OA19470A

OA19470A - RNAi agents for hepatitis B virus infection.

Info

Publication number: OA19470A
Application number: OA1201900037
Authority: OA
Inventors: David L. Lewis; David B. Rozema; Darren H. Wakefield; Zhen Ll; Rui ZHU; Christine I. Wooddell; Bruce D. Given; Tao Pei; Lauren J. Almeida
Original assignee: Arrowhead Pharmaceuticals, Inc.
Priority date: 2016-08-04
Filing date: 2017-08-04
Publication date: 2020-10-23

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 are 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

Described herein are RNAi agents for inhibiting expression of Hepatitis B Virus (HBV) (referred to herein as HBV RNAi agents or HBV RNAi tnggers) 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 complémentarité' 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 25 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 30 (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 5 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% complementary⁷ or 100% complementary· to the antisense 15 core stretch sequence. In some embodiments, the sense strand core stretch sequence contains a sequence 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 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 ol at least 16, at least 20 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 25 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 30 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) at 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 complementary 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 complementary' to the corresponding sense strand’s additional nucléotides, if présent.

As used herein, an extension comprises 1, 2, 3, 4, 5, or 6 nucléotides at the 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 complementary 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 complementary⁷ 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 mRNA 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' 10 extension of 1, 2, 3, 4, or 5 nucléotides in length. In other embodiments, an HBV RNAi agent compnses 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 15 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 descnbed 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 20 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 30 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 l, 2, 3, 4, 5, or 6 nucléotides in length. In some embodiments, one or more ofthe sense strand extension nucléotides comprise uracil or adenosine nucléotides or nucléotides which correspond to nucléotides in the HBV mRNA sequence. In some embodiments, the sense strand 5 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 10 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 15 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 20 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 25 RNAi agent sense strand includes the sequence of nucléotides 1-18,1-19,1-20,1-21, 1-22,1-

23, 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, 3-

24, 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, 6-

25. 6-26, 7-24, 7-25, 7-25, 8-25, 8-26 of any of the sequences m 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 ofthe two annealed strands are complementary (form a complementary base-pair). In some 5 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 10 of the two annealed strands from a pair (i.e. do not form an overhang) but are not complementary (i.e. form anon-complementary 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, 15 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 20 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 préserve activity of the compound in cells while at the same time increasing the sérum stability 25 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 30 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 N£ana), 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'-fluoro nucléotide), 2'-deoxy nucléotides (represented herein as dN), 2'-methoxy ethyl (2'-O-2-methoxylethyl) nucléotides (represented herein as NM or 2'-M0E), 2'-amino nucléotides, and 2'-alky 1 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-hydroxyniethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 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-sulfhydiyI, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (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 the nucléotides of an RNAi agent are modified nucléotides. As used herein, anRNAi agent wherein substantially ail of thenucléotides 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 10 strand having two or fewer (i.e., 0,1, or 2) nucléotides m 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-phosphate20 containing 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 25 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 30 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 thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methyl enehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH₂ 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, the 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 HBV 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 IDN0:l)).

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	X ORF
6	CTGTAGGCATAAATTGGTC	1781-1799	X ORF

In some embodiments, an HBV RNAi agent includes an antisense strand wherein position 19 10 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 18ofthe 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' -à 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.

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Genome Position of SEQ ID NO: t	1781-1799 1781-1799 1781-1799 1781-1799
Sense Sequence SEQ ID NO: (5’ 3 ) (19-mer)	57 NUGUAGGCAUAAAUUGGUC 58 NUGUAGGCAUAAAUUGGUU 59 NUGUAGGCAUAAAUUGGUA 60 NUGUAGGCAUAAAUUGGUN
Antisense Sequence SEQ ID NO: (5' > 3’) ( 19-mer) _________	30 GACCAAUUUAUGCCUACAN 31 AACCAAUUUAUGCCUACAN 32 UACCAAUUUAUGCCUACAN 33 NACCAAUUUAUGCCUACAN

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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 5 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 10 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 m 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 15 of the nucléotides in each of the unmodified sequences listed m 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 m 20 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 = cytidine-3'-phosphate;

G = guanosine-3'-phosphate;

U = uridine-3'-phosphate n = any 2'-OMe modified nucléotide a = 2'-O-methyladenosine-3 '-phosphate as = 2'-O-methyladenosine-3'-phosphorothioate c = 2'-O-methylcytidine-3-phosphate _cs = 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 t_s = 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^r-phosporothioate
Cf	= 2'-fluorocytidine-3'-phosphate
5 Cfs	= 2'-fluorocytidine-3'-phosphorothioate
Gf	= 2'-fl uoroguanosine-3'-phosphate
Gfs	= 2'-fl uoroguanosine-3 '-phosphorothioate
Tf	= 2'-fluoro-5'-methyluridine-3'-phosphate
Tfs	= 2'-fluoro-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
Nîana	= 2-F-Arabino nucléotide
NM	= 2'-methoxyethyl nucléotide
AM	= 2'-methoxyethyladenosine-3'-phosphate
AMs	= 2'-methoxyethyladenosine-3'-phosphorothioate
20 TM	= 2'-methoxy ethy Ithy midine-3'-phosphate
TMs	= 2'-methoxyethylthymidine-3'-phosphorothioate
R	= ribitol
(invdN)	= any inverted deoxyribonucleotide (3'-3' linked nucléotide)
(invAb)	= inverted (3'-3' linked) abasic deoxyribonucleotide, see Table 6
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 ordinary 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 (-0H) 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. (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), (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' and/or 3' end of the sequence.

103

Table 3. HBV RNAi Agent antisense strand sequences.

î , . _r , z., SEQ ID Unmodined sequence (5 —> 3 )

UACCAAUUUAUGCCUACAGGCCUUAU 149

UACCAAUUUAUGCCUACAGGCCU 150 |

UACCAAUUUAUGCCUACAGGC 151 |

UGUGAAGCGAAGUGCACACUU 152 |

fÜACCAAUUUAUGCCUACAGCCUCCGC 153 |

UACCAAUUUAUGCCUACAGCCUCCGC 153

UACCAAUUUAUGCCUACAGUU 154 |

[UACCAAUUUAUGCCUACAGGC 151 |

UACCAAUUUAUGCCUACAGGC 151 |

UACCAAUUUAUGCCUACAGUU 154 |

lAUUGAGAGAAGUCCACCACGA 155 I

lAUUGAGAGAAGUCCACCACGA 155 |

[AUUGAGAGAAGUCCACCACUU 156 1

|UUUGAGAGAAGUCCACCACGA 157 |

AAUUGAGAGAAGUCCACCACG 158 |

| AAUUGAGAGAAGUCCACCACG 158 |

|AAUUGAGAGAAGUCCACCAUU 159 1

UAUUGAGAGAAGUCCACCACG 160 |

|UACCAAUUUAUGCCUACAGGU 161 |

lUACCAAUUUAUGCCUACAGUU 154 |

lUACCAAUUUAUGCCUACAGCC 162 |

lUACCAAUUUAUGCCUACAGCCUU 163 |

IÛACCAAUUUAUGCCUACAGCCUC 164 |

™ SEQID Modifîed sequence (5 —> 3 )

