Compositions and methods for inhibiting expression of the LECT2 gene

Abstract

The present disclosure relates to double-stranded ribonucleic acid (dsRNA) compositions that target the LECT2 gene and methods of using such dsRNA compositions to alter (eg, inhibit) the expression of LECT2.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 895,217, filed September 3, 2019. The entire contents of each of the foregoing applications are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on August 27, 2020, is named A2038-7232WO_SL.Txt and is 221,086 bytes in size.

[0003] The present disclosure relates to the specific inhibition of expression of the LECT2 gene. [Background technology]

[0004] Amyloidosis is a group of diseases characterized by the deposition of insoluble fibrillar protein aggregates called amyloids in organs or tissues. Amyloids can be formed from mutant or wild-type proteins. One nomenclature for amyloid diseases uses an abbreviation for the protein that forms the amyloid deposits, preceded by the letter "A." Thus, for example, ALECT2 is an abbreviation for amyloidosis involving amyloid deposits formed from leukocyte cell-derived chemotactic factor-2 (ALECT2).

[0005] LECT2 amyloidosis (ALECT2) is one of the most recently discovered forms of amyloidosis. LECT2 amyloidosis has been observed in individuals with renal or hepatic amyloidosis. This form of amyloidosis may present with nephrotic syndrome or liver complications (e.g., hepatitis, e.g., chronic hepatitis). It may be particularly prevalent in Mexican Americans and / or individuals homozygous for the G allele, which encodes a valine at position 40 of the mature LECT2 protein (or position 58 of the unprocessed protein). Treatments for LECT2 amyloidosis are limited, and new treatments are needed. Summary of the Invention [Problem to be solved by the invention]

[0006] The present disclosure describes a method and iRNA composition for regulating the expression of LECT2 gene.In certain embodiments, the expression of LECT2 gene is reduced or inhibited using LECT2-specific iRNA.Such inhibition can be useful for treating disorders associated with LECT2 expression, such as amyloidosis, for example, LECT2 amyloidosis (ALECT2). [Means for solving the problem]

[0007] Therefore, the present invention provides compositions and methods for influencing the RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of LECT2 gene, for example, in cells or in subjects (e.g., mammals, such as human subjects).Also described are compositions and methods for treating disorders associated with the expression of LECT2 gene, such as LECT2 amyloidosis.

[0008] In some embodiments, the LECT2 amyloidosis is renal amyloidosis. In some embodiments, the LECT2 amyloidosis comprises amyloid deposits in the kidney. In some embodiments, the LECT2 amyloidosis is associated with a kidney disease (e.g., nephrotic syndrome). In some embodiments, the amyloidosis is associated with proteinuria. In some embodiments, proteinuria is absent. In some embodiments, the LECT2 amyloidosis is hepatic amyloidosis. In some embodiments, the LECT2 amyloidosis comprises amyloid deposits in the liver. In some embodiments, the LECT2 amyloidosis is associated with liver inflammation (e.g., hepatitis, e.g., chronic hepatitis). In some embodiments, the methods described herein are effective in inhibiting amyloid deposition or a syndrome associated with amyloid deposition (e.g., by preventing amyloid deposition or reducing amyloid deposition, e.g., by reducing the size, number, or extent of amyloid deposits).

[0009] As used herein, the terms "iRNA," "RNAi," "iRNA agent," "RNAi agent," or "iRNA molecule" refer to an agent containing RNA, as that term is defined herein, that mediates targeted cleavage of RNA transcripts, for example, via the RNA-induced silencing complex (RISC) pathway. In one embodiment, the iRNA described herein inhibits LECT2 expression in a cell or mammal.

[0010] The iRNA (e.g., dsRNA) included in the compositions featured herein includes an RNA strand (antisense strand) having a region that is substantially complementary to at least a portion of an mRNA transcript of a LECT2 gene (e.g., a mouse or human LECT2 gene), e.g., a region that is 30 nucleotides or less, generally 19-24 nucleotides in length (also referred to herein as a "LECT2-specific iRNA"). In some embodiments, the LECT2 mRNA transcript is a human LECT2 mRNA transcript, e.g., SEQ ID NO: 1. In some embodiments, the LECT2 mRNA transcript has an A to G substitution at nucleotide 373 of SEQ ID NO: 1. In some embodiments, the mRNA transcript encodes a valine at position 40 of the mature LECT2 protein (or amino acid 58 of the unprocessed protein). In some embodiments, the mRNA transcript encodes an isoleucine at position 40 of the mature LECT2 protein (or amino acid 58 of the unprocessed protein).

[0011] In some embodiments, the iRNA (e.g., dsRNA) described herein comprises an antisense strand having a region substantially complementary to a region of human LECT2 mRNA. In some embodiments, the human LECT2 mRNA has the sequence NM_002302.2 (SEQ ID NO: 1). In some embodiments, the human LECT2 mRNA has an A to G substitution at nucleotide 373 of SEQ ID NO: 1.

[0012] In other embodiments, iRNAs include dsRNAs having an RNA strand (antisense strand) with a region substantially complementary to a portion of LECT2 mRNA. In one embodiment, iRNAs include dsRNAs having an RNA strand (antisense strand) with a region substantially complementary to a portion of LECT2 mRNA, for example, human LECT2 mRNA (e.g., provided by NM_002302.2 (SEQ ID NO: 1), or human LECT2 mRNA with an A to G substitution at nucleotide 373 of SEQ ID NO: 1).

[0013] In one embodiment, an iRNA for inhibiting expression of the LECT2 gene comprises at least two sequences complementary to each other. The iRNA comprises a sense strand having a first sequence and an antisense strand having a second sequence. The antisense strand comprises a nucleotide sequence substantially complementary to at least a portion of the mRNA encoding the LECT2 transcript, and the complementary region is 30 nucleotides or less and at least 15 nucleotides in length. Typically, the iRNA is 19-24 nucleotides in length.

[0014] In some embodiments, the iRNA is 19-21 nucleotides in length. In some embodiments, the iRNA is 19-21 nucleotides in length and is formulated in a lipid, such as a lipid nanoparticle (LNP) formulation (e.g., an LNP11 formulation). In one embodiment, the iRNA targeting LECT2 is formulated in a stable nucleic acid lipid particle (SNALP).

[0015] In some embodiments, the iRNA is 21-23 nucleotides in length. In some embodiments, the iRNA is 21-23 nucleotides in length and is in the form of a conjugate, e.g., conjugated to one or more GalNAc derivatives described herein.

[0016] In some embodiments, the iRNA is about 15 to about 25 nucleotides in length, and in other embodiments, the iRNA is about 25 to about 30 nucleotides in length. When contacted with a cell expressing LECT2, an iRNA targeting LECT2 inhibits expression of the LECT2 gene (e.g., by at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%), as assayed by methods known to those of skill in the art or as described herein.

[0017] In one embodiment, an iRNA (e.g., a dsRNA) featured herein comprises or consists of a dsRNA first sequence selected from the group consisting of the sense sequences of Tables 2A-2B, 3A-3B, 6, or 7, and a second sequence selected from the group consisting of the corresponding antisense sequences of Tables 2A-2B, 3A-3B, 6, or 7, or a pharmaceutically acceptable salt thereof.

[0018] In some embodiments, an iRNA (e.g., dsRNA) featured herein comprises or consists of a sense and / or antisense sequence selected from those provided in Tables 2A-2B, 3A-3B, 6, or 7, or a pharmaceutically acceptable salt thereof. In some embodiments, an iRNA (e.g., dsRNA) featured herein comprises or consists of a sense and / or antisense sequence selected from those of AD-454781, AD-133461, AD-454746, AD-86459, or AD-86460, as disclosed herein in the Examples. In some embodiments, an iRNA (e.g., dsRNA) has a sense and / or antisense sequence selected from those of AD-454781, AD-133461, or AD-454746. In some embodiments, an iRNA (e.g., dsRNA) has a sense and / or antisense sequence of AD-454781.

[0019] The iRNA molecules featured herein can contain naturally occurring nucleotides or at least one modified nucleotide, including, but not limited to, 2'-O-methyl modified nucleotides, nucleotides with 5'-phosphorothioate groups, and terminal nucleotides linked to cholesteryl derivatives. Alternatively, the modified nucleotide can be selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, acyclic nucleotides, abasic nucleotides, glycol nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural base-containing nucleotides. Such modified sequences can be based, for example, on a first sequence of the iRNA selected from the group consisting of the sense sequences of Tables 2A, 3A, or 6 and a second sequence selected from the group consisting of the corresponding antisense sequences of Tables 2A, 3A, or 6.

[0020] In one embodiment, an iRNA (e.g., dsRNA) featured herein comprises a sense strand comprising a sequence selected from the group consisting of SEQ ID NO: 143, SEQ ID NO: 29, or SEQ ID NO: 23. In one embodiment, an iRNA (e.g., dsRNA) featured herein comprises an antisense strand comprising a sequence selected from the group consisting of SEQ ID NO: 144, SEQ ID NO: 30, or SEQ ID NO: 24. In one embodiment, an iRNA (e.g., dsRNA) comprises a sense strand comprising the sequence of SEQ ID NO: 143. In one embodiment, an iRNA (e.g., dsRNA) comprises an antisense strand comprising the sequence of SEQ ID NO: 144.

[0021] In one embodiment, an iRNA (e.g., dsRNA) featured herein comprises a sense strand comprising a sequence selected from the group consisting of SEQ ID NO: 370, SEQ ID NO: 294, or SEQ ID NO: 290. In one embodiment, an iRNA (e.g., dsRNA) featured herein comprises an antisense strand comprising a sequence selected from the group consisting of SEQ ID NO: 371, SEQ ID NO: 295, or SEQ ID NO: 291. In one embodiment, an iRNA (e.g., dsRNA) comprises a sense strand comprising the sequence of SEQ ID NO: 370. In one embodiment, an iRNA (e.g., dsRNA) comprises an antisense strand comprising the sequence of SEQ ID NO: 371.

[0022] In one embodiment, the iRNAs described herein target wild-type LECT2 RNA transcript variants, while in another embodiment, the iRNAs target mutant transcripts (e.g., LECT2 RNAs with allelic variants). For example, the iRNAs featured in this disclosure can target polymorphic variants of LECT2, such as single nucleotide polymorphisms (SNPs).

[0023] In some embodiments, the iRNA (dsRNA) targets (e.g., reduces) mRNA encoding valine at position 40 of the mature LECT2 protein (or amino acid 58 of the unprocessed protein). In some embodiments, the iRNA (e.g., dsRNA) targets (e.g., reduces) mRNA encoding isoleucine at position 40 of the mature LECT2 protein (or amino acid 58 of the unprocessed protein). In another embodiment, the iRNA (e.g., dsRNA) targets (e.g., reduces) both mRNA encoding valine and mRNA encoding isoleucine at position 40 of the mature LECT2 protein (or amino acid 58 of the unprocessed protein).

[0024] In another embodiment, the iRNA targets both wild-type and mutant LECT2 transcripts. In yet another embodiment, the iRNA targets a specific transcript variant of LECT2. In yet another embodiment, the iRNA agent targets multiple transcript variants.

[0025] In one embodiment, an iRNA featured in this disclosure targets a non-coding region of a LECT2 RNA transcript, for example, the 5' or 3' untranslated region of the transcript.

[0026] In some embodiments, the iRNA described herein is in the form of a conjugate, such as a carbohydrate conjugate, which can serve as targeting moiety and / or ligand, as described herein.In one embodiment, the conjugate is attached to the 3' end of the sense strand of dsRNA.In some embodiments, the conjugate is attached via a linker, for example, via a bivalent or trivalent branched linker.

[0027] In some embodiments, the conjugate comprises one or more N-acetylgalactosamine (GalNAc) derivatives. Such conjugates are also referred to herein as GalNAc conjugates. In some embodiments, the conjugate targets RNAi agents (e.g., dsRNA) to specific cells, such as liver cells, for example, hepatocytes. The GalNAc derivative can be linked via a linker, for example, a bivalent or trivalent branched linker. In certain embodiments, the conjugate is

[0028] [ka] is.

[0029] In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker, such as the linker shown in the schematic diagram below, where X is O or S.

[0030] [ka]

[0031] In some embodiments, X is O. In some embodiments, X is S.

[0032] In some embodiments, the RNAi agent is conjugated to L96, as defined in Table 1 and shown below.

[0033] [ka]

[0034] In some embodiments, the RNAi agent is conjugated to a ligand that targets the RNAi (e.g., dsRNA) to a desired organ (e.g., liver) or a specific cell type (e.g., hepatocyte). In some embodiments, the RNAi agent is conjugated to a ligand (e.g., GalNAc ligand, e.g., L96) that targets the RNAi agent (e.g., dsRNA) to the liver.

[0035] In one aspect, provided herein is a pharmaceutical composition for inhibiting the expression of the LECT2 gene in an organism, typically a human subject. The composition typically comprises one or more iRNAs described herein and a pharmaceutically acceptable carrier or delivery vehicle. In one embodiment, the composition is used to treat a disorder associated with LECT2 expression, such as amyloidosis, e.g., LECT2 amyloidosis.

[0036] In one embodiment, the iRNA provided herein is a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of LECT2, comprising a sense strand and an antisense strand, each of which is 15 to 30 base pairs in length, wherein the antisense strand is complementary to at least 15 nucleotides of a duplex target sequence disclosed in Tables 2A-2B, 3A-3B, 4A-4B, or 5-7, or a pharmaceutically acceptable salt thereof.

[0037] In some embodiments, the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides of a duplex target sequence disclosed in Tables 2A-2B, 3A-3B, 4A-4B, or 5-7. In some embodiments, the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of a duplex target sequence disclosed in Tables 2A-2B, 3A-3B, 4A-4B, or 5-7.

[0038] In some embodiments, the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides of a duplex target sequence disclosed in Tables 2A-2B, 3A-3B, 6, or 7. In some embodiments, the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of a duplex target sequence disclosed in Tables 2A-2B, 3A-3B, 6, or 7.

[0039] In some embodiments, the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of the target sequence of the duplex AD-454781, AD-133461, AD-454746, AD-86459, or AD-86460. In some embodiments, the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of the target sequence of the duplex AD-454781, AD-133461, AD-454746, AD-86459, or AD-86460.

[0040] In some embodiments, the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of the target sequence of the duplex AD-454781, AD-133461, or AD-454746. In some embodiments, the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of the target sequence of the duplex AD-454781, AD-133461, or AD-454746.

[0041] In some embodiments, the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides of the target sequence of duplex AD-454781. In some embodiments, the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of the target sequence of duplex AD-454781. In some embodiments, the antisense strand is complementary to every nucleotide of the target sequence of duplex AD-454781.

[0042] In some embodiments, the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides of a target sequence provided in Table 2A, 3A, or 6. In some embodiments, the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of a target sequence provided in Table 2A, 3A, or 7.

[0043] In some embodiments, the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of SEQ ID NO: 145, 31, or 25. In some embodiments, the antisense strand is complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of SEQ ID NO: 145. In some embodiments, the antisense strand is complementary to all nucleotides of SEQ ID NO: 145.

[0044] In a further aspect, the iRNA provided herein is a double-stranded RNAi (dsRNA) comprising a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a LECT2 RNA transcript, each strand having from about 14 to about 30 nucleotides, and wherein the double-stranded RNAi agent has Formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each N a and N a ' independently represent an oligonucleotide sequence containing 0-25 nucleotides that are either modified or unmodified, or a combination thereof, each sequence containing at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; each n p , n p 'n q , and n q ' independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications of three consecutive nucleotides; N b The modification of N is different from the modification of Y. b ' is different from Y' It is a double-stranded RNAi (dsRNA) represented by:

[0045] In some embodiments, the sense strand is conjugated to at least one ligand.

[0046] In some embodiments, i is 1; j is 1; or both i and j are 1.

[0047] In some embodiments, k is 1; l is 1; or both k and l are 1.

[0048] In some embodiments, XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

[0049] In some embodiments, the Y'Y'Y' motif occurs at positions 11, 12, and 13 of the antisense strand from the 5' end.

[0050] In some embodiments, Y' is 2'-O-methyl.

[0051] In some embodiments, the duplex region is 15-30 nucleotide pairs in length. In some embodiments, the duplex region is 17-23 nucleotide pairs in length. In some embodiments, the duplex region is 19-21 nucleotide pairs in length. In some embodiments, the duplex region is 21-23 nucleotide pairs in length.

