID NO: 93 and SEQ ID NO: 207);
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AD04434 (SEQ ID NO: 94 and SEQ ID NO: 207); AD04435 (SEQ ID NO: 95 and SEQ ID NO: 207); AD04436 (SEQ ID NO: 97 and SEQ ID NO: 207); AD04437 (SEQ ID NO: 96 and SEQ ID NO: 207); AD04438 (SEQ ID NO: 98 and SEQ ID NO: 226); AD04439 (SEQ ID NO: 99 and SEQ ID NO: 226); AD04440 (SEQ ID NO: 78 and SEQ ID NO: 227); AD04441 (SEQ ID NO: 77 and SEQ ID NO: 227); AD04442 (SEQ ID NO: 77 and SEQ ID NO: 228); AD04511 (SEQ ID NO: 100 and SEQ ID NO: 229); AD04581 (SEQ ID NO: 108 and SEQ ID NO: 211); AD04583 (SEQ ID NO: 109 and SEQ ID NO: 234); AD04584 (SEQ ID NO: 110 and SEQ ID NO: 234); AD04585 (SEQ ID NO: 111 and SEQ ID NO: 235); AD04586 (SEQ ID NO: 112 and SEQ ID NO: 235); AD04587 (SEQ ID NO: 109 and SEQ ID NO: 236); AD04588 (SEQ ID NO: 110 and SEQ ID NO: 237); AD04590 (SEQ ID NO: 113 and SEQ ID NO: 211); AD04591 (SEQ ID NO: 108 and SEQ ID NO: 238); AD04592 (SEQ ID NO: 74 and SEQ ID NO: 239); AD04593 (SEQ ID NO: 114 and SEQ ID NO: 240); AD04594 (SEQ ID NO: 115 and SEQ ID NO: 240); AD04595 (SEQ ID NO: 116 and SEQ ID NO: 241); AD04596 (SEQ ID NO: 117 and SEQ ID NO: 241); AD04597 (SEQ ID NO: 114 and SEQ ID NO: 242); AD04598 (SEQ ID NO: 115 and SEQ ID NO: 243); AD04599 (SEQ ID NO: 118 and SEQ ID NO: 220); AD04734 (SEQ ID NO: 119 and SEQ ID NO: 235); AD04771 (SEQ ID NO: 120 and SEQ ID NO: 244); AD04772 (SEQ ID NO: 121 and SEQ ID NO: 245); AD04773 (SEQ ID NO: 122 and SEQ ID NO: 244); AD04774 (SEQ ID NO: 123 and SEQ ID NO: 246); AD04775 (SEQ ID NO: 123 and SEQ ID NO: 247); AD04822 (SEQ ID NO: 124 and SEQ ID NO: 244); AD04871 (SEQ ID NO: 125 and SEQ ID NO: 251); AD04872 (SEQ ID NO: 126 and SEQ ID NO: 252); AD04873 (SEQ ID NO: 127 and SEQ ID NO: 252); AD04874 (SEQ ID NO: 128 and SEQ ID NO: 253); AD04875 (SEQ ID NO: 129 and SEQ ID NO: 254); AD04876 (SEQ ID NO: 130 and SEQ ID NO: 255); AD04962 (SEQ ID NO: 111 and SEQ ID NO: 244); or AD05164 (SEQ ID NO: 126 and SEQ ID NO: 273).