us Afs cCfaAfuU fuAfuGfcCfuAfc AfgGfccs us uAu_______61

usAfscCfaAfuUfuAfuGfcCfuAfcAfgGfcscsu ________62 |

us Afs csCfaAfuU fuAfuGfcCfuAfcAfgGfccsu__________63 |

usAfscsCfaAfuUfuAfuGfcCfuAfcAfgGfsc ________64 |

usGfsugaAfgCfGfaaguGfcAfcacsusu 65 |

us Afs cCfaAfuU fuAfuGfcCfuAfc AfgC fcsusccgc________66

vpusAfscCfaAfuUfuAfuGfcCfuAfcAfgCfcsusccgc_____67

usAfscsCfaAfuUfuAfuGfcCfuAfcAfgusu_____________68 |

us Afs es CfaAfuU fu AfuGfcCfu Afc Afggs c ________69

vpusAfscsCfaAfuUfuAfuGfcCfuAfcAfgGfsc _______70

vpusAfscsCfaAfuUfuAfuGfcCfuAfcAfgusu___________71

as U fsusGfaGfaGfaAfgUfcCfaCfcAfcGfsa ____________72

asUfsusGfaGfaGfaAfgUfcCfaCfcAfcgsa 73

asUfsusGfaGfaGfaAfgUfcCfaCfcAfcusu __________74

vpusUfsusGfaGfaGfaAfgUfcCfaCfcAfcGfsa__________75

asAfsusUfgAfgAfgAfaGfuCfcAfcCfaCfsg 76

asAfsusUfgAfgAfgAfaGfuCfcAfcCfacsg 77

asAfsusUfgAfgAfgAfaGfuCfcAfcCfausu ___________78

vpusAfsusUfgAfgAfgAfaGfuCfcAfcCfaCfsg 79___

usAfscsCfaAfuUfuAfuGfcCfuAfcAfgGfsu 80

usAfscCfaAfuUfuAfuGfcCfuAfcAfgsusu 81

us AfscsCfaAfuUfuAfuGfcCfuAfcAfgcsc _______82

us Afs es CfaAfuUfuAfuGfcC fuAfc Afgccusu____________83

usAfscsCfaAfuUfuAfuGfcCfuAfcAfgccusc 84

AS Strand ID

AM03508-AS

AM04441-AS

AM04442-AS

AM04443-AS

AM04661-AS

AM04768-AS

AM04769-AS

AM05011-AS

AM05012-AS

AM05013-AS

AM05014-AS

AM05052-AS

AM05053-AS

AM05054-AS

AM05055-AS

AM05056-AS

AM05057-AS

AM05058-AS

AM05060-AS

AM05351-AS

AM05608-AS

AM05609-AS

AM05610-AS

AM05611-AS

104

CM

CO

S Md

ND

WA

ND nd —H

ND ND

δ

o N0

168

169

Ch NO F^-*

o f—1

o r»

171

154

162

172

173

163

ko 1—M

4

160

174

160

ICI M

o

UACCAAUUUAUGCCUACAGCC_____

UACCAAUUUAUGCCUACAGCCUU

UACCAAUUUAUGCCUACAGCCUC

AUUGAGAGAAGUCCACCACUU_____

UUUGAGAGAAGUCCACCACUU_____

AUUGAGAGAAGUCCACCACGGUU

AUUGAGAGAAGUCCACCACGAGU

UAUUGAGAGAAGUCCACCACG_____

UAUUGAGAGAAGUCCACCACGUU

UAUUGAGAGAAGUCCACCACGAG

UAUUGAGAGAAGUCCACCACGA____

lAGAAAAUUGAGAGAAGUCCAC

[UACCAAUUUAUGCCUACAGUU_____

UACCAAUUUAUGCCUACAGCC

lUACCAAUUUAUGCCUACAGCUU

IUACCAAUUUAUGCCUACAGCCU

[UACCAAUUUAUGCCUACAGCCUU

[UACCAAUUUAUGCCUACAGCCUC

[UACCAAUUUAUGCCUACAGUU_____

[UAUUGAGAGAAGUCCACCACG

| UAUUGAGAGA AGUCC ACC AUU

[UAUUGAGAGAAGUCCACCACG_____

[UAUUGAGAGAAGUCCACCACUU____

[UAUUGAGAGAAGUCCACCACGA

85

86 |

δ

88 |

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asUfsusGfaGfaGfaAfgUfcCfaCfcAf

usAfsusUfgAfgAfgAfaGfuCfcAfcC

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us Afs csCfaAfuuuauGfcC fuAfcAfgi

<2? ,o < ,3 O £ ü g s i U 72 O & < 72 5

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us Afs csCfaAfuuuauGfcC fuAfcAfg<

us AfscsCfaAfuuuauGfcCfuAfcAfg<

us A CscsCfaAfuuuauGfcCfu Afc A fg<

cProus AfscsCfaAfuUfuAfuGfcCfiu

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us AfsusUfgAfgagaaGfuCfcAfcCfai

usAfsusUfgAfgagaaGfuCfcAfcCfa<

us Afs usU fg AfgagaaGfuCfc AfcCfat

CO MO

'613-AS

614-AS

SV-819:

1621-AS

Î623-AS 1

GO < CM c

Î628-AS 1

>631-AS

GO < CM CO NO

CO < co CO ND

CO < 4 CO

>635-AS

Î637-AS

CO < 1 00 CO NO

<

5849-AS

5850-AS

>851-AS

5852-AS

5853-AS

5854-AS

G0 < 1 ir oc

CO < c 'C X

5862-AS

5863-AS

G0 < 00 ir

5865-AS

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160

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157 1

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177 |

165 |

175 |

168 |

175 |

169 |

175 |

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171 |

179

180 |

152 |

181 1

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183 |

184 1

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187 |

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189	154 \|	162	061
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191	192 \|	193 \|	I 1761
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107

SEQ Unmodified sequence (5' —> 3’) _Νθ

UUGCCUGUAGGCAUAAAUUGGUAUT 275

UAUAUGCCUGUAGGCAUAAAUUGGUA 276

GCGGAGGCUGUAGGCAUAAAUUGGTA 277

CUGUAGGCAUAAAUUGGUAUU 278

GCCUGUAGGCAUAAAUUGGUA 279

GCCUGUAGGCAUAAAUUGGTA 280

AACUGUAGGCAUAAAUUGGUA 281

UCGUGGUGGACUUCUCUCAAU 282

AAGUGGUGGACUUCUCUCAAU 283

UCGUGGUGGACUUCUCUCAAT 284

CGUGGUGGACUUCUCUCAAUU 285

AAUGGUGGACUUCUCUCAAUU 286

CGUGGUGGACUUCUCUCAATT 287

GGACUUCUCUCAAUUUUCUAA 288

CGUGGUGGACUUCUCUCAAUA 289

UCGUGGUGGACUUCUCUCAAA 290

lACCUGUAGGCAUAAAUUGGUA 291

CUGUAGGCAUAAAUUGGUA 292

ACUGUAGGCAUAAAUUGGUA 293

Table 4. HBV RNAi agent sense strand sequences.