[0052] In some embodiments, the modification of the nucleotide is selected from the group consisting of locked nucleic acid (LNA), acyclic nucleotide, hexitol or hexose nucleic acid (HNA), cyclohexene nucleic acid (CeNA), glycol nucleic acid (GNA), 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and any combination thereof.

[0053] In some embodiments, the nucleotide modifications are 2'-O-methyl, 2'-fluoro, or both, and may be glycol nucleic acids (GNAs).

[0054] In some embodiments, the ligand comprises a carbohydrate.

[0055] In some embodiments, the ligand is attached via a linker.

[0056] In some embodiments, the linker is a bivalent or trivalent branched linker.

[0057] In some embodiments, the ligand is

[0058] [ka] is.

[0059] In some embodiments, the ligand and linker have the formula XXIV:

[0060] [ka] As shown in the figure.

[0061] In some embodiments, the ligand is attached to the 3' end of the sense strand.

[0062] In some embodiments, the dsRNA has (e.g., comprises) a nucleotide sequence (e.g., sense and / or antisense sequence) selected from the group of sequences provided in Tables 2A-2B, 3A-3B, 6, or 7.

[0063] In a further aspect, the iRNA provided herein is a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of LECT2, comprising a sense strand and an antisense strand that includes a region complementary to a LECT2 RNA transcript and that includes at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from one of the antisense sequences listed in any one of Tables 2A-2B, 3A-3B, 6, or 7, or a pharmaceutically acceptable salt thereof.

[0064] In some embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 3 nucleotides from one of the antisense sequences listed in any one of Tables 2A-2B, 3A-3B, 6, or 7. In some embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 2 nucleotides from one of the antisense sequences listed in any one of Tables 2A-2B, 3A-3B, 6, or 7. In some embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 1 nucleotide from one of the antisense sequences listed in any one of Tables 2A-2B, 3A-3B, 6, or 7.

[0065] In some embodiments, the sense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 3 nucleotides from one of the antisense sequences listed in any one of Tables 2A-2B, 3A-3B, 6, or 7. In some embodiments, the sense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 2 nucleotides from one of the antisense sequences listed in any one of Tables 2A-2B, 3A-3B, 6, or 7. In some embodiments, the sense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 1 nucleotide from one of the antisense sequences listed in any one of Tables 2A-2B, 3A-3B, 6, or 7.

[0066] In some embodiments, the sense and antisense sequences are selected from the duplexes AD-454781, AD-133461, AD-454746, AD-86459, or AD-86460 disclosed in the Examples. In some embodiments, the sense and antisense sequences are selected from the duplexes AD-454781, AD-133461, or AD-454746. In some embodiments, the sense and antisense sequences are of the duplex AD-454781. In some embodiments, the sense and antisense sequences are of a duplex disclosed herein that suppresses LECT2 mRNA expression by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, as assessed, for example, using the assays disclosed in the Examples provided herein.

[0067] In some embodiments, dsRNA comprises at least one modified nucleotide.In some embodiments, 5 or less nucleotides of the sense strand of dsRNA and 5 or less nucleotides of the antisense strand of dsRNA are unmodified nucleotide.In some embodiments, all nucleotides of the sense strand of dsRNA and all nucleotides of the antisense strand of dsRNA comprise modification.

[0068] In some embodiments, the at least one modified nucleotide is selected from the group consisting of: a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

[0069] In some embodiments, the modified nucleotide is selected from the group consisting of: 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, acyclic nucleotides, abasic nucleotides, glycol nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural base containing nucleotides.

[0070] In some embodiments, the dsRNA comprises 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, or both, and may be glycol nucleotides.

[0071] In some embodiments, the dsRNA comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more 2'-O-methyl modified nucleotides in the sense strand. In some embodiments, the dsRNA comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more 2'-O-methyl modified nucleotides in the antisense strand.

[0072] In some embodiments, the dsRNA comprises at least 1, 2, 3, 4 or more 2'-fluoro modified nucleotides in the sense strand. In some embodiments, the dsRNA comprises 2'-fluoro modified nucleotides at positions 7, 9, 10, 11, or a combination thereof in the sense strand. In some embodiments, the dsRNA comprises 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 in the sense strand. In some embodiments, the dsRNA comprises at least 1, 2, 3, 4, 5, 6, 7 or more 2'-fluoro modified nucleotides in the antisense strand. In some embodiments, the dsRNA comprises 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 9, 14, 16, or a combination thereof in the antisense strand. In some embodiments, the dsRNA comprises 2'-fluoro modified nucleotides at positions 2, 14, and 16 in the antisense strand. In some embodiments, the dsRNA comprises 2'-fluoro modified nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand. In some embodiments, the dsRNA comprises 2'-fluoro modified nucleotides at positions 2, 4, 8, 9, 12, 14, and 16 of the antisense strand.

[0073] In some embodiments, the dsRNA further comprises a glycol nucleotide. In some embodiments, the glycol nucleotide is present in the antisense strand. In some embodiments, the glycol nucleotide is present at position 7 of the antisense strand.

[0074] In some embodiments, the dsRNA comprises a phosphorothioate bond between positions 1 and 2, between positions 2 and 3, or both of the sense strand. In some embodiments, the dsRNA comprises a phosphorothioate bond between positions 1 and 2 and between positions 2 and 3 of the sense strand.

[0075] In some embodiments, the dsRNA comprises a phosphorothioate bond between positions 1 and 2, between positions 2 and 3, between positions 21 and 22, between positions 22 and 23, or a combination thereof, of the antisense strand. In some embodiments, the dsRNA comprises a phosphorothioate bond between positions 1 and 2, between positions 2 and 3, between positions 21 and 22, and between positions 22 and 23 of the antisense strand.

[0076] In some embodiments, the region of complementarity is at least 17 nucleotides in length. In some embodiments, the region of complementarity is between 19 and 23 nucleotides in length. In some embodiments, the region of complementarity is 21 nucleotides in length.

[0077] In some embodiments, each strand is 30 nucleotides or less in length. In some embodiments, each strand is between 21 and 23 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0078] In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide. In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides. In some embodiments, the dsRNA comprises a blunt end. In some embodiments, the dsRNA comprises a 3' overhang and a blunt end.

[0079] In some embodiments, the iRNA (e.g., dsRNA) described herein further comprises a ligand. In some embodiments, the ligand is a GalNAc ligand. In some embodiments, the ligand targets the iRNA (e.g., dsRNA) to the liver (e.g., hepatocytes). In some embodiments, the ligand is conjugated to the 3' end of the sense strand of the dsRNA.

[0080] In some embodiments, the complementary region consists of an antisense sequence selected from the antisense sequences provided in Tables 2A-2B, 3A-3B, 6, or 7. In some embodiments, the complementary region consists of an antisense sequence selected from those of AD-454781, AD-133461, AD-454746, AD-86459, or AD-86460 disclosed in the Examples. In some embodiments, the complementary region consists of an antisense sequence selected from those of AD-454781, AD-133461, or AD-454746. In some embodiments, the complementary region consists of the antisense sequence of the duplex AD-454781. In some embodiments, the complementary region consists of an antisense sequence selected from the duplexes disclosed herein, wherein the duplexes suppress LECT2 mRNA or protein expression by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, or 90%.

[0081] In some embodiments, the dsRNA comprises a sense strand comprising or consisting of a sense strand sequence selected from Tables 2A-2B, 3A-3B, 6, or 7, and an antisense strand comprising or consisting of an antisense sequence selected from Tables 2A-2B, 3A-3B, 6, or 7. In some embodiments, the dsRNA comprises or consists of a corresponding sense and antisense sequence pair selected from those duplexes disclosed in Tables 2A-2B, 3A-3B, 4A-4B, or 5-7. In certain embodiments, the dsRNA comprises or consists of a corresponding sense and antisense sequence pair selected from those duplexes disclosed in Tables 2A, 3A, or 6. In certain embodiments, the dsRNA comprises or consists of a corresponding sense and antisense sequence pair selected from those duplexes disclosed in Tables 2B, 3B, or 7. In certain embodiments, the dsRNA comprises or consists of a corresponding sense and antisense sequence pair selected from those duplexes disclosed in Tables 4A or 4B. In certain embodiments, the dsRNA comprises or consists of a pair of corresponding sense and antisense sequences selected from those duplexes disclosed in Table 5.

[0082] In one aspect, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of LECT2, the dsRNA comprising a sense strand and an antisense strand comprising a region complementary to a LECT2 RNA transcript, wherein the sense strand comprises the sequence of csasugugCfaCfAfUfugaaaacuguL96 (SEQ ID NO: 23) and all modifications, and the antisense strand comprises the sequence of asCfsaGfuu(Tgn)UfCfaaUfgUfgCfacaugscsg (SEQ ID NO: 24) and all modifications.

[0083] In one aspect, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of LECT2, the dsRNA comprising a sense strand and an antisense strand comprising a region complementary to a LECT2 RNA transcript, wherein the sense strand comprises the sequence of asusggucAfgAfUfCfuucaaaauaaL96 (SEQ ID NO: 29) and all modifications, and the antisense strand comprises the sequence of usUfsauuu(Tgn)gaagauCfuGfaccaususg (SEQ ID NO: 30) and all modifications.

[0084] In one aspect, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of LECT2, the dsRNA comprising a sense strand and an antisense strand comprising a region complementary to a LECT2 RNA transcript, wherein the sense strand comprises the sequence of gsgsucagAfuCfUfUfcaaaauaaauL96 (SEQ ID NO: 143) and all modifications, and the antisense strand comprises the sequence of asUfsuuaUfuUfUfgaagAfuCfugaccsgsg (SEQ ID NO: 144) and all modifications.

[0085] In one aspect, the present disclosure provides a cell containing at least one iRNA (e.g., dsRNA) disclosed herein. The cell is typically a mammalian cell, such as a human cell. In some embodiments, the cell is a liver cell (e.g., a hepatocyte).

[0086] In one aspect, the present disclosure provides human cells (e.g., human cells described herein) having reduced levels of LECT2 mRNA or LECT2 protein when compared to similar untreated cells, which may be reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0087] In some embodiments, the human cell was produced by a process comprising contacting a human cell (e.g., a human cell described herein) with dsRNA, e.g., a dsRNA described herein.

[0088] In one aspect, provided herein is a pharmaceutical composition for inhibiting expression of the LECT2 gene, comprising an iRNA (e.g., dsRNA) described herein.

[0089] In some embodiments of the pharmaceutical compositions described herein, the iRNA (e.g., dsRNA) is administered in an unbuffered solution. In some embodiments, the unbuffered solution is saline or water.

[0090] In some embodiments of the pharmaceutical compositions described herein, iRNA (e.g., dsRNA) is administered in a buffer solution. In some embodiments, the buffer solution comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In some embodiments, the buffer solution is phosphate buffered saline (PBS).

[0091] In some embodiments of the pharmaceutical compositions described herein, the iRNA (e.g., dsRNA) is targeted to the liver (e.g., hepatocytes).

[0092] In some embodiments of the pharmaceutical compositions described herein, the compositions are administered intravenously. In some embodiments of the pharmaceutical compositions described herein, the compositions are administered subcutaneously.

[0093] In some embodiments, the pharmaceutical composition comprises an iRNA (e.g., a dsRNA) described herein that comprises a ligand (e.g., a GalNAc ligand) that targets the iRNA (e.g., dsRNA) to a liver cell, e.g., a hepatocyte.

[0094] In some embodiments, the pharmaceutical composition comprises an iRNA (e.g., a dsRNA) described herein that includes a ligand (e.g., a GalNAc ligand), and the pharmaceutical composition is administered subcutaneously. In some embodiments, the ligand targets the iRNA (e.g., a dsRNA) to liver cells, e.g., hepatocytes.

[0095] In certain embodiments, pharmaceutical compositions, for example, compositions described herein, comprise lipid formulations.In some embodiments, RNAi agents are LNP formulations, for example, MC3 formulations.In some embodiments, LNP formulations target RNAi agents to specific cells, for example, liver cells (for example, hepatocytes).In some embodiments, lipid formulations are LNP11 formulations.In some embodiments, compositions are administered intravenously.

[0096] In another embodiment, the pharmaceutical composition is formulated for administration according to a dosage regimen described herein, for example, not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week. In another embodiment, administration of the pharmaceutical composition can be maintained for one month or more, for example, 1, 2, 3, or 6 months, or one year or more.

[0097] In another embodiment, a composition containing an iRNA featured in the present disclosure, such as a dsRNA targeting LECT2, is administered in combination with a second treatment for a disorder associated with LECT2 expression (e.g., LECT2 amyloidosis). The iRNA or a composition comprising an iRNA provided herein can be administered before, after, or simultaneously with the second treatment. In some embodiments, the iRNA is administered before the second treatment. In some embodiments, the iRNA is administered after the second treatment. In some embodiments, the iRNA is administered simultaneously with the second treatment.

[0098] In some embodiments, the second therapy is a non-iRNA therapeutic effective to treat a disorder or a symptom of a disorder.

[0099] In some embodiments, the disorder treated by the compositions or methods disclosed herein is LECT2 amyloidosis, which affects kidney function, for example, by amyloid deposition in the kidney. In some such embodiments, the iRNA is administered in conjunction with a treatment to support kidney function (e.g., dialysis). In some embodiments, the iRNA is administered in conjunction with a diuretic, angiotensin-converting enzyme (ACE) inhibitor, angiotensin receptor blocker, and / or dialysis, for example, to support or manage kidney function.

[0100] In some embodiments, the disorder treated by the compositions or methods disclosed herein is LECT2 amyloidosis, which involves amyloid deposition in the liver. In some such embodiments, the iRNA is administered in conjunction with a treatment that supports liver function.

[0101] In some embodiments, the disorder treated by the compositions or methods disclosed herein is LECT2 amyloidosis, and the iRNA is administered in conjunction with removal of all or part of an organ(s) affected by amyloidosis (e.g., resection of all or part of kidney or liver tissue affected by amyloidosis). Removal may also be performed in conjunction with replacement of all or part of the removed organ (e.g., in conjunction with kidney or liver transplantation).

[0102] In one aspect, provided herein is a method for inhibiting LECT2 expression in a cell, the method comprising: (a) introducing into the cell an iRNA (e.g., dsRNA) described herein; and (b) maintaining the cell of step (a) for a period of time sufficient to result in degradation of mRNA transcripts of the LECT2 gene, thereby inhibiting expression of the LECT2 gene in the cell.

[0103] In one aspect, provided herein is a method for inhibiting LECT2 expression in a cell (e.g., a cell described herein), comprising: (a) contacting, e.g., introducing, an iRNA (e.g., a dsRNA) described herein into the cell; and (b) maintaining the cell of step (a) for a sufficient time to result in a decrease in the level of LECT2 mRNA, LECT2 protein, or both LECT2 mRNA or LECT2 protein, thereby inhibiting expression of the LECT2 gene in the cell.

[0104] In one aspect, the present disclosure provides a method for reducing or inhibiting the expression of LECT2 gene in cells (e.g., liver cells, e.g., hepatocytes).The method comprises contacting cells with dsRNA as described herein, thereby inhibiting the expression of LECT2 gene.As used herein, "contacting" includes direct contacting of cells and indirect contacting of cells.For example, when a composition comprising RNAi is administered to a subject (e.g., intravenously or subcutaneously), cells in the subject (e.g., liver cells) can be contacted.

[0105] In some embodiments, the method comprises: (a) introducing into a cell double-stranded ribonucleic acid (dsRNA) comprising at least two sequences complementary to each other, the dsRNA having a sense strand with a first sequence and an antisense strand with a second sequence; the antisense strand having a complementary region that is substantially complementary to at least a portion of an mRNA encoding LECT2, the complementary region being 30 nucleotides or less, e.g., 15-30 nucleotides in length, and generally 19-24 nucleotides in length, such that upon contact with a cell expressing LECT2, the dsRNA inhibits expression of the LECT2 gene by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more; and (b) maintaining the cells of step (a) for a time sufficient to obtain degradation of the mRNA transcripts of the LECT2 gene, thereby reducing or inhibiting expression of the LECT2 gene in the cells. Includes:

[0106] In some embodiments of the method of inhibiting LECT2 expression in cells, the cells are treated ex vivo, in vitro, or in vivo. In some embodiments, the cells are hepatocytes.