35. The combination of any one of claims 19-34, wherein the second RNAi agent comprises an antisense strand and a sense strand having modified nucléotide sequences, the second RNAi agent having the duplex structure of AD03498 (SEQ ID NO: 61 and SEQ ID NO: 196); AD03499 (SEQ ID NO: 62 and SEQ ID NO: 195); AD03500 (SEQ ID NO: 63 and SEQ ID NO: 195); AD03501 (SEQ ID NO: 64 and SEQ ID NO: 195); AD03738 (SEQ ID NO: 66 and SEQ ID NO: 197); AD03739 (SEQ ID NO: 67 and SEQ ID NO: 197); AD03967 (SEQ ID NO: 64 and SEQ ID NO: 198); AD03968 (SEQ ID NO: 68 and SEQ ID NO: 198); AD03969 (SEQ ID NO: 64 and SEQ ID NO: 199); AD03970 (SEQ ID NO: 68 and SEQ ID NO: 203); AD03971 (SEQ ID NO: 69 and SEQ ID NO: 199); AD03972 (SEQ ID NO: 64 and SEQ ID NO: 200); AD03973 (SEQ ID NO: 64 and SEQ ID NO: 201); AD03974 (SEQ ID NO: 64 and
193
SEQ ID NO: 202); AD03975 (SEQ ID NO: 70 and SEQ ID NO: 199); AD03976 (SEQ ID NO: 71 and SEQ ID NO: 203); AD03977 (SEQ ID NO: 70 and SEQ ID NO: 201); AD03978 (SEQ IDNO: 70andSEQIDNO: 195); AD04176 (SEQIDNO: 80andSEQIDNO: 213); AD04177 (SEQ ID NO: 64 and SEQ ID NO: 214); AD04178 (SEQ ID NO: 68 and SEQ ID NO: 214); AD04412 (SEQ ID NO: 68 and SEQ ID NO: 215); AD04413 (SEQ ID NO: 68 and SEQ ID NO: 216); AD04414 (SEQ IDNO: 81 and SEQ IDNO: 215); AD04415 (SEQ IDNO: 68 and SEQ ID NO: 217); AD04416 (SEQ ID NO: 82 and SEQ ID NO: 218); AD04417 (SEQ ID NO: 83 and SEQ ID NO: 218); AD04418 (SEQ ID NO: 84 and SEQ ID NO: 218); AD04419 (SEQ IDNO: 85 and SEQ IDNO: 218); AD04420 (SEQ IDNO: 86 and SEQ IDNO: 218); AD04421 (SEQ ID NO: 87 and SEQ ID NO: 218); AD04570 (SEQ ID NO: 68 and SEQ ID NO: 230); AD04571 (SEQ ID NO: 101 and SEQ ID NO: 230); AD04572 (SEQ ID NO: 102 and SEQ ID NO: 230); AD04573 (SEQ ID NO: 103 and SEQ ID NO: 231); AD04574 (SEQ ID NO: 104 and SEQ ID NO: 231); AD04575 (SEQ ID NO: 105 and SEQ ID NO: 232); AD04576 (SEQ ID NO: 106 and SEQ ID NO: 232); AD04577 (SEQ ID NO: 68 and SEQ ID NO: 233); AD04578 (SEQ ID NO: 102 and SEQ ID NO: 232); AD04579 (SEQ ID NO: 71 and SEQ ID NO: 214); AD04580 (SEQ ID NO: 107 and SEQ ID NO: 214); AD04776 (SEQ ID NO: 102 and SEQ ID NO: 248); AD04777 (SEQ ID NO: 106 and SEQ ID NO: 249); AD04778(SEQ ID NO: 106 and SEQ ID NO: 250); AD04823 (SEQ ID NO: 82 and SEQ ID NO: 248); AD04881(SEQ ID NO: 131 and SEQ ID NO: 256); AD04882 (SEQ ID NO: 132 and SEQ ID NO: 257); AD04883 (SEQ ID NO: 133 and SEQ ID NO: 258); AD04884 (SEQ ID NO: 134 and SEQ ID NO: 259); AD04885 (SEQ ID NO: 135 and SEQ ID NO: 260); AD04963 (SEQ ID NO: 107 and SEQ ID NO: 216); AD04981 (SEQ ID NO: 136 and SEQ ID NO: 248); AD04982 (SEQ ID NO: 137 and SEQ ID NO: 248); AD04983 (SEQ ID NO: 138 and SEQ ID NO: 248); AD05069 (SEQ ID NO: 139 and SEQ ID NO: 261); AD05070 (SEQ ID NO: 140 and SEQ ID NO: 262); AD05071 (SEQ ID NO: 141 and SEQ ID NO: 263); AD05072 (SEQ ID NO: 68 and SEQ ID NO: 264); AD05073 (SEQ ID NO: 142 and SEQ ID NO: 265); AD05074 (SEQ ID NO: 143 and SEQ ID NO: 248); AD05075 (SEQ ID NO: 144 and SEQ ID NO: 266); AD05076 (SEQ ID NO: 145 and SEQ ID NO: 267); AD05077 (SEQ ID NO: 146 and SEQ ID NO: 268); AD05078 (SEQ ID NO: 147 and SEQ ID NO: 269); AD05147 (SEQ ID NO: 148 and SEQ ID NO: 270); AD05148 (SEQ ID NO: 140 and SEQ ID NO: 271); AD05149 (SEQ ID NO: 148 and SEQ ID NO: 272); or AD05165 (SEQ ID NO: 140 and SEQ ID NO: 274).
194
36. The combination of any one of claims 19-35, comprising a first RNAi agent having a structure of AD04872 (SEQ ID NO: 126 and SEQ ID NO: 252) and a second RNAi agent having a structure of AD05070 (SEQ ID NO: 140 and SEQ ID NO: 262).