/ , SEQID Modified sequence (5 —* 3 ) NO

(NAG25)uusgsccuguagGfCfAfuaaauugguaus(inydT)______________195

(NAG25)uauausgsccuguagGfCfAfuaaauuggu(inydA)_____________196

(NAG25)gcggagsgcuguagGfCfAfuaaauuggTM(inydA)____________197

(NAG25)scsuguagGfCfAfuaaauugguauus(inyAb)_______________198

(NAG25)sgsccuguagGfCfAfuaaauugguas(invAb)_________________199

(NAG25)sgsccuguagGfCfAfuaaauuggus(invdA)_________________200

(NAG25)sgsccuguagGfCfAfuaaauugguAMs(invAb)______________201

(NAG25)sgsccuguagGfCfAfuaaauuggTMAMs(inyAb)___________202

|(NAG25)sasacuguagGfCfAfuaaauugguas(invAb)_________________203

(NAG25)suscguggugGfAfCfuucucucaaus(invAb)________________²⁰⁴

(NAG25)sasaguggugGfAfCfuucucucaaus(invAb) ______________205

(NAG25)suscguggugGfAfCfuucucucaAMTMs(invAb)___________206

(NAG25)scsgugguggAfCfUfucucucaauus(invAb)________________207

(NAG25)sasaugguggAfCfUfucucucaauus(invAb)_________________²⁰⁸

(NAG25)scsgugguggAfCfUfucucucaaTMTMs(inyAb)____________209

(NAG25)sgsgacuucuCfUfCfaauuuucuaas(inyAb)_________________²¹⁰

(NAG25)scsgugguggAfCfUfucucucaauas(inyAb)_________________211

(NAG25)suscguggugGfAfCfuucucucaaas(inyAb)________________212

(NAG31)sasccuguagGfCfAfuaaauugguas(inyAb)_________________²13

+ ri Λ a C5 ? S a £ y Ü on c 0 S Q c/j Λ > C • w ω C<) o Z

ÎNAG25)s(invAb)scuguagGfCfAfuaaauugguas(inyAb)____________215

(NAG37)s(invAb)scuguagGfCfAfuaaauugguas(invAb)____________216

(NAG25)s(invAb)sacuguagGfCfAfuaaauugguas(invAb) 217

Strand ID

AM04444-SS

AM04445-SS

AM04767-SS

AM05010-SS

AM05015-SS

AM05016-SS

AM05017-SS

AM05018-SS

AM05019-SS

AM05034-SS

AM05046-SS

AM05047-SS

AM05048-SS

AM05049-SS

AM05050-SS

AM05051-SS

AM05063-SS

AM05064-SS

AM05346-SS

AM05347-SS

AM05606-SS

AM05607-SS

AM05615-SS

108

SEQ ID NO

294

283

295

296

297

298

299

300

301

r·*·* O ce

302

00 (N

303

304

305

306

307

308

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289

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AM06610-SS (NAG37)s(invAb)scuGfuAfgGfCfAfuAfaAfuUfgGfuasuus(invAb) 265 |CUGUAGGCAUAAAUUGGUAUU no

SEQ

(.ε <	□	<	P	<	<	<	<	p	<
>>	UGG	P P P	UGG	§ P	B P	B P	B P	U P P	§
Ο	Ρ	P	P	P	P		P	P	P
ο c	<	<	<	<	P	P	P	P	P
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ο	AG	P <	P <	P <	8	P P	P P	U P	P P
ε	Ρ	P	P	P	<	<	<	P	<
g	0	P	P	P	P	P	P	U	P
X	Ρ	P	P	P	P	P	P	<	P
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ο
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ο θ	Ν0	c	NO	NO	f'*»	p*.	f'*»	f*».	f'···.
w Ζ	η	CM	CM	CM	CM	CM	CM	CM	CM
ζΛ	us(invAb)	as(invAb)	us(invAb)	as(invAb)	as(invAb)		/—s	us (in v Ab)	as (in v Ab)
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ΓΠ	00	on	on	00	00	<	<	3	on
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111

The HBV RNAi agents described herein are formed by annealmg 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 differs 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 (from 5’ end -à 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 ofthe 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% complementanty 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 of the 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 linkinggroup whereinthe targeting group and/or linking group is covalently 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 30 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	AM05018-SS
AD03975	AM05013-AS	AM05015-SS
AD03976	AM05014-AS	AM05019-SS
AD03977	AM05013-AS	AM05017-SS
AD03978	AM05013-AS	ÂM04444-SS
AD04001	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
AD 04176	AM05351-AS	AM05346-SS
AD04177	AM04443-AS	AM05347-SS
AD 04178	AM050H-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
ADi »4428'	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
AD 04776	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
AD04875	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	AMO5855-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
ADOS 148	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 acell expressing an HBV 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 10 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 15 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 20 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 conjugale to which they are attached to improve cell-specific distribution and 25 cell-specific uptake of the conjugale. A targeting group can be monovalent, divalent, trivalent, tetravaient, or hâve higher valency. Représentative targeting groups include, without limitation, compounds with affinity to cell surface molécule, cell receptor ligands, hapten, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with affinity to cell surface molécules. In some embodiments, a targeting group is linked to an RNAi agent 30 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, 10 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 C1 carbon. In some embodiments, the galactose dérivative cluster is a galactose dérivative tnmer (also referred to as tri-antennary galactose dérivative or tri-valent galactose dérivative). In 30 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-antennary galactose dérivative or tetra-valent galactose dérivative). In some embodiments, the galactose dérivative cluster comprises fourN-acetyl-galactosamines.