[0107] In some embodiments, the cell is present in a subject in need of treatment, prevention, and / or management of a disorder associated with LECT2 expression.

[0108] In some embodiments, the disorder, as described herein, is LECT2 amyloidosis.

[0109] In some embodiments, expression of LECT2 is inhibited by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, for example, as determined by the methods described herein.

[0110] In some embodiments, LECT2 mRNA expression is inhibited by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, LECT2 protein expression is inhibited by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0111] In some embodiments, the iRNA (e.g., dsRNA) has an IC in the range of 0.0005-1 nM, e.g., between 0.001-0.2 nM, between 0.002-0.1 nM, between 0.005-0.075 nM, or between 0.01-0.05 nM. 50 In some embodiments, the iRNA (e.g., dsRNA) has an IC50 of 0.02 nM or less, e.g., between 0.0005 and 0.02 nM, between 0.001 and 0.02 nM, between 0.005 and 0.02 nM, or between 0.01 and 0.02 nM. 50 In some embodiments, the iRNA (e.g., dsRNA) has an IC in the range of 0.01 to 1 nM. 50 It has.

[0112] In some embodiments, the cell (e.g., hepatocyte) is a mammalian cell (e.g., human, non-human primate, or rodent cell). In one embodiment, the subject is a mammal (e.g., human) with LECT2 amyloidosis. In one embodiment, the introduced dsRNA reduces or inhibits the expression of LECT2 gene in the cell.

[0113] In one embodiment, the dsRNA inhibits the expression of the LECT2 gene or inhibits amyloid deposition (e.g., by preventing or reducing amyloid deposition, e.g., by reducing the size, number, or extent of amyloid deposits). Inhibition may include at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more inhibition compared to a reference (e.g., a control that is not treated or treated with a non-targeting dsRNA (e.g., a dsRNA that does not target LECT2)).

[0114] In some embodiments, inhibiting expression of the LECT2 gene reduces LECT2 protein levels in a biological sample (e.g., a serum sample) from the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0115] In another aspect, the present disclosure provides a method for treating the pathogenesis process (for example, amyloid deposition) associated with LECT2 expression.In one embodiment, the method comprises administering to a subject, for example, a patient who needs such treatment, an effective (for example, therapeutically or prophylactically effective) amount of the dsRNA provided herein.

[0116] In one aspect, provided herein is a method of treating and / or preventing a disorder associated with LECT2 expression (e.g., LECT2 amyloidosis), comprising administering to a subject in need of such treatment a therapeutically effective amount of an iRNA (e.g., dsRNA) described herein, or a composition comprising an iRNA (e.g., dsRNA) described herein.

[0117] In one aspect, provided herein is a method for treating a disorder associated with LECT2 expression (e.g., LECT2 amyloidosis) comprising administering to a subject in need of such treatment a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises a sense strand and an antisense strand 15-30 base pairs in length, and the antisense strand is complementary to at least 15 contiguous nucleotides of a LECT2 mRNA transcript, e.g., a human LECT2 mRNA transcript, e.g., SEQ ID NO: 1 or a nucleotide sequence having an A to G substitution at nucleotide 373 of SEQ ID NO: 1. In one embodiment, the iRNA (e.g., dsRNA) targets an mRNA encoding a valine at position 40 of the mature LECT2 protein (or amino acid 58 of the unprocessed protein).

[0118] In one embodiment, provided herein is a method of treating a subject with LECT2 amyloidosis, comprising administering to the subject a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises a sense strand and an antisense strand 15-30 base pairs in length, wherein the antisense strand is complementary to at least 15 contiguous nucleotides of a LECT2 mRNA transcript, e.g., a human LECT2 mRNA transcript, e.g., SEQ ID NO: 1, or a nucleotide sequence having an A to G substitution at nucleotide 373 of SEQ ID NO: 1. In one embodiment, the iRNA (e.g., dsRNA) targets an mRNA encoding a valine at position 40 of the mature LECT2 protein (or amino acid 58 of the unprocessed protein).

[0119] In some embodiments, administration of an iRNA targeting LECT2 alleviates or reduces the severity of at least one symptom of a disorder associated with LECT2 expression in a patient. In some embodiments, at least one sign or symptom of a disorder associated with LECT2 expression, such as amyloidosis, e.g., LECT2 amyloidosis, comprises a degree of amyloid deposits or the presence or level of LECT2 (e.g., the LECT2 gene, LECT2 mRNA, or LECT2 protein).

[0120] In one embodiment, the subject has LECT2 amyloidosis. In another embodiment, the subject is at risk of developing LECT2 amyloidosis.

[0121] In some embodiments, the subject is a human.

[0122] In some embodiments, a dsRNA or pharmaceutical composition, eg, a dsRNA or pharmaceutical composition described herein, is administered to a subject subcutaneously or intravenously.

[0123] In some embodiments, treating comprises preventing the progression of the disorder. In some embodiments, treating comprises inhibiting or reducing the expression or activity of LECT2 in cells, such as hepatocytes. In some embodiments, treating results in an average reduction of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% from baseline in LECT2 mRNA in cells.

[0124] In some embodiments, the methods described herein further include measuring the level of LECT2 (e.g., LECT2 gene, LECT2 mRNA, or LECT2 protein) in the subject. In some embodiments, measuring the level of LECT2 in the subject includes measuring the level of the LECT2 gene, LECT2 protein, or LECT2 mRNA in a biological sample (e.g., a tissue, blood, or serum sample) from the subject. In some embodiments, the methods described herein further include performing a blood test, an imaging test, or a liver or kidney biopsy. In some embodiments, measuring the level of LECT2 (e.g., LECT2 gene, LECT2 mRNA, or LECT2 protein) in the subject is performed before treatment with a dsRNA agent or pharmaceutical composition. In some embodiments, once the subject is determined to have a level of LECT2 (e.g., LECT2 gene, LECT2 mRNA, or LECT2 protein) higher than the reference level, a dsRNA agent or pharmaceutical composition is administered to the subject. In some embodiments, measuring the level of LECT2 (e.g., LECT2 gene, LECT2 mRNA, or LECT2 protein) in the subject is performed after treatment with a dsRNA agent or pharmaceutical composition.

[0125] In some embodiments, the iRNA (e.g., dsRNA) is formulated as an LNP formulation.

[0126] In some embodiments, the iRNA (e.g., dsRNA) is in the form of a GalNAc conjugate.

[0127] In some embodiments, the iRNA (e.g., dsRNA) is administered at a dose of 0.05 to 50 mg / kg.

[0128] In some embodiments, the iRNA (e.g., dsRNA) is administered at a concentration of 0.01 mg / kg to 5 mg / kg body weight of the subject.

[0129] In some embodiments, the iRNA (dsRNA) is formulated as an LNP formulation and administered at a dose of 0.05-5 mg / kg. In some embodiments, the iRNA (e.g., dsRNA) is formulated as an LNP formulation and administered at a dose of 0.1-0.5 mg / kg.

[0130] In some embodiments, the iRNA (e.g., dsRNA) is in the form of a GalNAc conjugate and is administered at a dose of 0.5-50 mg / kg. In some embodiments, the iRNA (e.g., dsRNA) is in the form of a GalNAc conjugate and is administered at a dose of 1-10 mg / kg.

[0131] In some embodiments, the method inhibits expression of the LECT2 gene or inhibits amyloid deposition (e.g., by preventing amyloid deposition or by reducing amyloid deposition, e.g., by reducing the size, number, or extent of amyloid deposits). Inhibition may include at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% inhibition compared to a reference (e.g., a control untreated or treated with a non-targeting dsRNA (e.g., a dsRNA that does not target LECT2)).

[0132] In some embodiments, the iRNA (e.g., dsRNA) has an IC in the range of 0.0005-1 nM, e.g., between 0.001-0.2 nM, between 0.002-0.1 nM, between 0.005-0.075 nM, or between 0.01-0.05 nM. 50 In some embodiments, the iRNA (e.g., dsRNA) has an IC50 of 0.02 nM or less, e.g., between 0.0005 and 0.02 nM, between 0.001 and 0.02 nM, between 0.005 and 0.02 nM, or between 0.01 and 0.02 nM. 50 In some embodiments, the iRNA (e.g., dsRNA) has an IC in the range of 0.01 to 1 nM. 50 It has.

[0133] In some embodiments, the methods described herein improve symptoms associated with a LECT2-related disorder (e.g., LECT2 amyloidosis). In some embodiments, the methods described herein inhibit expression of the LECT2 gene in a subject. In some embodiments, the methods described herein inhibit amyloid deposition (e.g., by preventing amyloid deposition or reducing amyloid deposition, e.g., by reducing the size, number, or extent of amyloid deposits).

[0134] In some embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered according to a dosing regimen. In some embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered repeatedly, e.g., according to a dosing regimen.

[0135] In some embodiments, the iRNA (e.g., dsRNA) or a composition comprising the iRNA is administered subcutaneously. In some embodiments, the iRNA is in the form of a GalNAc conjugate. In some embodiments, the iRNA (e.g., dsRNA) is administered at a dose of 0.5 to 50 mg / kg. In some embodiments, the iRNA (e.g., dsRNA) is in the form of a GalNAc conjugate and is administered at a dose of 1 to 10 mg / kg.

[0136] In one aspect, provided herein is a vector encoding at least one strand of an iRNA (e.g., a dsRNA) described herein.

[0137] In one aspect, provided herein is a vector encoding at least one strand of a dsRNA, wherein the dsRNA comprises a region complementary to at least a portion of an mRNA encoding LECT2, the dsRNA is 30 base pairs or less in length, and the dsRNA targets the mRNA for cleavage.

[0138] In some embodiments, the region of complementarity is at least 15 nucleotides in length. In some embodiments, the region of complementarity is 19-23 nucleotides in length. In some embodiments, the region of complementarity is 21-23 nucleotides in length.

[0139] In one aspect, a vector is provided for inhibiting the expression of the LECT2 gene in a cell. In one embodiment, the vector comprises an iRNA as described herein. In one embodiment, the vector comprises at least one regulatory sequence operably linked to a nucleotide sequence encoding at least one strand of the iRNA as described herein. In one embodiment, the vector comprises at least one strand of the LECT2 iRNA.

[0140] In one aspect, provided herein is a cell comprising a vector as described herein.

[0141] In one aspect, the present invention provides a cell containing a vector for inhibiting the expression of the LECT2 gene in a cell, wherein the vector comprises a regulatory sequence operably linked to a nucleotide sequence encoding at least one strand of an iRNA described herein.

[0142] All articles, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0143] Details of various embodiments of the disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.

[0144] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0145] [Figure 1] FIG. 1 shows the human LECT2 mRNA transcript sequence (reference sequence NM_002302.2 GI:59806344, archived April 17, 2013; SEQ ID NO: 1). [Figure 2] Figure 2 shows the sequences and chemistries of three exemplary LECT2 siRNAs: AD-454781, AD-133461, and AD-454746, designed to target regions of LECT2 mRNA in both humans and cynomolgus monkeys. For each siRNA, "F," shown in green, is a "2' fluoro" modification; OMe, shown in black, is a methoxy group; GNA, shown in purple, is glycol nucleic acid; and PS refers to a phosphonothioate linkage. Figure 2 discloses SEQ ID NOS: 897-902, respectively, in order of appearance. [Figure 3] Figure 3 shows the pharmacokinetics of three exemplary LECT2 siRNAs in a rodent AAV model. The relative levels of LECT2 mRNA in the liver and the relative levels (percent remaining) of circulating LECT2 protein in the plasma were quantified in the experimental (LECT2 siRNA) and control (PBS) groups at 14 days post-treatment. [Figure 4] 4A-4C show the dose response of three exemplary LECT2 siRNAs in suppressing LECT2 in cynomolgus monkeys compared to the PBS control. The relative plasma LECT2 levels (plasma LECT2 protein knockdown) were quantified by normalizing to the pre-treatment protein levels of each individual monkey. [Figure 5] Figures 5A-5B show the relative levels (fold difference in expression) of rat (Figure 5A) or mouse (Figure 5B) LECT2 mRNA in the liver 3, 6, or 12 months after initial treatment in the experimental (LECT2 siRNA) or control (PBS) groups. [Figure 6]Figures 6A-6B evaluate long-term LECT-2 knockdown in cynomolgus monkeys. In Figure 6A, the relative levels (fold difference in expression) of LECT2 mRNA in the liver of monkeys 6 months after initial treatment were quantified in the experimental (siRNA AD-81725) or control (PBS) groups. In Figure 6B, circulating plasma LECT2 protein levels (percent remaining protein) were measured in the experimental (siRNA AD-81725) and control (PBS) groups before treatment and each month for 6 months after the first dose. DETAILED DESCRIPTION OF THE INVENTION

[0146] iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). This paper describes iRNA and the method of using them to modulate (e.g., inhibit) the expression of LECT2 gene. Also provided are compositions and methods for treating disorders related to LECT2 expression, such as amyloidosis (e.g., LECT2 amyloidosis).

[0147] The iRNAs of the compositions featured herein comprise an RNA strand (antisense strand) having a region of 30 nucleotides or less, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, that is substantially complementary to at least a portion of the mRNA transcript of the LECT2 gene (also referred to herein as a "LECT2-specific iRNA"). The use of such iRNAs allows for targeted degradation of the mRNA of genes involved in disorders associated with LECT2 expression, as described herein. Very low doses of LECT2-specific iRNAs can specifically and efficiently mediate RNAi, resulting in significant inhibition of LECT2 gene expression. iRNAs targeting LECT2 can specifically and efficiently mediate RNAi, resulting in significant inhibition of LECT2 gene expression, which can be assessed, for example, in cell-based assays.

[0148] The following description discloses methods for making and using compositions containing iRNA to modulate (e.g., inhibit) expression of the LECT2 gene, as well as compositions and methods for treating disorders associated with expression of the LECT2 gene.

[0149] Embodiments of the pharmaceutical compositions featured herein include an iRNA having an antisense strand that includes a region of 30 nucleotides or less in length, generally 19-24 nucleotides in length, that is substantially complementary to at least a portion of an RNA transcript of the LECT2 gene.

[0150] In some aspects, featured herein are pharmaceutical compositions containing a LECT2 iRNA and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the LECT2 gene, and methods of using the pharmaceutical compositions to treat disorders associated with expression of the LECT2 gene (e.g., LECT2 amyloidosis).

[0151] I. Definition For convenience, the meanings of certain terms and phrases used in the specification, examples, and appended claims are provided below. If there is an apparent discrepancy between the use of a term in other parts of this specification and its definition provided in this section, the definition in this section shall control.

[0152] As used herein, "LECT2" refers to leukocyte chemotactic factor 2 (also known as leukocyte-derived chemotaxin 2, chondromodulin-II, chm-II, or chm2). See, for example, Yamagoe S et al. Genomics, 1998 Mar 15; 48(3):324-9. LECT2 was first identified as a novel neutrophil chemotactic protein and is identical to chondromodulin II, a growth stimulatory factor for chondrocytes and osteoblasts. The human LECT2 gene has been mapped to chromosome 5q31.1-q32. Ibid.

[0153] The sequence of human LECT2 mRNA transcript can be found in NM_002302.2 (SEQ ID NO: 1), the sequence of mouse LECT2 mRNA can be found in NM_010702.1 and NM_010702.2, and the sequence of rat LECT2 mRNA can be found in NM_001108405.1.

[0154] The human LECT2 protein is a secreted 16 kDa protein. LECT2 protein is secreted from the liver. It has high sequence similarity to the chondromodulin repeat region of chicken myb-induced myeloid 1 protein (www.genecards.org / cgi-bin / carddisp.pl?gene=LECT2; accessed August 29, 2013). Polymorphisms in the LECT2 gene are associated with rheumatoid arthritis. Ibid.

[0155] LECT2 is expressed in various tissues, including the brain, stomach, and liver. Koshimizu, Y & Ohtomi, M. (2010) Brain Res. 1311:1-11. In a study using indirect immunoperoxidase staining to examine LECT2 expression in normal and diseased human organs and tissues other than the liver, LECT2 was generally found to be expressed in blood vessels, endothelial cells, and smooth muscle cells, adipocytes, brain neurons, apical squamous epithelial cells, parathyroid cells, sweat and sebaceous gland epithelium, Hassall's corpuscles, and some mononuclear cells in immune hematopoietic tissues. The protein was generally negative, but occasionally stained positive in osteoblasts, chondrocytes, cardiac and skeletal muscle cells, smooth muscle cells of the gastrointestinal tract, and epithelial cells of some tissues. Nagai et al. (1998) Pathol Int. 48(11):882-6.