37. The combination of any one of claims 19-36, comprising a first RNAi agent having a structure of AD04872 (SEQ ID NO: 126 and SEQ ID NO: 252) and a second RNAi agent having a structure of AD04982 (SEQ ID NO: 137 and SEQ ID NO: 248).
38. The combination of any one of claims 19-35, comprising a first RNAi agent having a structure of AD04872 (SEQ ID NO: 126 and SEQ ID NO: 252) and a second RNAi agent having a structure of AD04776 (SEQ ID NO: 102 and SEQ ID NO: 248).
39. The combination of any one of claims 19-35, comprising a first RNAi agent having a structure of AD04585 (SEQ ID NO: 111 and SEQ ID NO: 235) and a second RNAi agent having a structure of AD04580 (SEQ ID NO: 107 and SEQ ID NO: 214).
40. The combination of any one of claims 19-39, wherein the ratio of the first RNAi agent to the second RNAi agent in the composition is about 1:1 to about 5:1.
41. The combination of any one of claims 19-40, further comprising lamivudine, tenofovir, tenofovir alafenamide, tenofovir disoproxil, or entecavir.
42. A composition comprising the RNAi agent of any one of claims 71-88 or the combination of any one of claims 19-41, wherein the composition further comprises a pharmaceutically acceptable excipient.
43. The RNAi agent of any one of claims 1-18, the combination of any one of claims 1941, or the composition of claim 42 for use in a method of inhibiting expression of a Hepatitis B Virus gene in a subject.
44. The RNAi agent of any one of claims 1-18, the combination of any one of claims 1941, or the composition of claim 42 for use in a method of treating an HBV infection and/or a disease, disorder, or condition associated with an HBV infection in a subject.
45. The RNAi agent, combination, or composition of claim 44, wherein the disease, disorder, or condition associated with HBV infection is a chronic liver disease or disorder, liver inflammation, fibrotic condition, a proliférative disorder, hepatocellular carcinoma, Hepatitis D virus infection, or acute HBV infection.
46. The combination or the composition of claim 45, wherein the first RNAi agent and the second RNAi agent are for administration to the subject at a ratio of about 1:1 to about 5:1.
47. A method of making an RNAi agent, comprising annealing sense strand comprising a nucleobase sequence according to any one of SEQ ID NO: 282-284, 286-290, 296, 297, 300,
195
302, 306-315, and 318-321, to an antisense strand at least partially complementary to the sense strand.
48. The method of claim 47, wherein the antisense strand comprises a nucleobase sequence according to any one of SEQ IDNO: 152, 155-160, 165-169, 170-171, and 174-181.
49. The method of claim 47 or 48, wherein the sense strand comprises a sequence according to any one of SEQ ID NO: 204-206, 208-212, 219-223, 226, 229, 234-247, 251-255, and 273.
50. The method of any one of claims 47-49, wherein the antisense strand comprises a sequence according to any one of SEQ ID NO: 72-76, 78, 79, 88-92, 98-100, and 108-130.
51. Use of the RNAi agent of any one of claims 1-18 or the combination of any one of claims 19-41, for the manufacture of a médicament for treating a disease, disorder, or condition associated with the HBV infection.
52. The use of claim 51 wherein the disease, disorder, or condition associated with the HBV infection is a chronic liver disease or disorder.
53. The use of claim 51 wherein the disease, disorder, or condition associated with the HBV infection is liver inflammation.
54. The use of claim 51 wherein the disease, disorder, or condition associated with the HBV infection is a fibrotic condition.
55. The use of claim 51 wherein the disease, disorder, or condition associated with the HBV infection is a proliférative disorder.
56. The use of claim 51 wherein the disease, disorder, or condition associated with the HBV infection is hepatocellular carcinoma.
57. The use of claim 51 wherein the disease, disorder, or condition associated with the HBV infection is a Hepatitis D virus infection.
58. The use of claim 51, wherein the composition further is for co-administration to the subject with one or more additional therapeutics.
59. The use of claim 58, wherein the one or more additional therapeutics comprises an antiviral agent.
60. The use of claim 58, wherein the one or more additional therapeutics comprises lamivudine, tenofovir, tenofovir alafenamide, tenofovir disoproxil, or entecavir.
61. The use of claim 58, wherein the RNAi agent or combination is for administration in an effective amount sufficient to reduce the level ofHBsAg, HBeAg, and/or sérum HBV DNA, in a subject by at least about 40% relative to the subject's respective expression level prior to administration of the composition.
OA1201900396 2016-08-04 2017-08-04 RNAi agents for hepatitis B virus infection OA19828A (en)

Applications Claiming Priority (3)

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US62/534,733 2017-07-20
US62/540,639 2017-08-03

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