As used herem, a galactose denvative trimer contains three galactose denvatives, each linked 5 to a central branch point. As used herein, a galactose denvative tetramer contains four galactose denvatives, each linked to a central branch point. The galactose denvatives can be attached to the central branch point through the C-l carbons of the saccharides. In some embodiments, the galactose denvatives 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, 10 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 PE& spacer. The branch point can be any small molécule which permits attachment of three galactose denvatives 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 15 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 denvative tetramer, which can be, for example, an N-acetyl20 galactosamine 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 25 galactose dérivative cluster is comprised of a galactose denvative trimer, which can be, for example, an N-acetyl-galactosamine trimer, or galactose denvative 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 5 facilitâtes covalent linkage of the agent to a targeting group or delivery polymer or delivery 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 10 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 15 (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, 20 alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, 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 unmodifîed, 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 30 to, those depicted in Table 3. Any of the HBV RNAi agent duplexes listed in Table 5, whether modified or unmodifîed, 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.

121

122

123

124

125

126

127

128

129

130

131

132

133

134

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. F or example, in some 5 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

H-H h-h 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), alipid, areversibly modified poly mer or peptide, or areversibly 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 ofthe 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 that

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 of the target mRNA or an inhibition in expression the target gene. In one embodiment, the method includes administering an HBV RNAi agent linked to a targeting ligand as described herein, to a subj ect 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 ot 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 15 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 20 of pharmaceutical compositions is administered to a subject in need of such treatment.

prévention or management. In some embodiments, administration of any of the 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 RNA agent can be used to treat at least one symptomin 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 inore 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 herem. dire 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, the 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 acceptability 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, delivery⁷enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, 10 solvents, sugars, suspendmg agents, sustained release matnces, 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. It 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 many 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 30 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 canbe used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, 15 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 RNA agents can be formulated 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 25 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 30 pharmaceutical compositions. Such additional components include, but are not limited to: antipruntics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc.). It is also envisioned that cells, tissues or isolated organs that express or comprise the herein defined RNA agents may be used as “pharmaceutical compositions.”

140

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 patient, 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 dispensers. The pharmaceutical 25 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 bemg 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 15 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 a therapeutically or effective 20 amount of one or more ofthe 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 25 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 bemg 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 30 cells, and/ortissue ofthe 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 5 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 10 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 sérum HBV DNA in a subject to whom a described HBV RNAi agent has been administered is reduced by 15 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. 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 20 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, non30 limiting examples.

Examples

Exaniple 1. Synthesis ofHBVRNAi 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 5 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 Â or 600Â, obtained from Prime Synthesis, Aston, PA, USA). Ail RNA and 2'-modified phosphoramidites were purchased from 10 Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the following 2'-O-methyl phosphoramidites were used: (5'-O-dimethoxytrityl-N⁶-(benzoyl)-2'-O-rnethyl-adenosine-3'-O(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, 5'-O-dimethoxy-lntyl-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,N15 diisopropylamino) phosphoramidite, and 5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2cyanoethyl-N,N-diisopropylarnmo) 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-diisopropylamino) phosphoramidites were purchased from ChemGenes (Wilmington, MA, USA). Targeting ligand containing 20 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-tetrazole (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 (2'0Me), and 60 sec (2'F). In order 25 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 deproteciion of support bound oligomer. After fmalization of the solid 30 phase synthesis, the diied solid support was treated with a 1:1 volume solution of 40 wt. % methylamine in water and 28% ammoniumhydroxide solution (Aldrich) for 1.5 hours at 30°C. The solution was evaporated and the solid residue was reconstituted in water (see below).

144

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 the 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 water 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 were lyophilized and stored at -15 to -25°C. Duplex concentration was determined by measuring the solution absorbance on aUV-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.

Example 2. pHBV model mice.

Six to eight-week-old female NOD.CB17-Prkdscid/NcrCrl (NOD-SCID) mice were transiently transfected m 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 an HBV RNA agent or control. MC-HBV1.3 is a plasmid-derived minicircle that contains the same terminally redondant human hepatitis B virus sequence HBV 1.3 as in plasmid pHBV1.3 and in the HBV1.3.32 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-HBV1.3 in Ringer’s Solution in a total volume of 10% of the animaTs body weight 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, p 17351737.). 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 allowed to coagulate at ambient température for 20 mm. 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 were 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 were determined with a GS HBsAg EIA 3.0 kit (Bio-Rad Laboratories, Inc., Redmond, WA) as described by the manufacturer. Recombinant HBsAg protein, ayw subtype, also diluted in nonfat milk m 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-treatment’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-treatment” 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 (DiaSonn) 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 account 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 dividmg 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 with éthanol and the entire solution applied to a column. After washing, 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), was used to create a six log standard curve. Samples with 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 5 probes to quantitate HBV and pHCR UbC-SEAP were included in each reaction.

Example 3. HBVRNAi agents in pHBVmodel 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 10 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 havmg the duplex numbers shown in Table 7, below. The injections were performed between the skin 15 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, and day 29, and sérum Hepatitis B surface antigen (HBsAg) levels were determined pursuant to the procedure set forth in Example 2, 20 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 AD04178, 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 5 genome shown in Tables 1 and 2, above. Each of the HBV RNA 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 was pooled and then DNA was 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 RNA 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 was 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 ofthe sense strand, as shown m Tables 4 and 5. The HBV RNAi agents administered included those listed m

Table 9, below. 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, 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
AD045 84	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, AD04591, AD04434, AD04583, AD04584, AD04585, AD04586, AD04588, and AD04438 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. The HBV RNAi agents shown in Table 9, directly above, each showed substantiel 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.

150

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 5 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 reflected 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 HB V RNAi 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 w'eekly 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 weeklv 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 of 200 μΐ containing the amount of HBV RNAi agent(s) formulated 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 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 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 AD04585 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 m 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 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 14. Each of the 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 on day -I pnor 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 5 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 complementaiy to theX ORF at positions 1781-1799 ofthe HBV genome, as shown in Tables 1 and 2), which 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 10 targets m the X ORF of the HBV genome, similarly showed 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 15 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 RNA 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 in pHBVmice: additional dose response and combination 5 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 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 17. Each of the HBV RNAi agents included 15 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 20 day 29 (n=3).

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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 showed a réduction in HBsAg as compared to the PBS control across ail measured time points.

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 5 calculated as normalized HBeAg level of the control (PBS) group/normalized HBeAg level of the respected RNAi agent(s) group (i.e., 1.000/HBeAg level). The data m 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 of the HBV genome) with AD04982 (which includes an antisense strand sequence that is at least partially 10 complementary to the X ORF at positions 1781-1799 of the HBV genome). showed 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 1he sum of the fold decrease of HBeAg in AD04872 and AD04982 administered 15 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, which 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 were 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 was 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: fiirther dose response and combination studies.

The pHBV mouse model described in Example 2, above, was used. Mice were divided into 15 various groups as set forth in Table 21, below, and each mouse was administered a single 200 μΐ subeutaneous 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 AD05070	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.