[0156] The human LECT2 gene encodes 151 amino acids, including an 18-amino acid signal peptide. The secreted protein has 133 residues. A G / A polymorphism (codon change GTC to ATC) at nucleotide 172 in exon 3 of the gene has been identified, resulting in the presence of either a valine or an isoleucine at position 58 of the unprocessed protein (or position 40 of the mature protein). The overall frequency of the G allele is 0.477, ranging from 0.6 to 0.7 in individuals of European descent. See Benson, MD et al. (2008) Kidney International, 74: 218-222; Murphy, CL et al. (2010) Am J Kidney Dis, 56(6):1100-1107. Patients with LECT2 amyloidosis are typically homozygous for the G allele. Without wishing to be bound by theory, it has been suggested that the substitution of a buried isoleucine (A allele) side chain with valine (G allele) may destabilize the protein and possibly account for the amyloidogenic tendency of this LECT2 variant. Murphy, CL et al. (2010) Am J Kidney Dis, 56(6):1100-1107.

[0157] As used herein, "LECT2 amyloidosis" or "ALECT2" includes amyloidosis associated with the deposition of amyloid or amyloid fibrils containing a LECT2 protein (e.g., any polymorphic variant of a LECT2 protein) or a portion of a LECT2 protein. The LECT2 protein may be a mutant (e.g., mutant) LECT2 protein. Amyloidosis may be systemic or localized. In some embodiments, LECT2 amyloidosis is associated with amyloid deposition in the kidney and / or liver.

[0158] "G", "C", "A", "T" and "U" each generally represent the nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively.However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to the modified nucleotide or surrogate replacement moiety, as further described below.Those skilled in the art will fully recognize that guanine, cytosine, adenine and uracil can be substituted with other moieties without substantially changing the base pairing properties of the oligonucleotide that contains the nucleotide with such a replacement moiety.For example, but not limited to, a nucleotide that contains inosine as its base can form base pairs with the nucleotide that contains adenine, cytosine or uracil.Therefore, the nucleotide that contains uracil, guanine or adenine can be substituted with the nucleotide that contains inosine, for example, in the nucleotide sequence of the dsRNA that is featured in the present disclosure.In another example, adenine and cytosine in any location in an oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU wobble base pair with target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in this disclosure.

[0159] As used herein, the terms "iRNA," "RNAi," "iRNA agent," or "RNAi agent" refer to an agent that contains RNA, as that term is defined herein, and mediates targeted cleavage of an RNA transcript, e.g., via the RNA-induced silencing complex (RISC) pathway. In one embodiment, the iRNA described herein results in inhibition of ALECT2 expression. Inhibition of ALECT2 expression can be assessed based on a reduction in ALECT2 mRNA levels or a reduction in ALECT2 protein levels. As used herein, a "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the ALECT2 gene, including the mRNA that is the product of RNA processing of the primary transcript. The target portion of the sequence is at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion. For example, the target sequence is generally 9 to 36 nucleotides in length, e.g., 15 to 30 nucleotides in length, including all subranges therebetween. By way of non-limiting example, the target sequence may be 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, The amino acid sequence may be 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.

[0160] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0161] As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence in relation to a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions, as will be understood by those skilled in the art. Such conditions can be, for example, stringent conditions, such as 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 o C or 70 o The incubation time may include incubation at 4°C for 12-16 hours, followed by washing. Other conditions may be applied, such as physiologically relevant conditions that may be encountered in an organism. Those skilled in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences, depending on the ultimate application of the hybridized nucleotides.

[0162] Complementary sequences in iRNA, for example, in dsRNA described herein, include base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence across the entire length of one or both nucleotide sequences.Such sequences can be referred to herein as "fully complementary" to each other.However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences can be completely complementary, or they can form one or more, but generally no more than 5, 4, 3 or 2 mismatched base pairs during hybridization for a duplex of up to 30 base pairs, while retaining the ability to hybridize under conditions most relevant to their final application, such as inhibiting gene expression through the RISC pathway.However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, these overhangs shall not be considered mismatches in determining complementarity. For example, a dsRNA comprising one oligonucleotide that is 21 nucleotides in length and another oligonucleotide that is 23 nucleotides in length comprises a 21 nucleotide sequence in which the longer oligonucleotide is perfectly complementary to the shorter oligonucleotide, and may further be referred to as "perfectly complementary" for purposes described herein.

[0163] As used herein, a "complementary" sequence may also include, or be formed entirely from, non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, so long as the above requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

[0164] The terms "complementary," "fully complementary," and "substantially complementary" herein can be used in reference to base matching between the sense and antisense strands of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as understood in the context of their use.

[0165] As used herein, a polynucleotide "substantially complementary to at least a portion" of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of a target mRNA (e.g., an mRNA encoding an ALECT2 protein). For example, a polynucleotide is complementary to at least a portion of a LECT2 mRNA if its sequence is substantially complementary to an uninterrupted portion of an mRNA encoding LECT2. As another example, a polynucleotide is complementary to at least a portion of a LECT2 mRNA if its sequence is substantially complementary to an uninterrupted portion of an mRNA encoding LECT2.

[0166] The term "double-stranded RNA" or "dsRNA," as used herein, refers to an iRNA comprising an RNA molecule or molecular complex having a hybridized duplex region comprising two antiparallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations with respect to the target RNA. The duplex region can be of any length that allows for specific degradation of the desired target RNA, for example, via the RISC pathway, but is typically in the range of 9-36 base pairs in length, e.g., 15-30 base pairs in length. Considering a duplex of 9 to 36 base pairs, the duplex may be any duplex within this range, e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36, and any subrange therebetween, e.g., but not limited to, 15 to 30 base pairs, 15 to 26 base pairs, 15 to 23 base pairs, 15 to 22 base pairs, 15 to 21 base pairs, 15 to 20 base pairs, 15 to 19 base pairs, 15 to 18 base pairs, 15 to 17 base pairs, 18 to 30 base pairs, and the like. dsRNA can be any length between 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNA generated in cells by processing with Dicer and similar enzymes is generally 19-22 base pairs in length. One strand of the double-stranded region of dsDNA contains a sequence that is substantially complementary to a region of the target RNA. The two strands that form the duplex structure can be derived from a single RNA molecule with at least one self-complementary region, or can be formed from two or more separate RNA molecules.When the double-stranded region is formed from two strands of a single molecule, the molecule can have a double-stranded region separated by a single strand of nucleotides (referred to herein as a "hairpin loop") between the 3'-end of one strand and the 5'-end of each of the other strands that form the double-stranded structure. The hairpin loop can contain at least one unpaired nucleotide; in some embodiments, the hairpin loop can contain at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules can, but do not need to, be covalently linked. When the two strands are covalently linked by means other than a hairpin loop, the linking structure is called a "linker." The term "siRNA" is also used herein to refer to dsRNA as described above.

[0167] In another embodiment, an iRNA agent can be a "single-stranded siRNA" introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides long and chemically modified. The design and testing of single-stranded siRNAs is described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150: 883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein (e.g., the sequences provided in Tables 2A-2B, 3A-3B, 6, or 7) can be used as single-stranded siRNAs as described herein or chemically modified by the methods described in Lima et al., (2012) Cell 150: 883-894.

[0168] Those skilled in the art will recognize that the term "RNA molecule" or "ribonucleic acid molecule" includes not only naturally occurring or occurring RNA molecules, but also RNA analogs and derivatives containing one or more ribonucleotide / ribonucleoside analogs or derivatives described herein or known in the art. Strictly speaking, a "ribonucleoside" contains a nucleoside base and a ribose sugar, and a "ribonucleotide" is a ribonucleoside having one, two, or three phosphate moieties. However, the terms "ribonucleoside" and "ribonucleotide" can be considered equivalent as used herein. RNA can be modified in the nucleobase structure, ribose structure, or ribose-phosphate backbone structure, for example, as described herein below. However, molecules containing ribonucleoside analogs or derivatives must retain the ability to form duplexes. As non-limiting examples, the RNA molecule can also include at least one modified ribonucleoside containing, but not limited to, a 2'-O-methyl modified nucleoside, a nucleoside containing a 5' phosphorothioate group, a terminal nucleoside linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, an acyclic nucleoside, a glycol nucleotide, a 2'-deoxy-2'-fluoro modified nucleoside, a 2'-amino modified nucleoside, a 2'-alkyl modified nucleoside, a morpholino nucleoside, a phosphoramidate or non-natural base containing nucleoside, or any combination thereof. Alternatively, the RNA molecule can contain at least two modified ribonucleosides, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty or more, up to the full length of the dsRNA molecule. The modification need not be the same for each of such multiple modified ribonucleosides in the RNA molecule.In one embodiment, modified RNAs contemplated for use in the methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure, enabling or mediating the specific degradation of a target RNA, e.g., via the RISC pathway.

[0169] In one aspect, modified ribonucleosides include deoxyribonucleosides. In such examples, iRNA agents can include, for example, one or more deoxynucleosides, including deoxynucleoside overhangs, or one or more deoxynucleosides within the double-stranded portion of a dsRNA. In certain embodiments, the RNA molecule includes, for example, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more (but not 100%) percentages of deoxyribonucleosides in one or both strands. In other embodiments, the term "iRNA" does not encompass double-stranded DNA molecules (e.g., naturally occurring double-stranded DNA molecules or DNA molecules containing 100% deoxynucleosides).

[0170] In one embodiment, RNA interference agents comprise single-stranded RNAs that interact with target RNA sequences and direct the cleavage of target RNAs. Without wishing to be bound by theory, long double-stranded RNAs introduced into cells are degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer is a ribonuclease III-like enzyme that processes dsRNA into short interfering RNAs of 19-23 base pairs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). When bound to the appropriate target mRNA, one or more endonucleases in RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect, the present disclosure relates to a single-stranded RNA that promotes the formation of a RISC complex to cause target gene silencing.

[0171] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of an iRNA, such as a dsRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. A dsRNA can include an overhang of at least one nucleotide; alternatively, the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang can include or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of the overhang can be present at the 5'-end, the 3'-end, or both ends of either the antisense strand or the sense strand of a dsRNA.

[0172] In one embodiment, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at the 3'-end and / or 5'-end. In one embodiment, the sense strand of the dsRNA has a 1-10 nucleotide overhang at the 3'-end and / or 5'-end. In another embodiment, one or more nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0173] The term "blunt end" or "blunt-end" used herein in relation to dsRNA means that there is no unpaired nucleotide or nucleotide analog at given end of dsRNA, that is, there is no nucleotide overhang.One or both ends of dsRNA can be blunt.If both ends of dsRNA are blunt, dsRNA is called blunt-end.For clarity, "blunt-end" dsRNA is blunt at both ends, that is, there is no nucleotide overhang at both ends of molecule.In most cases, this molecule is double-stranded throughout its entire length.

[0174] The term "antisense strand" or "guide strand" refers to the strand of iRNA, for example, dsRNA, that comprises a region that is substantially complementary to a target sequence.As used herein, the term "complementary region" refers to the region on the antisense strand that is substantially complementary to a sequence, for example, a target sequence, as defined herein.If the complementary region is not completely complementary to the target sequence, mismatches can be located in the internal or terminal regions of the molecule.In some embodiments, the complementary region comprises 0, 1, or 2 mismatches.

[0175] The term "sense strand" or "passenger strand," as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as that term is defined herein.

[0176] As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles.SNALP refers to lipid vesicles that coat a reduced aqueous interior containing nucleic acid such as iRNA or a plasmid into which iRNA is transcribed.SNALP is described, for example, in U.S. Patent Application Publication No. 2006 / 0240093, U.S. Patent Application Publication No. 2007 / 0135372, and International Patent Application Publication No. 2009 / 082817.These applications are incorporated herein by reference in their entirety.

[0177] "Introduction into a cell," when referring to iRNA, means facilitating or resulting in uptake or absorption into a cell, as understood by those skilled in the art. Absorption or uptake of iRNA can occur via diffusive or active cellular processes alone, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; iRNA can also be "introduced into a cell," where the cell is part of a living organism. In such cases, introduction into a cell includes delivery to the organism. For example, for in vivo delivery, iRNA can be injected into a tissue site or administered systemically. In vivo delivery is also possible via β-glucan delivery systems, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Patent Application Publication No. 2005 / 0281781, which are incorporated herein by reference in their entireties. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein below or are known in the art.

[0178] As used herein, the term "modulate expression" refers to at least partial "inhibition" or partial "activation" of LECT2 gene expression in cells treated with an iRNA composition described herein compared to the expression of LECT2 in control cells. Control cells include untreated cells or cells treated with a non-targeting control iRNA.

[0179] The terms "activate," "enhance," "upregulate expression," "increase expression," and the like, as they refer to the LECT2 gene, refer herein to at least partial activation of expression of the LECT2 gene, as manifested by an increase in the amount of LECT2 mRNA that can be isolated or detected from a first cell or group of cells that have been treated to transcribe and increase expression of the LECT2 gene, compared to a second cell or group of cells that is substantially identical to the first cell or group of cells that have not been so treated (control cells).

[0180] In one embodiment, the expression of the LECT2 gene is activated by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administering an iRNA described herein. In some embodiments, the expression of the LECT2 gene is activated by at least about 60%, 70%, or 80% by administering an iRNA described herein. In some embodiments, the expression of the LECT2 gene is activated by at least about 85%, 90%, or 95% or more by administering an iRNA described herein. In some embodiments, the expression of the LECT2 gene is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more in cells treated with an iRNA described herein compared to expression in untreated cells. Activation of expression by small dsRNA is described, for example, in Li et al., 2006 Proc. Natl. Acad. Sci. USA 103:17337-42, and U.S. Patent Application Publication Nos. 2007 / 0111963 and 2005 / 226848, each of which is incorporated herein by reference.

[0181] The terms "silence," "inhibit expression," "downregulate expression," "suppress expression," and the like, when referring to the LECT2 gene, refer herein to at least partial suppression of LECT2 gene expression, as assessed, for example, based on LECT2 mRNA expression, LECT2 protein expression, or another parameter functionally linked to LECT2 gene expression. For example, inhibition of LECT2 expression can be represented by a reduction in the amount of LECT2 mRNA that can be isolated or detected from a first cell or group of cells in which the LECT2 gene is transcribed and that has been treated so that expression of the LECT2 gene is inhibited compared to a control. The control can be a second cell or group of cells that is substantially identical to the first cell or group of cells, except that the second cell or group of cells has not been so treated (control cells). The degree of inhibition is usually expressed as a percentage of the control level, e.g.,

[0182]

number

[0183] It is expressed as:

[0184] Alternatively, the degree of inhibition can be given in terms of a parameter functionally linked to LECT2 gene expression, such as the reduction in the amount of protein encoded by the LECT2 gene.The reduction of a parameter functionally linked to LECT2 gene expression can also be expressed as a percentage of the control level.In principle, LECT2 gene silencing can be determined in any cell that expresses LECT2, either constitutively or by genome engineering, and by any suitable assay.

[0185] For example, in certain instances, expression of the LECT2 gene is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administering an iRNA disclosed herein. In some embodiments, expression of the LECT2 gene is suppressed by at least about 60%, 65%, 70%, 75%, or 80% by administering an iRNA disclosed herein. In some embodiments, expression of the LECT2 gene is suppressed by at least about 85%, 90%, 95%, 98%, 99%, or more by administering an iRNA described herein.

[0186] In the context of the present disclosure, the terms "treat," "treatment," and the like refer to preventing, reducing, or alleviating at least one symptom associated with a disorder associated with LECT2 expression, or slowing or reversing the progression or predicted progression of such a disorder. For example, the methods featured herein, when used to treat LECT2 amyloidosis, can help inhibit amyloid deposition, reduce or prevent one or more symptoms of amyloidosis, or reduce the risk or severity of an associated condition (e.g., nephrotic syndrome or hepatitis). Thus, unless the context clearly indicates otherwise, the terms "treat," "treatment," and the like are intended to encompass prophylaxis, e.g., prevention of a disorder and / or symptoms of a disorder associated with LECT2 expression.

[0187] "Lower" in the context of a disease marker or symptom means any decrease, e.g., a statistically or clinically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. The decrease can be to a level accepted as being within the normal range for individuals without such disorder.