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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 of 200 μΐ containing the amount ofHBV 5 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 21. 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. As shown in 10 Table 14 above, four (4) mice in each group were 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 were 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 ofHBV 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

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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 RNA 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 RNA agents tested included 10 those having the duplex numbers shown in Table 23, below, 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 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 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, above, included an antisense strand sequence that is at least partially complementary 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. HBVRNAi Agents 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 AD04585	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 AD04580	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 of 200 μΐ containing the amount ofHBV RNAi agent(s) formulated in phosphate buffered saline, or 200 μΐ of phosphate buffered saline 15 without an HBV RNAi agent, as set forth in Table 24. Each of the HBV RNAi agents included

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 m 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 reflected 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
1	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

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 w⁷as 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 m 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.

166

Example 11. HBVRNAi Agents in pHBVmice: 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 μΐ subeutaneous 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 was given a subeutaneous administration of 200 μΐ containing the amount ofHBV RNAi agent(s) formulated in phosphate buffered saline, or 200 μΐ 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. subeutaneous injections) into the loose skin over the neck and shoulder area. Three (3) mice in each group were tested (n=3).

167

Sérum was 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 were 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 was 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 5 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 RNA agents shown m 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 10 réduction.

Additionally, Sérum Hepatitis B e-antigen (HBeAg) levels were also assessed for ail groups except Groups L and M. Samples from the mice m each respective group were first pooled, and the resulting sérum samples were assayed in singlet. Data from the experiment is shown 15 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 were determined for each of the groups in Table 27 from sérum samples collected on days 8, 15, 22, 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:

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 ofHBV DNA.

Example 12. HBV RNAi Agents in pHBV 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 of 200 μΐ containing the amount ofHBV 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 of the 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 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 were determined pursuant to the procedure set forth in Example 2, above. Data from the experiment is shown 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 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.

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 pHBV 1.3 plasmid within the binding srte 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 posrtions 1780 and 1781 of the HBV genome drsclosed herein is expected to be ineffectrve in silencing expression.

The mice were divided into various groups including those set forth in Table 33, below, and the mice were given 200 μΐ subcutaneous injections pursuant to the dosing regimen set forth in the following Table:

172

Table 33. Dosing groups ofX 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 of 200 pl containing the amount of HBV 5 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 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. 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 10 in each group were tested (n=3).

Sérum was 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 was 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 reflected 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 mg/kg 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

HBsAg. Additionally, each of Groups 2 through 5 provided substantiel 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, was used. Mice were divided into various groups including those 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 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 mgkg AD04872 + 1.0 mg/kg AD05070	Single injection on day 1
5	2.0 mgkg AD04872 + 2.0 mg/kg AD05070 o o	Single injection on day 1

Each mouse was given a subcutaneous administration of 200 μΐ containing the amount ofHBV

RNAi agent(s) formulated in phosphate buffered saline, or 200 μΐ 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, subeutaneous 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 was collected on day 1 (pre-dose), 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 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 15 AD04872 (which includes an antisense strand that is at least partially complementary to the S open readmg 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 20 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.

Additionally, sérum HBV DNA levels were 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 25 was pooled and then DNA was 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 m 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 inpHBV mice.

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 μΐ subcutaneous injection pursuant to the dosing regimen set forth in Table 38:

Table 38. Dosing groups of pHBV mice for Example 15.

Group	RNA 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 of 200 μΐ containing the amount ofHBV 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 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) mrce in each group were tested (n=3).

Sérum was collected on day -1 prior to admrnistration, 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 were 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 ofHBV 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

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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.

178

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 significant 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, was used. Mice were divided into various groups as set forth in Table 40, below, and each mouse was 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 mgkg AD04872	Single injection on day 1
3	3.2 mg/kg AD04872	Single injection on day 1 and day 22
4	3.0 mgkg AD04872 + 0.8 mgkg AD05070	Single injection on day 1
5	3.0 mg'kg AD04872 + 0.8 mgkg AD05070	Single injection on day 1 and day 22
6	3.0 mgkg 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 mgkg AD05070	Single injection on day 1
9	2.7 mgkg 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

179

Each mouse was given a subcutaneous administration of 200 μΐ containing the amount ofHBV 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, 5 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 was collected prior to administration, and then on day 8, day 15, day 22, and day 29, 10 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	l.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 was further reduced in ail groups that were re-dosed on day 22.

Additionally, Sérum Hepatitis B e-antigen (HBeAg) levels w^rere also assessed. For the day 8 measurement, the sérum samples for ail six mice in each group were pooled, and the resulting samples w^zere assayed m 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 were 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 of HBV 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 m HBeAg as compared to the saline control across ail measured time points. HBeAg expression was further reduced in ail groups that were re-dosed on day 22.

Further, sérum HBV DNA levels were 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 was 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. HBVRNAi Agents 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 mg/kg AD04963	Single injection on day 1 and day 8
5	5 mg/kg AD04872 + 1 mg/kg AD04963	Single injection on day 1 and day 8
6	2.5 mg/kg 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 mg/kg 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 mg/kg AD04872 + 1 mg/kg 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 of200 μΐ containing the amount ofHBV 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 of the 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 ofHBV 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 RNA 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 m place 5 of AD04872. These greater réductions were 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 (Yecuris) were transplanted with human hépatocytes when they were

1-2 months old. The human hépatocytes were allowed to repopulate the liver for approximately 6 months with periodic NTBC treatment to discourage growth of mouse hépatocytes. At 9 months of âge the mice were given an intravenous inoculation of 4 x 10^sgenomes/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 were one year old at the start of treatment with HBV RNAi agents, thus nearing the end of their life span.

Pre-treatment sérum samples were 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 were 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-tenninal 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 were 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, pursuant to the procedure set forth in Example 2, above. Data are presented m 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 daims. Other aspects, advantages, and modifications are within the scope of the following daims.

Claims

1. An RNAi agent for inhibiting expression of a Hepatitis B Virus gene comprising an antisense strand and a sense strand, wherein the antisense strand comprises a sequence having at least about 85% identity to any of the antisense strand sequences selected from the group consisting of SEQ ID NOs: 7-33 and 149-194.

2. The RNAi agent of claim 1, wherein the antisense strand comprises any of the antisense strand sequences selected from the group consisting of SEQ ID NOs: 7-33 and 149-194.

3. The RNAi agent of claim 1 or claim 2, wherein at least one nudeotide of the sense strand and/or at least one nudeotide of the antisense strand of the RNAi agent is a modified nudeotide and/or has a modified internucleoside linkage.

4. The RNAi agent of any of claims 1-3, wherein the sense strand and the antisense strand are each 21-26 nucléotides in length.

5. The RNAi agent of any of claims 1-4, wherein the sense strand comprises at least 16 contiguous nucléotides of any of the sense strand sequences selected from the group consisting of SEQ ID NOs: 34-60 and 275-328 , and wherein the sense strand has a région of at least about 85% complementarity over the contiguous 16 nucléotides to the antisense strand.

6. The RNAi agent of any of claims 1-5, wherein ail or substantially ail of the nucléotides in both the sense strand and the antisense strand of the RNAi agent are modified nucléotides.

7. The RNAi agent of claim 1, wherein the antisense strand comprises the nudeotide sequence of any of the modified sequences selected from the group consisting of SEQ IDNOs: 14-194.

8. The RNAi agent of claim 1 or claim 7, wherein the sense strand comprises the nudeotide sequence of any of the modified sense sequences selected from the group consisting of SEQ ID NOs: 275-328.

9. A composition for inhibiting the expression of a HBV gene comprising an RNAi agent of any of claims 1-8, wherein the RNAi agent is conjugated to a targeting ligand.

10. The composition of claim 9, wherein the targetmg ligand comprises N-acetylgalactosamine.

11. The composition of claim 10, wherein the targetmg 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,

188

AMENDED SHEET (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.

12. The composition of any of claims 9-11, wherein the targeting ligand is conjugated to the sense strand of the RNAi agent.

13. The composition of any of claims 9-12, wherein the composition further comprises a pharmaceutically acceptable excipient.

14. The composition of any of claims 9-13, wherein the RNAi agent has the duplex structure of AD03498; AD03499; AD03500; AD03501; AD03738; AD03739; AD03967; AD03968; AD03969; AD03970; AD03971; AD03972; AD03973; AD03974; AD03975; AD03976; AD03977; AD03978; AD04001; AD04002; AD04003; AD04004; AD04005; AD04006; AD04007; AD04008; AD04009; AD04010; AD04176; AD04177; AD04178; AD04412; AD04413; AD04414; AD04415; AD04416; AD04417; AD04418; AD04419; AD04420; AD04421; AD04422; AD04423; AD04425; AD04426; AD04427; AD04428; AD04429; AD04430; AD04431; AD04432; AD04433; AD04434; AD04435; AD04436; AD04437; AD04438; AD04439; AD04440; AD04441; AD04442; AD04511; AD04570; AD04571; AD04572; AD04573; AD04574; AD04575; AD04576; AD04577; AD04578; AD04579; AD04580; AD04581; AD04583; AD04584; AD04585; AD04586; AD04587; AD04588; AD04590; AD04591; AD04592; AD04593; AD04594; AD04595; AD04596; AD04597; AD04598; AD04599; AD04734; AD04771; AD04772; AD04773; AD04774; AD04775; AD04776; AD04777; AD04778; AD04822; AD04823; AD04871; AD04872; AD04873; AD04874; AD04875; AD04876; AD04881; AD04882; AD04883; AD04884; AD04885; AD04962; AD04963; AD04981; AD04982; AD04983; AD05069; AD05070; AD05071; AD05072; AD05073; AD05074; AD05075; AD05076; AD05077; AD05078; AD05147; AD05148; AD05149; AD05164; or AD05165.

15. The composition of claim 14, wherein the RNAi agent has the duplex structure of AD04580; AD04585; AD04776; AD04872; AD04962; AD04963; AD04982; or AD05070.

16. The composition of any of claims 9-15, wherein the composition further comprises a second RNAi agent comprising a sense strand and an antisense strand for inhibiting expression of a Hepatitis B Virus gene.

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AMENDED SHEET

17. The composition of claim 16, wherein the second RNAi agent comprises an antisense sequence having at least about 85% identity to any of the antisense strand sequences selected from the group consisting of SEQ ID NOs: 7-33 and 149-194.

18. The composition of claim 17, wherein the antisense strand of the second RNAi agent comprises at least 16 contiguous nucléotides of any of the antisense sequences selected from the group consisting of SEQ ID NOs: 7-33 and 149-194.

19. The composition of claim 18, wherein the antisense strand of the second RNAi agent comprises the nucléotides at positions 2 through 18 (5'->3Q ofany ofthe antisense strand sequences selected from the group consisting of SEQ ID NOs: 7-33 and 149-194.

20. The composition of any of claims 16-19, 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. The composition of any of claims 16-19, wherein the first RNAi agent targets the X ORF of an HBV gene, and the second RNAi agent targets the S ORF of an HBV gene.

22. The composition of any of claims 16-20, wherein the first RNAi agent is selected from the group consisting of: AD04001; AD04002; AD04003; AD04004; AD04005; AD04006; AD04007; AD04008; AD04009; AD04010; AD04422; AD04423; AD04425; AD04426; AD04427; AD04428; AD04429; AD04430; AD04431; AD04432; AD04433; AD04434; AD04435; AD04436; AD04437; AD04438; AD04439; AD04440; AD04441; AD04442; AD04511; AD04581; AD04583; AD04584; AD04585; AD04586; AD04587; AD04588; AD04590; AD04591; AD04592; AD04593; AD04594; AD04595; AD04596; AD04597; AD04598; AD04599; AD04734; AD04771; AD04772; AD04773; AD04774; AD04775; AD04822; AD04871; AD04872; AD04873; AD04874; AD04875; AD04876; AD04962; and AD05164; and the second RNAi agent is selected from the group consisting of: AD03498; AD03499; AD03500; AD03501; AD03738; AD03739; AD03967; AD03968; AD03969; AD03970; AD03971; AD03972; AD03973; AD03974; AD03975; AD03976; AD03977; AD03978; AD04176; AD04177; AD04178; AD04412; AD04413; AD04414; AD04415; AD04416; AD04417; AD04418; AD04419; AD04420; AD04421; AD04570; AD04571; AD04572; AD04573; AD04574; AD04575; AD04576; AD04577; AD04578; AD04579; AD04580; AD04776; AD04777; AD04778; AD04823; AD04881; AD04882; AD04883; AD04884; AD04885; AD04963; AD04981; AD04982; AD04983; AD05069; AD05070; AD05071; AD05072; AD05073; AD05074; AD05075; AD05076; AD05077; AD05078; AD05147; AD05148; AD05149; and AD05165.

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AMENDED SHEET

23. The composition of any of claims 16-20 or 22, wherein the first RNAi agent is AD04872 and the second RNAi agent is AD05070.

24. The composition of any of claims 16-20 or 22, wherein the first RNAi agent is AD04872 and the second RNAi agent is AD04982.

25. The composition of any of claims 16-20 or 22, wherein the first RNAi agent is AD04872 and the second RNAi agent is AD04776.

26. The composition of any of claims 16-20, wherein the first RNAi agent is AD04585 and the second RNAi agent is AD04580.

27. The composition of any of claims 9-26, further comprising one or more additional therapeutics.

28. The composition of claim 27, wherein the additional therapeutic is lamivudine, tenofovir, tenofovir alafenamide, tenofovir disoproxil, or entecavir.