[0188] As used herein, the phrases "therapeutically effective amount" and "prophylactically effective amount" refer to the amount that provides therapeutic benefit in the treatment, prevention or management of any disorder or pathological process associated with LECT2 expression.The specific amount that is therapeutically effective can vary depending on factors known in the art, such as the type of disorder or pathological process, the patient's medical history and age, the stage of disorder or pathological process, and the administration of other therapeutic agents.

[0189] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of an iRNA and a pharmaceutically acceptable carrier. As used herein, a "pharmacologically effective amount," "therapeutically effective amount," or simply "effective amount" refers to that amount of an iRNA effective to produce the intended pharmacological, therapeutic, or preventative result. For example, in a method for treating a disorder associated with LECT2 expression (e.g., LECT2 amyloidosis), an effective amount includes an amount effective to reduce one or more symptoms associated with LECT2 amyloidosis, an amount effective to inhibit amyloid deposition (e.g., LECT2 amyloid deposition), or an amount effective to reduce the risk of developing a condition associated with LECT2 amyloidosis. For example, if a given clinical treatment is considered effective if there is at least a 10% reduction in a measurable parameter associated with the disease or disorder, then a therapeutically effective amount of a drug for treating that disease or disorder is the amount necessary to achieve at least a 10% reduction in that parameter. For example, a therapeutically effective amount of an iRNA targeting LECT2 can reduce LECT2 mRNA levels or LECT2 protein levels by a measurable amount, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0190] The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. This term specifically excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include pharmaceutically acceptable excipients, such as, but not limited to, inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents, and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, and corn starch and alginic acid are suitable disintegrants. Binders may include starch and gelatin, while lubricants, if present, are generally magnesium stearate, stearic acid, or talc. If desired, tablets can be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract. The agents contained in the drug formulation are further described herein below.

[0191] When referring to a numerical value or numerical range, the term "about" means that the referenced numerical value or numerical range is approximate within experimental variability (or within statistical experimental error), and thus the numerical value or numerical range may vary, for example, between 1% and 15% of the stated number or numerical range.

[0192] II. iRNA Agents Described herein are iRNA agents that modulate (eg, inhibit) expression of the LECT2 gene.

[0193] In some embodiments, the iRNA agent activates expression of a LECT2 gene in a cell or mammal.

[0194] In some embodiments, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of the LECT2 gene in a cell or subject (e.g., a mammal, e.g., a human), wherein the dsRNA comprises an antisense strand having a region of complementarity that is complementary to at least a portion of an mRNA formed upon expression of the LECT2 gene, wherein the region of complementarity is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and wherein the dsRNA inhibits expression of the LECT2 gene, e.g., by at least 10%, 20%, 30%, 40%, or 50%, upon contact with a cell expressing the LECT2 gene.

[0195] The modulation (e.g., inhibition) of the expression of the LECT2 gene can be assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods such as Western blot. The expression of the LECT2 gene in cell cultures such as COS cells, HeLa cells, primary hepatocytes, HepG2 cells, primary cultured cells, or biological samples from subjects can be assayed by measuring LECT2 mRNA levels, for example, by bDNA or TaqMan assay, or by measuring protein levels, for example, by immunofluorescence analysis using Western blotting or flow cytometry techniques.

[0196] dsRNA contains two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a region of complementarity that is substantially complementary, and generally perfectly complementary, to a target sequence derived from the sequence of the mRNA formed during expression of the LECT2 gene. The other strand (the sense strand) contains a region that is complementary to the antisense strand so that the two strands hybridize to form a duplex structure when combined under appropriate conditions. Typically, the duplex structure is 15 to 30 base pairs (inclusive), more commonly 18 to 25 base pairs (inclusive), even more commonly 19 to 24 base pairs (inclusive), and most commonly 19 to 21 base pairs (inclusive) in length. Similarly, the region of complementarity to the target sequence is 15 to 30 (inclusive), more usually 18 to 25 (inclusive), even more usually 19 to 24 (inclusive), and most usually 19 to 21 (inclusive) nucleotides in length.

[0197] In some embodiments, the dsRNA is 15-20 nucleotides in length (inclusive), and in other embodiments, the dsRNA is 25-30 nucleotides in length (inclusive). As those skilled in the art will recognize, the targeted region of an RNA targeted for cleavage is often a portion of a larger RNA molecule, often an mRNA molecule. In relevant cases, a "portion" of an mRNA target is a contiguous sequence of the mRNA target long enough to serve as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). dsRNA with duplexes as short as 9 base pairs can mediate RNAi-directed RNA cleavage under some circumstances. In most cases, the target is at least 15 nucleotides in length, e.g., 15-30 nucleotides in length.

[0198] Those skilled in the art will also recognize that the duplex region is the primary functional portion of a dsRNA, e.g., a 9-36 duplex region, e.g., 15-30 base pairs. Thus, in one embodiment, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA, to the extent that it targets a desired RNA for cleavage, e.g., becomes processed into a 15-30 base pair functional duplex. Thus, those skilled in the art will recognize that, in one embodiment, an miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting LECT2 expression is not generated in a target cell by cleavage of a larger dsRNA.

[0199] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs.DsRNA can be synthesized by standard methods known in the art, for example, by using automated DNA synthesizer, such as that commercially available from Biosearch, Applied Biosystems, Inc., as will be further discussed below.

[0200] In one embodiment, the LECT2 gene is a human LECT2 gene. In another embodiment, the LECT2 gene is a mouse or rat LECT2 gene.

[0201] In certain embodiments, the dsRNA comprises a sense strand comprising or consisting of a sense sequence selected from the sense sequences provided in Tables 2A-2B, 3A-3B, 6, or 7, and an antisense strand comprising or consisting of an antisense sequence selected from the antisense sequences provided in Tables 2A-2B, 3A-3B, 6, or 7.

[0202] In one embodiment, the dsRNA comprises at least a sense and an antisense nucleotide sequence, whereby the sense strand is selected from the sequences provided in Tables 2A-2B, 3A-3B, 6 or 7, and the corresponding antisense strand is selected from the sequences provided in Tables 2A-2B, 3A-3B, 6 or 7.

[0203] In these embodiments, one of the two sequences is complementary to the other of the two sequences, and one sequence is substantially complementary to the sequence of the mRNA produced by the expression of LECT2 gene.Therefore, dsRNA comprises two oligonucleotides, one oligonucleotide is described as sense strand, and the second oligonucleotide is described as corresponding antisense strand.As described elsewhere herein and known in the art, the complementary sequences of dsRNA can be contained as the self-complementary region of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

[0204] Those skilled in the art are well aware that the dsRNA with 20-23 base pairs, especially with 21 base pairs of double-stranded structure, is praised as being particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888).However, some people find that shorter or longer RNA double-stranded structure can also be effective.

[0205] In the above-described embodiments, due to the nature of the oligonucleotide sequences provided in Tables 2A-2B, 3A-3B, 6, or 7, the dsRNAs described herein can comprise at least one strand at least 19 nucleotides in length. It can be reasonably expected that shorter duplexes having one of the sequences in Tables 2A-2B, 3A-3B, 6, or 7, minus just a few nucleotides at one or both ends, will be similarly effective compared to the dsRNAs described above.

[0206] In some embodiments, the dsRNA has a subsequence of at least 15, 16, 17, 18, 19, 20 or more contiguous nucleotides from one of the sequences in Tables 2A-2B, 3A-3B, 6 or 7.

[0207] In some embodiments, the dsRNA has an antisense sequence comprising at least 15, 16, 17, 18, or 19 contiguous nucleotides of the antisense sequence provided in Tables 2A-2B, 3A-3B, 6, or 7, and a sense sequence comprising at least 15, 16, 17, 18, or 19 contiguous nucleotides of the corresponding sense sequence provided in Tables 2A-2B, 3A-3B, 6, or 7.

[0208] In some embodiments, the dsRNA comprises an antisense sequence comprising at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of the antisense sequence provided in Tables 2A-2B or 3A-3B, and a sense sequence comprising at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides of the corresponding sense sequence provided in Tables 2A-2B, 3A-3B, 6, or 7.

[0209] In some embodiments, the dsRNA comprises an antisense sequence comprising at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of the antisense sequence provided in Table 2A or 2B, and a sense sequence comprising at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides of the corresponding sense sequence provided in Table 2A or 2B.

[0210] In some embodiments, the dsRNA comprises an antisense sequence comprising at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of the antisense sequence provided in Table 3A or 3B, and a sense sequence comprising at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides of the corresponding sense sequence provided in Table 3A or 3B.

[0211] In some embodiments, the dsRNA comprises an antisense sequence comprising at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of the antisense sequence provided in Table 6 or 7, and a sense sequence comprising at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides of the corresponding sense sequence provided in Table 6 or 7.

[0212] In some such embodiments, the dsRNA comprises only a portion of the sequence provided in Tables 2A-2B, 3A-3B, 6, or 7, but is equally effective in inhibiting the level of LECT2 expression as a dsRNA comprising the full-length sequence provided in Tables 2A-2B, 3A-3B, 6, or 7. In some embodiments, the dsRNA differs in inhibiting the expression level of the LECT2 gene by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% inhibition compared to a dsRNA comprising the entire sequence disclosed herein.

[0213] The iRNAs provided in Tables 2A-2B, 3A-3B, 4A-4B, or 5-7 identify sites in the LECT2 transcript that are susceptible to RISC-mediated cleavage. Accordingly, the present disclosure further characterizes iRNAs that target within one of such sequences. As used herein, an iRNA is said to target within a specific site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that specific site. Such iRNAs generally comprise at least 15 contiguous nucleotides from one of the sequences provided in Tables 2A-2B, 3A-3B, 4A-4B, or 5-7 coupled to an additional nucleotide sequence obtained from a region contiguous to the selected sequence in the LECT2 gene.

[0214] Target sequences are generally 15-30 nucleotides long, although there is wide variation in the suitability of specific sequences within this range for directing cleavage of any given target RNA. While various software packages and the guidelines described herein provide guidance for identifying optimal target sequences for any given gene target, an empirical approach can also be taken, in which a "window" or "mask" of a given size (for example, 21 nucleotides) is placed literally or graphically (including, for example, in silico) on the target RNA sequence to identify sequences within a size range that can serve as target sequences. By incrementally moving the sequence "window" one nucleotide upstream or downstream of the initial target sequence position, subsequent potential target sequences can be identified until a complete set of possible sequences has been identified for any given target size selected. This process, combined with systematic synthesis and testing of the identified sequences (using assays described herein or known in the art), identifies sequences that can optimally identify RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, for example, the sequences identified in Tables 2A-2B, 3A-3B, 6, or 7 represent effective target sequences, but it is contemplated that further optimization of inhibitory efficiency can be achieved by incrementally "window-walking" one nucleotide upstream or downstream of a given sequence to identify sequences with comparable or better inhibitory properties.

[0215] It is further contemplated that further optimization can be achieved by testing any identified sequences, e.g., in Tables 2A-2B, 3A-3B, 6, or 7, by either adding or removing nucleotides to generate longer or shorter sequences, and then walking up or down the longer or shorter size windows of the target RNA from that point. Furthermore, combining this approach with testing the effectiveness of iRNAs based on the target sequence in inhibition assays known in the art or described herein can result in further improvements in the efficiency of inhibition. Furthermore, such optimized sequences can be adjusted, for example, by introducing modified nucleotides described herein or known in the art, adding or changing overhangs, or other modifications known in the art and / or discussed herein, to further optimize the molecule as an expression inhibitor (e.g., increasing serum stability or circulatory half-life, increasing thermostability, enhancing transmembrane delivery, targeting to specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.).

[0216] The iRNAs described herein can contain one or more mismatches to the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches. When the antisense strand of an iRNA contains mismatches to the target sequence, it is preferable that the mismatched region is not located in the center of the complementary region. When the antisense strand of an iRNA contains mismatches to the target sequence, it is preferable that the mismatches be limited to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, for a 23-nucleotide iRNA agent RNA strand complementary to a region of the LECT2 gene, the RNA strand generally does not contain mismatches within the central 13 nucleotides. Using methods described herein or known in the art, it can be determined whether an iRNA containing mismatches to the target sequence is effective in inhibiting LECT2 gene expression. Considering the effectiveness of iRNAs with mismatches in inhibiting LECT2 gene expression is important, especially when the specific complementary region in the LECT2 gene is known to have polymorphic sequence variations within the population.

[0217] In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, typically 1 or 2, nucleotides. dsRNAs with at least one nucleotide overhang have unexpectedly superior inhibitory properties compared to their blunt-ended counterparts. In yet another embodiment, the RNA (e.g., dsRNA) of the iRNA is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in this disclosure can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. Modifications include, for example, (a) terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, reverse ligation, etc.), 3'-end modifications (conjugation, DNA nucleotides, inverted ligation, etc.), (b) base modifications, such as substitution with a stabilizing base, a destabilizing base, or a base that base-pairs with an expanded repertoire of partners, removal of a base (abasic nucleotide), or a conjugated base, (c) sugar modifications (e.g., at the 2'- or 4'-position, or an acyclic sugar) or sugar substitution, and (d) backbone modifications, such as modification or replacement of a phosphodiester linkage. Specific examples of RNA compounds useful in the present disclosure include, but are not limited to, RNAs containing modified backbones or RNAs that do not contain natural internucleoside linkages. RNAs with modified backbones include, among others, RNAs that do not have a phosphorus atom in the backbone. For purposes of this specification, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in the internucleoside backbone can also be considered oligonucleosides. In certain embodiments, a modified RNA has a phosphorus atom in its internucleoside backbone.

[0218] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and 3'-alkylene phosphonates and other alkyl phosphonates, including chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reverse polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0219] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717 No. 5,321,131; No. 5,399,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,47 No. 6,925; No. 5,519,126; No. 5,536,821; No. 5,541,316; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5,587,361; No. 5,625,050; No. 6,028,188; No. 6,124,445; No. 6,160,109; No. 6,169,170; No. 6,172, No. 209; No. 6,239,265; No. 6,277,603; No. 6,326,199; No. 6,346,614; No. 6,444,423; No. 6,531,590; No. 6 ,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent RE39464, each of which is incorporated herein by reference.

[0220] Modified RNA backbones that do not contain phosphate atoms therein have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 moieties.

[0221] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; and 5,470,967. ; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is incorporated herein by reference.

[0222] In other RNA mimics suitable or intended for use in iRNA, both the sugar and internucleoside linkages, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0223] Some embodiments featured in this disclosure include RNAs with phosphorothioate backbones and oligonucleotides with heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (also known as methylene (methylimino) or MMI backbones), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- (the natural phosphodiester backbone is represented as --O--P--O--CH2--) of the above-referenced U.S. Patent No. 5,489,677, and oligonucleotides with amide backbones of the above-referenced U.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structure of the above-referenced U.S. Patent No. 5,034,506.

[0224] Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl and alkynyl. Exemplary suitable modifications include O[(CH) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA includes at the 2' position one of the following: C1 to C 10The modification may include one of the following: lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group for improving the pharmacokinetic properties of iRNA, or group for improving the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the O(CH)ON(CH), also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE, group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH--O--CH--N(CH).

[0225] In other embodiments, the iRNA agent includes one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides (or nucleosides). In certain embodiments, the sense strand or antisense strand, or both the sense and antisense strands, include fewer than five acyclic nucleotides per strand (e.g., 4, 3, 2, or 1 acyclic nucleotide per strand). The one or more acyclic nucleotides can be found, for example, in the double-stranded region of the sense or antisense strand, or both strands; at the 5' end, the 3' end, or both the 5' and 3' ends of the sense or antisense strand, or both strands of the iRNA agent. In one embodiment, the one or more acyclic nucleotides are present in positions 1-8 of the sense strand or antisense strand, or both. In one embodiment, the one or more acyclic nucleotides are found in the antisense strand at positions 4-10 (e.g., positions 6-8) from the 5' end of the antisense strand. In another embodiment, the one or more acyclic nucleotides are found in one or both 3' overhangs of the iRNA agent.