29. The composition of claim 27, wherein the additional therapeutic is interferon.

30. A method for inhibiting expression of a Hepatitis B Virus gene in a subject, comprising administering to the subject the RNAi agent of any of claims 1-9.

31. A method for inhibiting expression of a Hepatitis B Virus gene in a subject, comprising administering to the subject the composition of any of claims 9-29.

32. The method of claim 30 or claim 31, wherein the subject has or is at risk of having an HBV infection.

33. A method for treating HBV infection and/or a disease, disorder, or condition associated with HBV infection in a subject, comprising administering to a subject an effective amount of the RNAi agent of any of claims 1-8.

34. A method for treating HBV infection and/or a disease, disorder, or condition associated with HBV infection in a subject, comprising administering to a subject an effective amount of the composition of any of claims 9-29.

35. The method of claim 31, 32, or 34, wherein the composition comprises an effective amount of two RNAi agents for inhibiting the expression of a Hepatitis B Virus gene.

36. The method of any of any of claims 31, 32, 34, or 35, wherein the effective amount of the composition is sufficient to reduce the level of HBsAg in a subject by at least about 40% relative to the subject’s HBsAg expression level prior to administration of the composition.

37. The method of any of any of claims 31, 32, 34, or 35, wherein the effective amount of the composition is sufficient to reduce the level of HBeAg in a subject by at least about

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AMENDEDSHEET

40% relative to the subject’s HBeAg expression level prior to administration of the composition.

38. The method of any of any of claims 31, 32, 34, or 35, wherein the effective amount of the composition is sufficient to reduce the sérum HBV DNA level in a subject by at least about 40% relative to the subject’s sérum HBV DNA level prior to administration of the composition.

39. The method of claims 33 or 34, 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.

40. The method of any of claims 31, 32, 34, 35, 36, 37, 38, or 39, wherein the first RNAi agent and the second RNAi agent are administered in a ratio of about 1:1.

41. The method of any of claims 31, 32, 34, 35, 36, 37, 38, or 39, wherein the first RNAi agent and the second RNAi agent are administered in a ratio of about 2:1.

42. The method of any of claims 31, 32, 34, 35, 36, 37, 38, or 39, wherein the first RNAi agent and the second RNAi agent are administered in a ratio of about 3:1.

43. The method of any of claims 31, 32, 34, 35, 36, 37, 38, or 39, wherein the first RNAi agent and the second RNAi agent are administered in a ratio of about 4:1.

44. The method of any of claims 31, 32, 34, 35, 36, 37, 38, or 39, wherein the first RNAi agent and the second RNAi agent are administered in a ratio of about 5:1.

45. The method of any of claims 31, 32, 34, 35, 36, 37, 38, or 39, wherein the effective amount of the first RNAi agent is between about 0.5 mg/kg and about 5 mg/kg, and the effective amount of the second RNAi agent is between about 0.5 mg/kg and about 5 mg/kg.

46. The method of claim 30, wherein the effective amount of the RNAi agent is between about 0.5 mg/kg and about 5 mg/kg.

47. Use of the RNAi agent of any of claims 1 -8 for the treatment of HBV infection.

48. Use of the composition of any of claims 9-29 for the treatment ofHBV infection.

49. Use of the composition of any of claims 9-29 for the manufacture of a médicament for treatment of HBV infection.

50. An RNAi agent for inhibiting expression of a Hepatitis B Virus gene comprising an antisense strand and a sense strand, wherein the RNAi agent has a duplex structure comprising a sense strand or an antisense strand that is identical or substantially identical

192

AMENDED SHEET to the respective sense or antisense strand in the duplex structure from the group consisting of: AD03498; AD03499; AD03500; AD03501; AD03738; AD03739; AD03967; AD03968; AD03969; AD03970; AD03971; AD03972; AD03973; AD03974; AD03975; AD03976; AD03977; AD03978; AD04001; AD04002; AD04003; AD04004; AD04005; AD04006; AD04007; AD04008; AD04009; AD04010; AD04176; AD04177; AD04178; AD04412; AD04413; AD04414; AD04415; AD04416; AD04417; AD04418; AD04419; AD04420; AD04421; AD04422; AD04423; AD04425; AD04426; AD04427; AD04428; AD04429; AD04430; AD04431; AD04432; AD04433; AD04434; AD04435; AD04436; AD04437; AD04438; AD04439; AD04440; AD04441; AD04442; AD04511; AD04570; AD04571; AD04572; AD04573; AD04574; AD04575; AD04576; AD04577; AD04578; AD04579; AD04580; AD04581; AD04583; AD04584; AD04585; AD04586; AD04587; AD04588; AD04590; AD04591; AD04592; AD04593; AD04594; AD04595; AD04596; AD04597; AD04598; AD04599; AD04734; AD04771; AD04772; AD04773; AD04774; AD04775; AD04776; AD04777; AD04778; AD04822; AD04823; AD04871; AD04872; AD04873; AD04874; AD04875; AD04876; AD04881; AD04882; AD04883; AD04884; AD04885; AD04962; AD04963; AD04981; AD04982; AD04983; AD05069; AD05070; AD05071 ; AD05072; AD05073; AD05074; AD05075; AD05076; AD05077; AD05078; AD05147; AD05148; AD05149; AD05164; or AD05165.

51. The RNAi agent of claim 50, wherein the RNAi agent has a duplex structure comprising a sense strand that is identical or substantially identical to the respective sense in the duplex structure from the group consisting of: AD03498; AD03499; AD03500; AD03501; AD03738; AD03739; AD03967; AD03968; AD03969; AD03970; AD03971; AD03972; AD03973; AD03974; AD03975; AD03976; AD03977; AD03978; AD04001; AD04002; AD04003; AD04004; AD04005; AD04006; AD04007; AD04008; AD04009; AD04010; AD04176; AD04177; AD04178; AD04412; AD04413; AD04414; AD04415; AD04416; AD04417; AD04418; AD04419; AD04420; AD04421; AD04422; AD04423; AD04425; AD04426; AD04427; AD04428; AD04429; AD04430; AD04431; AD04432; AD04433; AD04434; AD04435; AD04436; AD04437; AD04438; AD04439; AD04440; AD04441; AD04442; AD04511; AD04570; AD04571; AD04572; AD04573; AD04574; AD04575; AD04576; AD04577; AD04578; AD04579; AD04580; AD04581; AD04583; AD04584; AD04585; AD04586; AD04587; AD04588; AD04590; AD04591 ; AD04592; AD04593; AD04594; AD04595; AD04596; AD04597; AD04598; AD04599; AD04734; AD04771; AD04772; AD04773; AD04774; AD04775; AD04776; AD04777; AD04778;

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AMENDED SHEET

AD04822; AD04823; AD04871; AD04872; AD04873; AD04874; AD04875; AD04876;

AD04881; AD04882; AD04883; AD04884; AD04885; AD04962; AD04963; AD04981;

AD04982; AD04983; AD05069; AD05070; AD05071; AD05072; AD05073; AD05074;

AD05075; AD05076; AD05077; AD05078; AD05147; AD05148; AD05149; AD05164;

or AD05165.

52. The RNAi agent of claim 51, wherein the RNAi agent has a duplex structure comprising an antisense strand that is identical or substantially identical to the respective antisense strand in the duplex structure from the group consisting of: AD03498; AD03499; AD03500; AD03501; AD03738; AD03739; AD03967; AD03968; AD03969; AD03970; AD03971; AD03972; AD03973; AD03974; AD03975; AD03976; AD03977; AD03978; AD04001 ; AD04002; AD04003; AD04004; AD04005; AD04006; AD04007; AD04008; AD04009; AD04010; AD04176; AD04177; AD04178; AD04412; AD04413; AD04414; AD04415; AD04416; AD04417; AD04418; AD04419; AD04420; AD04421; AD04422; AD04423; AD04425; AD04426; AD04427; AD04428; AD04429; AD04430; AD04431; AD04432; AD04433; AD04434; AD04435; AD04436; AD04437; AD04438; AD04439; AD04440; AD04441; AD04442; AD04511; AD04570; AD04571; AD04572; AD04573; AD04574; AD04575; AD04576; AD04577; AD04578; AD04579; AD04580; AD04581; AD04583; AD04584; AD04585; AD04586; AD04587; AD04588; AD04590; AD04591; AD04592; AD04593; AD04594; AD04595; AD04596; AD04597; AD04598; AD04599; AD04734; AD04771; AD04772; AD04773; AD04774; AD04775; AD04776; AD04777; AD04778; AD04822; AD04823; AD04871; AD04872; AD04873; AD04874; AD04875; AD04876; AD04881 ; AD04882; AD04883; AD04884; AD04885; AD04962; AD04963; AD04981; AD04982; AD04983; AD05069; AD05070; AD05071; AD05072; AD05073; AD05074; AD05075; AD05076; AD05077; AD05078; AD05147; AD05148; AD05149; AD05164; or AD05165.

53. The RNAi agent of claim 52, wherein the RNAi agent has a duplex structure comprising a sense strand and an antisense strand that are both identical or substantially identical to the respective sense and antisense strand in the duplex structure from the group consisting of: AD03498; AD03499; AD03500; AD03501; AD03738; AD03739; AD03967; AD03968; AD03969; AD03970; AD03971; AD03972; AD03973; AD03974; AD03975; AD03976; AD03977; AD03978; AD04001; AD04002; AD04003; AD04004; AD04005; AD04006; AD04007; AD04008; AD04009; AD04010; AD04176; AD04177; AD04178; AD04412; AD04413; AD04414; AD04415; AD04416; AD04417; AD04418; AD04419;

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AMENDED SHEET

AD04420; AD04421; AD04422; AD04423; AD04425; AD04426; AD04427; AD04428;

AD04429; AD04430; AD04431 ; AD04432; AD04433; AD04434; AD04435; AD04436;

AD04437; AD04438; AD04439; AD04440; AD04441; AD04442; AD04511; AD04570;

AD04571; AD04572; AD04573; AD04574; AD04575; AD04576; AD04577; AD04578;

AD04579; AD04580; AD04581; AD04583; AD04584; AD04585; AD04586; AD04587;

AD04588; AD04590; AD04591; AD04592; AD04593; AD04594; AD04595; AD04596;

AD04597; AD04598; AD04599; AD04734; AD04771; AD04772; AD04773; AD04774;

AD04775; AD04776; AD04777; AD04778; AD04822; AD04823; AD04871; AD04872;

AD04873; AD04874; AD04875; AD04876; AD04881; AD04882; AD04883; AD04884;

AD04885; AD04962; AD04963; AD04981 ; AD04982; AD04983; AD05069; AD05070; AD05071; AD05072; AD05073; AD05074; AD05075; AD05076; AD05077; AD05078; AD05147; AD05148; AD05149; AD05164; or AD05165

54. The RNAi agent of claims 50-53, wherein the substantial identity is exactly, at least, or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

55. The RNAi agent of any of claims 50-53, wherein the substantial identity is at least, or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

56. The RNAi agent of any of claims 50-53, wherein the substantial identity is about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

57. The RNAi agent of any of claims 50-53, wherein the substantial identity is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

58. A composition comprising the RNAi agent of any of claims 50-57.

59. A composition comprising at least two RNAi agents of any of claims 50-57.

60. The RNAi agent of any of claims 1-8 or 50-57, wherein the RNAi agent comprises at least one overhang.

61. The RNAi agent of any of claim 60, wherein the RNAi agent comprises an overhang at the 3 ' end of the antisense strand.

62. The RNAi agent of claim 61, wherein the RNAi agent comprises an overhang at the 3' end of the antisense strand and the 3 ' end of the sense strand.

63. The RNAi agent of any of claims 1-8 or 50-57, wherein the RNAi agent comprises one or two blunt ends.

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AMENDED SHEET

64. The RNAi agent of any of daims l-8 or 50-57, wherein the RNAi agent comprises one or two frayed ends.

65. The RNAi agent of any of daims l-8, 50-57, or 60-64, wherein the sense strand comprises at least one inverted abasic nucleoside.

66. A composition comprising one or more RNAi agents, wherein the one or more RNAi agents comprise:

(a) an antisense strand comprising a nucléotide sequence of any one ofthe following: SEQ ID NO:l00, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:171, SEQ ID NO: 179 and SEQ ID NO: 180; and (b) a sense strand comprising a nucléotide sequence of any one ofthe following: SEQ ID NO:229, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:273, SEQ ID NO:302 and SEQ ID NO:319.

67. The composition of claim 66, wherein the one or more RNAi agents further comprise a targeting ligand that is conjugated to the one or more RNAi agents.

68. The composition of claim 67, wherein the targeting ligand is (NAG25), (NAG25)s, (NAG31), (NAG31)s, (NAG37), or (NAG37)s.

69. The composition of any one of daims 66-68 further comprising an additional RNAi agent, wherein the additional RNAi agent comprises: an antisense strand comprising a nucléotide sequence of any one of the following: SEQ ID NO:140, SEQ ID NO:137, SEQ ID NO: 102, SEQ ID NO: 162 and SEQ ID NO: 188, and a sense strand comprising a nucléotide sequence of any one of the following: SEQ ID NO:248, SEQ ID NO:294, SEQ ID NO:262, SEQ ID NO:271, SEQ ID NO:274, and SEQ ID NO:328.