[0226] The term "acyclic nucleotide" or "acyclic nucleoside," as used herein, refers to any nucleotide or nucleoside having an acyclic sugar, e.g., an acyclic ribose. Exemplary acyclic nucleotides or nucleosides can include a nucleobase, e.g., a naturally occurring or modified nucleobase (e.g., a nucleobase described herein). In certain embodiments, the bond between any of the ribose carbons (C1, C2, C3, C4, or C5), independently or in combination, is absent in a nucleotide. In one embodiment, the bond between the C2-C3 carbons of the ribose ring is absent, e.g., in an acyclic 2'-3'-seco-nucleotide monomer. In other embodiments, the bond between C1-C2, C3-C4, or C4-C5 is absent (e.g., in a 1'-2', 3'-4', or 4'-5'-seco nucleotide monomer). Exemplary acyclic nucleotides are disclosed in U.S. Pat. No. 8,314,227, the entire contents of which are incorporated herein by reference. For example, the acyclic nucleotide can include any of the monomers D through J in Figures 1-2 of U.S. Patent No. 8,314,227. In one embodiment, the acyclic nucleotide includes the following monomers:

[0227] [ka]

[0228] wherein the base is a nucleobase, e.g., a naturally occurring or modified nucleobase (e.g., a nucleobase described herein).

[0229] In certain embodiments, acyclic nucleotides can be modified or derivatized, particularly by coupling the acyclic nucleotide to another moiety, such as a ligand (e.g., GalNAc, cholesterol ligand), alkyl, polyamine, sugar, polypeptide.

[0230] In other embodiments, the iRNA agent includes one or more acyclic nucleotides and one or more LNAs (e.g., LNAs described herein). For example, one or more acyclic nucleotides and / or one or more LNAs can be present in the sense strand, the antisense strand, or both. The number of acyclic nucleotides in one strand can be the same or different from the number of LNAs in the opposite strand. In certain embodiments, the sense strand and / or antisense strand include fewer than five LNAs (e.g., four, three, two, or one LNA) located in the double-stranded region or 3' overhang. In other embodiments, one or two LNAs are located in the double-stranded region or 3' overhang of the sense strand. Alternatively, or in combination, the sense strand and / or antisense strand include fewer than five acyclic nucleotides (e.g., four, three, two, or one acyclic nucleotide) in the double-stranded region or 3' overhang. In one embodiment, the sense strand of an iRNA agent includes one or two LNAs in the 3' overhang of the sense strand and one or two acyclic nucleotides in the duplex region of the antisense strand of the iRNA agent (e.g., positions 4-10 (e.g., 6-8) from the 5' end of the antisense strand).

[0231] In other embodiments, inclusion of one or more acyclic nucleotides (alone or in addition to one or more LNAs) in an iRNA agent results in one or more (or all) of: (i) reduced off-target effects; (ii) reduced involvement of the passenger strand in RNAi; (iii) increased specificity of the guide strand for its target mRNA; (iv) reduced microRNA off-target effects; (v) increased stability; or (vi) increased resistance to degradation of the iRNA molecule.

[0232] Other modifications include 2'-methoxy (2'-OCH), 2'-5 aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide, or in 2'-5' linked dsRNA and the 5' position of the 5' terminal nucleotide. iRNAs can also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,42 Nos. 5,658,873; 5,670,633; and 5,700,920, several of which are commonly owned with the present application, and each of which is incorporated herein by reference.

[0233] iRNAs may also contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and uracil. and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-dazaadenine and 3-deazaguanine and 3-deazaadenine.

[0234] Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present disclosure. These include 5-substituted pyrimidines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST, and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are exemplary base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0235] Representative United States patents that teach certain preparations of the above-described modified nucleobases as well as other modified nucleobases include, but are not limited to, the above-mentioned U.S. Patent Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469 ... Nos. 5,594,121; 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, as well as U.S. Pat. No. 5,750,692, which is also incorporated herein by reference.

[0236] The RNA of an iRNA can also be modified to include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acids (also referred to herein as "locked nucleotides"). In one embodiment, a locked nucleic acid is a nucleotide having a modified ribose moiety, e.g., the ribose moiety includes an extra bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose into a 3'-endo structure. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum, increase thermal stability, and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0237] Representative United States patents that teach the preparation of locked nucleic acids include, but are not limited to, the following: United States Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; 7,399,845, and 8,314,227, each of which is incorporated herein by reference in its entirety.Exemplary LNAs include, but are not limited to, 2',4'-C methylene bicyclonucleotides (see, for example, Wengel et al., International PCT Application Publication Nos. WO 00 / 66604 and WO 99 / 14226).

[0238] In other embodiments, an iRNA agent includes one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. G-clamp nucleotides are modified cytosine analogs that confer the ability to hydrogen bond with both the Watson-Crick and Hoogsteen faces of complementary guanines within a duplex. See, e.g., Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to a complementary oligonucleotide. Inclusion of such nucleotides in an iRNA molecule can result in increased affinity and specificity for a nucleic acid target, complementary sequence, or template strand.

[0239] Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3''-phosphate, inverted base dT (idT), and the like. Disclosure of this modification is described in PCT Application WO 2011 / 005861.

[0240] iRNA motifs In one embodiment, the sense strand sequence has formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'(I) can be expressed by [In the formula, i and j are each independently 0 or 1; p and q each independently represent 0 to 6; each N a independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two different modified nucleotides; each N b independently represent an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p and n q independently represent overhanging nucleotides; N b and Y do not have the same modification; and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably, all YYY are 2'-F modified nucleotides.

[0241] In one embodiment, N a and / or N b includes alternating pattern modifications.

[0242] In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand.For example, when the RNAi agent has a double-stranded region of 17 to 23 nucleotides in length, the YYY motif can occur at or near the cleavage site of the sense strand (for example, at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13), and counting starts from the first nucleotide from the 5' end; or it can start from the first paired nucleotide in the double-stranded region from the 5' end.

[0243] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 1. Thus, the sense strand has the formula: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib); 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic); or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id) It can be expressed as:

[0244] When the sense strand is represented by formula (Ib), N brepresents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0245] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0246] When the sense strand is represented by formula (Id), each N b represents an oligonucleotide sequence containing, independently, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6. Each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0247] Each of X, Y and Z can be the same as or different from one another.

[0248] In other embodiments, i is 0 and j is 0, and the sense strand has the formula: 5'n p -N a -YYY-N a -n q 3'(Ia) It can be expressed as:

[0249] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0250] In one embodiment, the antisense strand sequence of the RNAi is represented by formula (II); 5'n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3'(II) can be expressed by [In the formula, k and l are each independently 0 or 1; p' and q' are each independently 0 to 6; each N a ' independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two different modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p ' and n q ' independently represents an overhanging nucleotide; N b ' and Y' do not have the same modification; and X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one of three identical modifications on three consecutive nucleotides].

[0251] In one embodiment, N a ' and / or N b ' includes alternating pattern modifications.

[0252] The Y'Y'Y' motif occurs at or near the cleavage site of the antisense strand. For example, if the RNAi agent has a duplex region 17 to 23 nucleotides long, the Y'Y'Y' motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting starting from the first nucleotide from the 5' end; or starting from the first paired nucleotide in the duplex region from the 5' end. Preferably, the Y'Y'Y' motif occurs at positions 11, 12, or 13.

[0253] In one embodiment, the Y'Y'Y' motif is all 2'-Ome modified nucleotides.

[0254] In one embodiment, k is 1 and l is 0, k is 0 and l is 1, or both k and l are 1.

[0255] Thus, the antisense strand has the following formula: 5'n q '-N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p '3'(IIb); 5'n q '-N a '-Y'Y'Y'-N b '-X'X'X'-n p '3' (IIc); or 5'n q '-N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p '3' (IId) It can be expressed as:

[0256] When the antisense strand is represented by formula (IIb), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a' represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0257] When the antisense strand is represented by formula (IId), each N b ' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6.

[0258] In other embodiments, k is 0 and l is 0, and the antisense strand has the formula: 5'n p '-N a '-Y'Y'Y'-N a '-n q '3'(Ia) It can be expressed as:

[0259] When the antisense strand is represented by formula (IIa), each N a ' represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0260] Each of X', Y' and Z' can be the same as or different from one another.

[0261] Each nucleotide of sense strand and antisense strand can be independently modified with LNA, HNA, CeNA, GNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl or 2'-fluoro.For example, each nucleotide of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.Each X, Y, Z, X', Y' and Z' can specifically represent 2'-O-methyl modification or 2'-fluoro modification.

[0262] In one embodiment, the sense strand of the RNAi agent can contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the duplex region is 21 nt, counting may start from the first nucleotide from the 5' end or from the first paired nucleotide within the duplex region from the 5' end, and Y represents a 2'-F modification. The sense strand can further contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region; XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0263] In one embodiment, the antisense strand can be a Y'Y'Y' motif occurring at positions 11, 12, or 13 of the strand, counting may start from the first nucleotide from the 5' end or from the first paired nucleotide in the duplex region from the 5' end, where Y' represents a 2'-O-methyl modification. The antisense strand can further include an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the duplex region, where X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0264] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the formulas (IIa), (IIb), (IIc), and (IId), respectively.

[0265] Thus, the RNAi agent used in the methods of the present disclosure can include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi duplex can have the formula (III): Sense:5'n p -N a -(XXX)iN b -YYY-N b -(ZZZ)jN a -n q 3' Antisense: 3'n p '-Na'-(X'X'X')kNb '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III) is represented by [In the formula, i, j, k, and l are each independently 0 or 1; p, p', q, and q' each independently represent 0 to 6; each N a and N a ' independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; During the ceremony, each n p ',n p , n q ', and n q each independently may or may not be present, and represents an overhanging nucleotide; and XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides].

[0266] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or i and j are both 0; or i and j are both 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or k and l are both 0; or k and l are both 1.

[0267] Exemplary combinations of sense and antisense strands that form RNAi duplexes include the following formulas: 5'n p -N a-YYY-N a -n q 3' 3'n p '-N a '-Y'Y'Y'-N a 'n q '5' IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'n p -N a '-Y'Y'Y'-N b '-Z'Z'Z'-Na'-nq'5' (IIIb) 5'n p -N a -XXX-N b -YYY-N a -n q 3' 3'n p -N a '-X'X'X'-N b '-Y'Y'Y'-Na'-n q '5' (IIIc) 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' 3'n p -N a '-X'X'X'-N b '-Y'Y'Y'-N b '-Z'Z'Z'-Na'-n q '5' (IIId)

[0268] When the RNAi agent is represented by formula (IIIa), each N a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0269] When the RNAi agent is represented by formula (IIIb), each N b represents an oligonucleotide sequence containing, independently, 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0270] When the RNAi agent is represented by formula (IIIc), each N b , N b ' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0271] When the RNAi agent is represented as formula (IIId), each N b , N b ' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N a N' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b and N b ' each independently includes an alternating pattern of modifications.

[0272] Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc) and (IIId) can be the same as or different from each other.

[0273] When an RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides can be base-paired with one of the Y' nucleotides. Alternatively, at least two of the Y nucleotides are base-paired with the corresponding Y' nucleotide, or all three of the Y nucleotides are base-paired with the corresponding Y' nucleotide.

[0274] When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can be base-paired with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides are base-paired with the corresponding Z' nucleotide, or all three of the Z nucleotides are base-paired with the corresponding Z' nucleotide.

[0275] When the RNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides can be base-paired with one of the X' nucleotides, or at least two of the X nucleotides can be base-paired with the corresponding X' nucleotide, or all three of the X nucleotides can be base-paired with the corresponding X' nucleotide.

[0276] In one embodiment, the modification on the Y nucleotide is different from the modification on the Y' nucleotide, the modification on the Z nucleotide is different from the modification on the Z' nucleotide, and / or the modification on the X nucleotide is different from the modification on the X' nucleotide.

[0277] In one embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or a 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n pIn yet another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage. a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p In another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

[0278] In one embodiment, when the RNAi agent is represented by formula (IIIa), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

[0279] In one embodiment, the RNAi agent is a multimer comprising at least two double strands represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the double strands are connected by a linker. The linker can be cleavable or non-cleavable. The multimer may further comprise a ligand. Each of the double strands can target the same gene or two different genes; or each of the double strands can target the same gene at two different target sites.

[0280] In one embodiment, the RNAi agent is a multimer comprising 3, 4, 5, 6 or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. The multimer may further comprise a ligand. Each duplex can target the same gene or two different genes; or each duplex can target the same gene at two different target sites.

[0281] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5' end, and one or both of the 3' ends may be conjugated to a ligand. Each agent can target the same gene or two different genes; or each agent can target the same gene at two different target sites.

[0282] iRNA conjugates The iRNA agent disclosed herein can be in the form of conjugate.Conjugate can be attached to any suitable position in iRNA molecule, for example, the 3'-end or 5'-end of sense strand or antisense strand.Conjugate can be attached via linker.

[0283] In some embodiments, an iRNA agent described herein is chemically linked to one or more ligands, moieties, or conjugates that can confer functionality, for example, by affecting (e.g., enhancing) the activity, cellular distribution, or cellular uptake of the iRNA. Such moieties include, but are not limited to, lipid moieties, e.g., cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), and the like. 20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylaminocarbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0284] In one embodiment, the ligand changes the distribution, targeting or life span of the iRNA agent that it is incorporated into.In some embodiments, the ligand provides enhanced affinity to selected targets, for example, molecules, cells or cell types, compartments, for example, cell or organ compartments, tissues, organs or regions of the living body, for example, compared to species that do not have such ligand.Typical ligands are not involved in double-stranded pairing in double-stranded nucleic acid.

[0285] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, for example, synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0286] The ligand can also include a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid, or protein, e.g., an antibody that binds to a specific cell type, such as a kidney cell. The targeting group can be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, biotin, or an RGD peptide or RGD peptidomimetic.

[0287] In some embodiments, the ligand is a GalNAc ligand comprising one or more N-acetylgalactosamine (GalNAc) derivatives. In some embodiments, the GalNAc ligand is used to target iRNA to the liver (e.g., hepatocytes). Further description of GalNAc ligands is provided in the section entitled "Carbohydrate Conjugates."

[0288] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (e.g., TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bismidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0289] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules with specific affinity for a co-ligand, or antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, e.g., lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-κB.

[0290] The ligand can be, for example, a substance, e.g., a drug, that can increase cellular uptake of the iRNA agent by, for example, disrupting the cytoskeleton of the cell, e.g., by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0291] In some embodiments, the ligands that bind to the iRNAs described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipid-soluble substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing multiple phosphorothioate linkages are also known to bind to serum proteins, and therefore, short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate linkages in the backbone, are also amenable to the present disclosure as ligands (e.g., PK-modulating ligands). Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0292] Ligand-conjugated oligonucleotides of the present disclosure can be synthesized by using an oligonucleotide bearing a pendant reactive functional group, such as that resulting from the attachment of a linking molecule to an oligonucleotide (described below). This reactive oligonucleotide can be reacted directly with a commercially available ligand, a ligand synthesized with any of a variety of protecting groups, or a ligand having a linking moiety attached to it.

[0293] The oligonucleotides used in the conjugates of the present disclosure can be conveniently and routinely produced through the well-known technique of solid phase synthesis.The equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, Calif.).Any other means for such synthesis known in the art can additionally or alternatively be used.It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0294] In the ligand-conjugated oligonucleotides and sequence-specific linked nucleosides bearing ligand-molecules of the present disclosure, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already bearing linking molecules, ligand-nucleotide or nucleoside-conjugate precursors already bearing ligand moieties, or building blocks bearing non-nucleoside ligands.

[0295] When using a nucleotide-conjugate precursor that already has a linking moiety, the synthesis of the sequence-specific linked nucleoside is typically completed, and then a ligand molecule is reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present disclosure are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to standard phosphoramidites and non-standard phosphoramidites that are commercially available and commonly used in oligonucleotide synthesis.

[0296] lipid conjugates In one embodiment, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecule can typically bind to serum proteins such as human serum albumin (HSA). HSA-binding ligands allow the conjugate to be distributed to target tissues. For example, the target tissue can be the liver, including liver parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, nephroxin or aspirin can be used. Lipid or lipid-based ligands can (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) be used to adjust the binding to serum proteins, such as HSA.

[0297] Lipid-based ligand can be used to modulate, for example, control (for example, inhibit) the binding of conjugate to target tissue.For example, the lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to kidney, and therefore is less likely to be removed from the body.The lipid or lipid-based ligand that binds weaker to HSA can be used to target conjugate to kidney.

[0298] In one embodiment, lipid-based ligand binds to HSA.For example, the ligand can bind to HSA with sufficient affinity, so that the distribution of the conjugate to non-renal tissues is enhanced.However, the affinity is usually not strong enough to reverse the HSA-ligand binding.

[0299] In another embodiment, the lipid-based ligand binds weakly or not at all to HSA, resulting in enhanced distribution of the conjugate to the kidney. Other moieties that target kidney cells can also be used in place of or in addition to the lipid-based ligand.

[0300] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, for example, proliferating cells.These are particularly useful for treating disorders characterized by unwanted cell proliferation, for example, malignant or non-malignant, for example, cancer cells.Exemplary vitamins include vitamins A, E, and K.Other exemplary vitamins include vitamin B, for example, folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by cancer cells.Also included are HSA and low-density lipoprotein (LDL).

[0301] Cell-penetrating agents In another aspect, the ligand is a cell-penetrating agent, for example, a helical cell-penetrating agent. In one embodiment, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennapetia. When the agent is a peptide, it can be modified, including peptidyl mimics, invertomers, non-peptide or pseudo-peptide linkages, and the use of D-amino acids. The helical agent is typically an α-helical agent and can have a lipophilic and lipophilic phase.

[0302] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. The attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, for example, by enhancing cellular recognition and uptake. The peptide or peptidomimetic portion can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0303] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 903). RFGF analogs containing a hydrophobic MTS [e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 904)] can also be targeting moieties. The peptide moiety can be a "delivery" peptide capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences from the HIV Tat protein [GRKKRRQRRRPPQ (SEQ ID NO: 905)] and the Drosophila Antennapedia protein [RQIKIWFQNRRMKWKK (SEQ ID NO: 906)] have been found to function as delivery peptides. The peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage display library or a one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Typically, the peptide or peptidomimetic tethered to the dsRNA agent via the incorporated monomer unit is a cell-targeting peptide, such as an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

[0304] The RGD peptides used in the compositions and methods of the present disclosure can be linear or cyclic, and can be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissue(s).RGD-containing peptides and peptidomimetics can contain D-amino acids, as well as synthetic RGD mimics.In addition to RGD, other moieties can be used to target integrin ligands.Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0305] RGD peptide moieties can be used to target specific cell types, for example, tumor cells such as endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can facilitate targeting of dsRNA agents to tumors in various other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, RGD peptides facilitate targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic and can be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissues. For example, glycosylated RGD peptides can target iRNA agents to α- V It can be delivered to tumor cells that express β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0306] A "cell-penetrating peptide" can penetrate cells, such as microbial cells such as bacterial or fungal cells, or mammalian cells such as human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or seropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bisected amphipathic peptide such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0307] Carbohydrate conjugates In some embodiments of the compositions and methods of the present disclosure, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use, as described herein. As used herein, "carbohydrate" refers to a compound that is either a carbohydrate itself, composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound having, as part thereof, a carbohydrate moiety composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include sugars of C5 or higher (e.g., C5, C6, C7, or C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0308] In one embodiment, the carbohydrate conjugate comprises a monosaccharide. In one embodiment, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates comprising one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in U.S. Patent No. 8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, the GalNAc conjugate serves as a ligand that targets iRNA to specific cells. In some embodiments, the GalNAc conjugate targets iRNA to liver cells, for example, by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

[0309] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be linked via a linker, for example, a bivalent or trivalent branched-chain linker. In some embodiments, the GalNAc conjugate is conjugated to the 3'-end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3'-end of the sense strand) via a linker, for example, a linker described herein.

[0310] In some embodiments, the GalNAc conjugate is:

[0311] [ka]

[0312] In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker, as shown in the schematic diagram below, where X is O or S.

[0313] [ka]

[0314] In some embodiments, the RNAi agent is conjugated to L96, as defined in Table 1 and shown below.

[0315] [ka]

[0316] In some embodiments, L96 is:

[0317] [ka]

[0318] In some embodiments, the carbohydrate conjugate for use in the compositions and methods of the present disclosure is selected from the group consisting of:

[0319] [ka] [ka] [ka] [ka] [ka]

[0320] Other exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to:

[0321] [ka]

[0322] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands described above, such as, but not limited to, a PK modulator and / or a cell-penetrating peptide.

[0323] In one embodiment, the iRNA of the present disclosure is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the present disclosure include, but are not limited to, the following:

[0324] [ka] [ka]

[0325] Linker In some embodiments, the conjugates or ligands described herein can be attached to iRNA oligonucleotides with various linkers that can be cleavable or non-cleavable.

[0326] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, e.g., covalently bonds two parts of a compound. A linker is typically a direct bond or an atom, such as oxygen or sulfur, a unit, such as NR, C(O), C(O)NH, SO, SO, SONH, or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkenyl, alkylheteroarylalkenyl, alkylheteroarylalkenyl, alkylheteroarylalkenyl, alkylheteroarylalkyn ...alkynyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkylheteroarylalkynyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkylheteroarylalkynyl, alkylheteroarylalken

[0044] The methylene groups include heteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclyl, alkynylheterocyclicalkenyl, alkynylheterocyclicalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, wherein one or more methylenes can be interrupted or terminated by O, S, S(O), SO, N(R), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, and R is hydrogen, acyl, aliphatic, or substituted aliphatic.In one embodiment, the linker is about 1 to 24 atoms, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18 atoms, 7 to 17, 8 to 17, 6 to 16, 7 to 16, or 8 to 16 atoms.

[0327] In one embodiment, the dsRNA of the present disclosure is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formulas (XXXI)-(XXXIV):

[0328] [ka]

[0329] [In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C independently represent 0 to 20 for each occurrence, and the repeat units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C are each independently, for each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5Cis independently, for each occurrence, absent, alkylene, substituted alkylenecholine, and one or more methylenes are O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C are each independently, for each occurrence, absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -CH(O)-CH(R a )-NH-, CO, CH=NO,

[0330] [ka]

[0331] or heterocyclyl];

[0332] L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand, i.e., for each occurrence, a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R a is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are particularly useful for use with RNAi agents to inhibit expression of target genes, such as those of formula (XXXV).

[0333] [ka]

[0334] [In the formula, L 5A , L 5B and L 5C represents a monosaccharide such as a GalNAc derivative].

[0335] Examples of GalNAc derivatives conjugated with suitable divalent and trivalent branched linker groups include, but are not limited to, the structures shown above as Formulas II, VII, XI, X, and XIII.

[0336] A cleavable linker is one that is sufficiently stable outside a cell but is cleaved upon entry into a target cell to release the two moieties held together by the linker. In preferred embodiments, the cleavable linker is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 60-fold, 70-fold, 80-fold, 90-fold or more, or at least about 100-fold faster in the target cell than in the target cell or under a first reference condition (which may, for example, be selected to mimic or represent intracellular conditions), in the subject's blood, or under a second reference condition (which may, for example, be selected to mimic or represent conditions found in blood or serum).

[0337] Cleavable linking groups are sensitive to cleaving agents, such as pH, redox potential, or the presence of degradable molecules.Generally, cleaving agents are more common in cells than in serum or blood, or are found at higher levels or activity.Examples of such degrading agents include redox agents that are selective for specific substrates or do not have substrate specificity, such as oxidases or reductases or reducing agents present in cells, such as mercaptans, which can degrade redox cleavable linking groups by reduction; esterases; endosomes or agents that can generate an acidic environment, such as those that cause a pH of 5 or less; enzymes that can hydrolyze or degrade acid cleavable linking groups by acting as general acids, peptidases (which can be substrate specific), and phosphatases.

[0338] Cleavable linkers, such as disulfide bonds, can be pH-sensitive. Human serum has a pH of 7.4, while the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers have cleavable linkers that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand intracellularly or into a desired compartment of the cell.

[0339] Linker can comprise a cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can depend on the cell to be targeted.For example, liver targeting ligand can be linked to cationic lipid through a linker that comprises an ester group.Liver cells are rich in esterase, therefore, linker is more efficiently cleaved in liver cells than in cells that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testicular cells.

[0340] Linkers containing peptide bonds can be used when targeting cell types that are rich in peptidases, such as liver cells and synovial cells.

[0341] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability (or conditions) of a degrading agent to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative susceptibility to cleavage between a first condition and a second condition can be determined, with the first condition being selected to exhibit cleavage in target cells and the second condition being selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, cells, cell culture, organ or tissue culture, or whole animals. It is useful to perform initial evaluations under cell-free or culture conditions and confirm them with further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0342] Redox-cleavable linking groups In one embodiment, the cleavable linker is a redox-cleavable linker that is cleaved upon reduction or oxidation. An example of a reductively cleavable linker is a disulfide linker (-SS-). To determine whether a candidate cleavable linker is a suitable "reductively cleavable linker," or whether it is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, the methods described herein can be found. For example, the candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, e.g., target cells. The candidate can also be evaluated under conditions selected to mimic blood or serum conditions. For example, the candidate compound is cleaved in blood by a maximum of about 10%. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular medium and compared to conditions selected to mimic extracellular medium.

[0343] Phosphate-Based Cleavable Linkers In another embodiment, the cleavable linker comprises a phosphate-based cleavable linker that is cleaved by a substance that degrades or hydrolyzes the phosphate group. An example of a substance that cleaves phosphate groups within a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0344] Acid-cleavable linking group In another embodiment, the cleavable linker comprises an acid-cleavable linker. An acid-cleavable linker is a linker that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linker is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or less), or by a substance such as an enzyme that can act as a general acid. Within a cell, specific low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linker. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, the carbon attached to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0345] Ester-Based Cleavable Linkers In another embodiment, the cleavable linker comprises an ester-based cleavable linker. Ester-based cleavable linkers are cleaved in cells by enzymes such as esterases and amidases. Examples of ester-based cleavable linkers include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linkers have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0346] Peptide-Based Cleavable Linkers In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linker. Peptide-based cleavable linkers are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linkers are peptide bonds formed between amino acids to generate oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to generate peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to generate peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable linkers have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.Representative United States patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,48 No. 6,603; No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,8 No. 24,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5, No. 112,963; No. 5,214,136; No. 5,245,022; No. 5,254,469; No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,098; No. 5 No. 5,510,475; No. 5,512,667; No. 5,514,785; No. 5,565,552; No. 5,567,810; No. Nos. 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, the entire contents of each of which are incorporated herein by reference.

[0347] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the above modifications may be incorporated in a single compound, or even at a single nucleoside within an iRNA. The present disclosure also includes iRNA compounds that are chimeric compounds.

[0348] A "chimeric" iRNA compound, or "chimera" in the context of this disclosure, is an iRNA compound, e.g., dsRNA, that contains two or more chemically distinct regions, each composed of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for the target nucleic acid. Additional regions of the iRNA can serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. As a result, comparable results can often be achieved with shorter iRNAs when using chimeric dsRNAs compared to phosphorothioate-deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if desired, associated nucleic acid hybridization techniques known in the art.

[0349] In certain instances, the RNA of an iRNA can be modified with a non-ligand group. Numerous non-ligand molecules have been conjugated to iRNAs to enhance their activity, cellular distribution, or cellular uptake, and procedures for such conjugation are available in the scientific literature. Such non-ligand moieties include lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule to be conjugated using an appropriate coupling or activating reagent. The conjugation reaction can be carried out in solution phase, with the RNA still bound to the solid support, or after cleavage of the RNA. Purification of the RNA conjugate by HPLC typically yields a pure conjugate.

[0350] iRNA delivery The delivery of iRNA to a subject in need thereof can be achieved in many different ways.In vivo delivery can be achieved directly by administering a composition containing iRNA, for example, dsRNA, to a subject.Alternatively, delivery can be achieved indirectly by administering one or more vectors that encode iRNA and direct its expression.These alternatives will be further discussed below.

[0351] Direct Delivery Generally, any method for delivering nucleic acid molecules can be adapted for use with iRNA (see, e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and International Publication No. WO 94 / 02595, the entire contents of which are incorporated herein by reference). However, there are three important factors to consider for successful in vivo delivery of iRNA molecules: (a) the biological stability of the delivered molecule, (2) prevention of nonspecific effects, and (3) accumulation of the delivered molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, such as direct injection or implantation into a tissue (for example, a tumor) or by local administration of a preparation. Local administration to the treatment site maximizes the local concentration of the drug, limits exposure of the agent to systemic tissues that may be harmed by or otherwise degrade the drug, and allows for a lower total dose of the iRNA molecule to be administered. Several studies have shown that local administration of iRNA can successfully knock down gene products. For example, intraocular delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ., et al (2004) Retina 24:132-138) and subretinal injection in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) both prevented neovascularization in experimental models of age-related macular degeneration. Furthermore, direct intratumoral injection of dsRNA into mice can reduce tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and prolong survival of tumor-bearing mice (Kim, WJ., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523).RNA interference has also been demonstrated by localized delivery to the CNS via direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, GT., et al (2004) Neuroscience 129:521-528; Thakker, ER., et al (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya,Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lung via intranasal administration (Howard, KA., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). To administer iRNA systemically for the treatment of disease, the RNA can be modified or, alternatively, delivered using a drug delivery system; both methods act to prevent the rapid degradation of dsRNA by endonucleases and exonucleases in vivo.

[0352] The modification of RNA or pharmaceutical carrier can also allow iRNA composition to be targeted to target tissue, and can avoid undesired off-target effects.iRNA molecule can be modified by chemical conjugation to other groups, for example, lipid or carbohydrate groups as described herein.Such conjugates can be used to target iRNA to specific cells, for example, liver cells, for example, hepatocytes.For example, GalNAc conjugates or lipid (for example, LNP) formulations can be used to target iRNA to specific cells, for example, liver cells, for example, hepatocytes.

[0353] Lipophilic groups such as cholesterol enhance cellular uptake and prevent degradation. For example, systemic injection of iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety into mice resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO., et al (2006) Nat. Biotechnol. 24:1005-1015). In alternative embodiments, iRNA can be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cation delivery systems promote the binding of iRNA molecules (which are negatively charged) and also enhance interaction with the negatively charged cell membrane, allowing for efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can be induced to bind to iRNA or form vesicles or micelles that encapsulate iRNA (see, e.g., Kim SH., et al. (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents degradation of iRNA when administered systemically. Methods for making and administering cationic RNA complexes are well within the capabilities of those skilled in the art (see, e.g., Sorensen, DR., et al (2003) J. Mol. Biol 327:761-766; Verma, UN., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entireties).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, D.R., et al (2003), supra; Verma, U.N., et al (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T.S., et al (2006) Nature 441:111-11), cardiolipin (Chien, P.Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M.E., et al (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D.A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, the iRNA is complexed with cyclodextrin for systemic administration. Methods and pharmaceutical compositions for administering iRNA and cyclodextrin are described in U.S. Patent No. 7,427,60, which is incorporated herein by reference in its entirety.

[0354] iRNA-encoding vector In another embodiment, iRNA targeting the LECT2 gene can be expressed from a transcription unit inserted into a DNA or RNA vector (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Application Publication No. 00 / 22113; Conrad, International PCT Application Publication No. 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (on the order of hours to weeks) or persistent (weeks to months or longer), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, and can be integrative or non-integrative vectors. The transgene can also be constructed to allow it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0355] Individual strands or multiple strands of iRNA can be transcribed from the promoter on the expression vector.For example, when expressing two separate strands to produce dsRNA, two separate expression vectors can be simultaneously introduced into target cells (for example, by transfection or infection).Alternatively, both individual strands of dsRNA can be transcribed by the promoter located on the same expression plasmid.In one embodiment, dsRNA is expressed as an inverted repeat connected by a linker polynucleotide sequence, so that dsRNA has a stem and loop structure.

[0356] iRNA expression vectors are typically DNA plasmids or viral vectors. Recombinant constructs for expressing the iRNAs described herein can be generated using expression vectors compatible with eukaryotic cells, such as vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from numerous commercial sources. Typically, such vectors contain convenient restriction sites for inserting the desired nucleic acid segment. Delivery of the iRNA expression vector can be systemic, for example, by intravenous or intramuscular administration, by administration to target cells explanted from the patient and then reintroduced into the patient, or by any other means that allows introduction into the desired target cells.

[0357] iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections targeting different regions of a target RNA for iRNA-mediated knockdown over a period of a week or more are also contemplated by the present disclosure. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfections can be signaled with a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable transfection of cells ex vivo can be ensured using a marker that confers resistance to certain environmental factors (e.g., antibiotics and drugs) on the transfected cells, such as hygromycin B resistance.

[0358] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, such as, but not limited to, lentiviral vectors, Moloney murine leukemia virus, and the like; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopox, e.g., vaccinia virus vectors, or avipox, e.g., canarypox or fowlpox; and (j) helper-dependent or gutless adenovirus. Replication-deficient viruses can also be advantageous. Different vectors may or may not be integrated into the cellular genome. If desired, the construct can contain viral sequences for transfection. Alternatively, the construct can be incorporated into a vector that allows episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are described further below.

[0359] Vectors useful for delivery of iRNA contain sufficient regulatory elements (promoters, enhancers, etc.) for expression of the iRNA in the desired target cells or tissues. Regulatory elements can be selected to provide for either constitutive or regulated / inducible expression.

[0360] The expression of iRNA can be precisely controlled, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24).Such inducible expression systems suitable for controlling dsRNA expression in cells or mammals include, for example, ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG).Those skilled in the art can select appropriate regulatory / promoter sequences based on the intended use of iRNA transgene.

[0361] In certain embodiments, viral vectors containing nucleic acid sequences encoding iRNAs can be used. For example, retroviral vectors can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for correct packaging of the viral genome and integration into host cell DNA. The nucleic acid sequences encoding iRNAs are cloned into one or more vectors, which facilitates delivery of the nucleic acid to patients. More details about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy include Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, HIV-based vectors described in U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, which are incorporated herein by reference.

[0362] Adenoviruses are also contemplated for use in delivering iRNA. Adenoviruses are particularly attractive vehicles, for example, for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia and cause a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), provide an overview of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994), demonstrated the use of adenovirus vectors to introduce genes into the respiratory epithelia of rhesus monkeys. Other examples of the use of adenovirus in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94 / 12649; and Wang, et al., Gene Therapy 2:775-783 (1995). Suitable AV vectors for expressing iRNAs featured in this disclosure, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

[0363] The use of adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoter, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in this disclosure, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher KJ et al. (1996), J. Virol., 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; International Patent Application Publication No. 94 / 13788; and International Patent Application Publication No. 93 / 24641, the entire disclosures of which are incorporated herein by reference.

[0364] Another exemplary viral vector is a poxvirus such as vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowlpox or canarypox.

[0365] The tropism of viral vectors can be modified by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by appropriately replacing different viral capsid proteins.For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc.AAV vectors can be engineered to target different cells by engineering the vector to express different capsid protein serotypes; for example, see Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0366] The pharmaceutical preparation of the vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0367] III. Pharmaceutical Compositions Containing iRNA In one embodiment, the present disclosure provides a pharmaceutical composition comprising an iRNA described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing iRNA are useful for treating diseases or disorders associated with the expression or activity of the LECT2 gene (e.g., LECT2 amyloidosis). Such pharmaceutical compositions are formulated based on the mode of delivery. For example, the composition can be formulated for systemic administration via parenteral delivery, e.g., intravenous (IV) delivery. In some embodiments, the compositions provided herein (e.g., LNP formulations) are formulated for intravenous delivery. In some embodiments, the compositions provided herein (e.g., compositions comprising GalNAc conjugates) are formulated for subcutaneous delivery.

[0368] The pharmaceutical compositions featured herein are administered at a dosage sufficient to inhibit expression of the LECT2 gene. Generally, suitable doses of iRNA range from 0.01 to 200.0 milligrams per kg of recipient body weight per day, typically from 1 to 50 mg per kg of body weight per day. For example, dsRNA can be administered at 0.05 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg per dose. The pharmaceutical composition can be administered once daily, or the iRNA can be administered in two, three, or more subdoses at appropriate intervals throughout the day, or delivery via continuous infusion or controlled-release formulations can be used. In this case, the amount of iRNA contained in each subdose must be correspondingly smaller to achieve the total daily dosage. The dosage unit can also be formulated for delivery over several days, for example, using a conventional sustained-release formulation that provides sustained release of iRNA over several days. Sustained-release formulations are well known in the art and are particularly useful for delivering drugs to specific sites, such as those that can be used with the drugs of the present disclosure. In this embodiment, the dosage unit contains a multiple of the corresponding daily dose.

[0369] The effect of a single dose on LECT2 levels can be long-lasting, with subsequent doses administered no more than 3, 4, or 5 days apart, or no more than 1, 2, 3, or 4 weeks apart.

[0370] Those skilled in the art will understand that certain factors, including but not limited to, the severity of the disease or disorder, previous treatment, the overall health and / or age of the subject, and other existing diseases, can affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. The effective dosage and in vivo half-life of each iRNA encompassed by the disclosure can be estimated using conventional methods or based on in vivo tests using appropriate animal models.

[0371] A suitable animal model, for example, a mouse containing a transgene expressing human LECT2, can be used to determine a therapeutically effective dose and / or an effective dosage regimen of LECT2 siRNA.

[0372] The present disclosure also includes pharmaceutical compositions and formulations containing the iRNA compounds featured herein.The pharmaceutical compositions of the present disclosure can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated.Administration can be topical (for example, by transdermal patch), pulmonary, for example, by inhalation or insufflation of powder or aerosol, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral.Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous, for example, via implanted device; or intracranial, for example, intraparenchymal, intrathecal or intraventricular administration.

[0373] iRNA can be delivered in a manner that targets a specific tissue, such as a tissue that produces red blood cells. For example, iRNA can be delivered to the bone marrow, liver (e.g., hepatocytes of the liver), lymph nodes, spleen, lung (e.g., pleura of the lung), or spine. In one embodiment, iRNA is delivered to the bone marrow.

[0374] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful. Suitable topical formulations include those in which the iRNAs featured in this disclosure are mixed with topical delivery agents, such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNAs featured in this disclosure can be encapsulated within liposomes or complexed therewith, particularly cationic liposomes. Alternatively, the iRNAs can be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1-20 These include alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0375] Liposomal formulation In addition to microemulsions, which have been studied and used for drug formulation, there are many other organized surfactant structures. These include monolayers, micelles, bilayers, and vesicles. Vesicles such as liposomes have attracted great interest due to their specificity and the duration of action they provide in terms of drug delivery. As used in this disclosure, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or multiple bilayers.

[0376] Liposomes are unilamellar or multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with the cell wall. Non-cationic liposomes cannot fuse efficiently with the cell wall, but they are taken up by macrophages in vivo.

[0377] To pass through intact mammalian skin, lipid vesicles must pass through a series of pores, each less than 50 nm in diameter, under the influence of an appropriate transdermal gradient. Therefore, it is desirable to use liposomes that are highly deformable and can pass through such pores.

[0378] Additional advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water- and lipid-soluble drugs; and liposomes can protect encapsulated drugs within their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Important considerations in the preparation of liposome formulations are lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

[0379] Liposomes are useful for transporting and delivering active ingredients to the site of action. Because liposome membranes are structurally similar to biological membranes, when liposomes are applied to tissues, they begin to fuse with cell membranes, and as the fusion of liposomes and cells progresses, the liposome contents are emptied into the cells where the active agent can act.

[0380] Liposomal formulations have been the focus of extensive research as a mode of delivery for many drugs. There is growing evidence that liposomes offer several advantages over other formulations for topical administration. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to deliver a wide range of drugs, both hydrophilic and hydrophobic, to the skin.

[0381] Several reports have detailed the ability of liposomes to deliver drugs, including high-molecular-weight DNA, to the skin. Painkillers, antibodies, hormones, and compounds containing high-molecular-weight DNA have been administered to the skin. The majority of applications have resulted in targeting of the upper epidermis.

[0382] Liposomes are broadly classified into two types. Cationic liposomes are positively charged liposomes that interact with negatively charged DNA molecules to form stable complexes. The positively charged DNA / liposome complexes bind to the negatively charged cell surface and are internalized into endosomes. Due to the acidic pH within the endosome, the liposomes rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0383] pH-sensitive or negatively charged liposomes entrap DNA rather than complex with it. Because both DNA and lipids are similarly charged, repulsion occurs rather than complexation. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0384] One major type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholine. Neutral liposome compositions can be formed, for example, from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0385] Several studies have evaluated the topical delivery of liposomal drug formulations to the skin. Application of interferon-containing liposomes to the skin of guinea pigs resulted in a reduction in cutaneous herpes ulcers, while delivery of interferon by other means (e.g., as a solution or emulsion) was ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Furthermore, additional studies have tested the effectiveness of interferon administered as part of a liposomal formulation relative to administration using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0386] Nonionic liposomal systems have also been tested to determine their usefulness for delivering drugs to the skin, particularly in systems containing nonionic surfactants and cholesterol. Nonionic liposomal formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine A to the dermis of mouse skin. The results showed that such nonionic liposomal systems were effective in promoting the deposition of cyclosporine A into different layers of the skin (Hu et al. STP Pharma. Sci., 1994, 4, 6, 466).

[0387] Liposomes also include "sterically stabilized" liposomes, which term, as used herein, refers to liposomes containing one or more specialized lipids that, when incorporated into the liposome, enhance circulation lifetime compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of liposome (A) is monosialoganglioside G M1or (B) those that are derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without wishing to be bound by any particular theory, it is believed in the art that for sterically stabilized liposomes containing at least gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes is due to reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0388] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) describe the use of monosialoganglioside G M1 reported the ability of galactocerebroside sulfate and phosphatidylinositol to improve the blood half-life of liposomes. These findings were explained by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Patent No. 4,837,028 and WO 88 / 04924, both by Allen et al., reported the ability of (1) sphingomyelin and (2) ganglioside G M1 or liposomes containing galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. WO 97 / 13499 (Lim et al.) discloses liposomes containing 1,2-sn-dimyristoylphosphatidylcholine.

[0389] Many liposomes containing lipids derivatized with one or more hydrophilic polymers and methods for their preparation are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) reported the preparation of liposomes containing a nonionic surfactant, 2C, containing a PEG moiety. 1215Gdescribed liposomes containing PEG- or PEG-stearate-derivatized phosphatidylethanolamine (PE). Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols resulted in a significant increase in blood half-life. Synthetic phospholipids modified by the attachment of carboxyl groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes containing phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have a significantly increased blood circulation half-life. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended these observations to other PEG-derivatized phospholipids, such as DSPE-PEG, formed from a combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes with covalently bound PEG moieties on their outer surface are described in Fisher's European Patent No. 0445131 B1 and International Publication No. WO 90 / 04384. Liposomal compositions containing 1 to 20 mole percent PE derivatized with PEG, and methods for their use, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. 0496813 B1). Liposomes containing many other lipid-polymer conjugates are disclosed in International Publication No. 91 / 05545 and U.S. Patent No. 5,225,212 (both Martin et al.) and International Publication No. 94 / 20073 (Zalipsky et al.). Liposomes containing PEG-modified ceramide lipids are described in International Publication No. 96 / 10391 (Choi et al.). U.S. Patent No. 5,540,935 (Miyazaki et al.) and U.S. Patent No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on the surface.

[0390] Numerous liposomes containing nucleic acids are known in the art. Thierry et al., International Publication No. 96 / 40062, discloses a method for encapsulating high molecular weight nucleic acids in liposomes. Tagawa et al., U.S. Patent No. 5,264,221, discloses protein-bound liposomes, and claims that the contents of such liposomes can contain dsRNA. Rahman et al., U.S. Patent No. 5,665,710, describes a specific method for encapsulating oligodeoxynucleotides in liposomes. Love et al., International Publication No. 97 / 04787, discloses liposomes containing dsRNA targeted to raf gene.

[0391] Transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets that are so deformable that they can easily penetrate pores smaller than the droplets. Transfersomes are adaptable to the environment in which they are used; for example, they are self-optimizing (adapting to the shape of skin pores), self-repairing, often reach their target without fragmentation, and are often self-loading. To create transfersomes, surface-edge active agents, usually surfactants, can be added to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0392] Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the properties of many different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for classifying the various surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0393] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceuticals and cosmetics and are usable over a wide range of pH values. Their HLB values ​​generally range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this class. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0394] If surfactant molecule carries negative charge when dissolved or dispersed in water, surfactant is classified as anionic.Anionic surfactants include carboxylates, such as soaps, acyl lactylates, acyl amides of amino acids, sulfuric acid esters, such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates, such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.The most important members of anionic surfactant class are alkyl sulfates and soaps.

[0395] If the surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used compounds in this class.

[0396] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0397] The use of surfactants in drug products, formulations and emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 28).

[0398] nucleic acid lipid particles In one embodiment, the LECT2 dsRNA featured in this disclosure is fully encapsulated in a lipid formulation, forming, for example, SPLP, pSPLP, SNALP, or other nucleic acid-lipid particles. As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle comprising SPLP. As used herein, the term "SPLP" refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALP and SPLP typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALP and SPLP exhibit long circulation life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the administration site), making them extremely useful for systemic administration. SPLPs include "pSPLP," which comprises an encapsulated condensing agent-nucleic acid complex, as described in PCT Application WO 00 / 03683. The particles of the present disclosure typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic. Furthermore, when present in the nucleic acid-lipid particles of the present disclosure, the nucleic acid is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication WO 96 / 40964.

[0399] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) is within the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0400] Examples of cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleyl Carbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or similar. The compound may be (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxolan-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

[0401] In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles.The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61 / 107,998, filed October 23, 2008, which is incorporated herein by reference.

[0402] In one embodiment, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane:10% DSPC:40% cholesterol:10% PEG-C-DOMG (mol percent), have a particle size of 63.0±20 nm, and an siRNA / lipid ratio of 0.027.

[0403] Non-cationic lipids are anionic or neutral lipids, including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (P ... Non-cationic lipids include oleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. The non-cationic lipid can be about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% if cholesterol is included, of the total lipid present in the particle.

[0404] The conjugated lipid that inhibits particle aggregation can be a polyethylene glycol (PEG)-lipid, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C)8. The conjugated lipid that inhibits particle aggregation can be 0 mol% to about 20 mol% or about 2 mol% of the total lipid present in the particle.

[0405] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol at about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

[0406] In some embodiments, the iRNA is formulated in a lipid nanoparticle (LNP).

[0407] LNP01 In one embodiment, lipid-dsRNA nanoparticles (e.g., LNP01 particles) can be prepared using the lipidoid ND98·4HCl (MW1487) (U.S. Patent Application No. 12 / 056,230, incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-ceramide C16, 100 mg / ml. The ND98, cholesterol, and PEG-ceramide C16 stock solutions can then be combined, for example, in a 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate, pH 5) so that the final ethanol concentration is approximately 35-45% and the final sodium acetate concentration is approximately 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using a heated barrel extruder such as Lipex Extruder (Northern Lipids, Inc.). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. The buffer can be exchanged with phosphate buffered saline at, for example, about pH 7, for example, about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

[0408] [ka]

[0409] LNP01 formulations are described, for example, in International Application Publication No. WO 2008 / 042973, which is incorporated herein by reference.

[0410] Further exemplary lipid-dsRNA formulations are provided in the table below.

[0411] [Table 1-1] [Table 1-2]

[0412] DSPC: Distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-dimyristoylglycerol (C14-PEG or PEG-C14) (PEG with an average molecular weight of 2000) PEG-DSG: PEG-distyrylglycerol (C18-PEG or PEG-C18) (PEG with an average molecular weight of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with an average molecular weight of 2000)

[0413] Formulations containing SNALP (1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) are described in WO 2009 / 127060, filed April 15, 2009, and incorporated herein by reference.

[0414] Formulations containing XTC are described, for example, in U.S. Provisional Application No. 61 / 148,366, filed January 29, 2009; U.S. Provisional Application No. 61 / 156,851, filed March 2, 2009; U.S. Provisional Application No. 61 / 185,712, filed June 10, 2009; U.S. Provisional Application No. 61 / 228,373, filed July 24, 2009; U.S. Provisional Application No. 61 / 239,686, filed September 3, 2009; and International Application No. PCT / US2010 / 022614, filed January 29, 2010, which are incorporated herein by reference.

[0415] Formulations containing MC3 are described, for example, in U.S. Provisional Application No. 61 / 244,834, filed September 22, 2009, and International Application No. PCT / US10 / 28224, filed June 10, 2010, which are incorporated herein by reference.

[0416] Formulations containing ALNY-100 are described, for example, in International Patent Application No. PCT / US09 / 63933, filed November 10, 2009, which is incorporated herein by reference.

[0417] C12-200 containing formulations are described in U.S. Provisional Application No. 61 / 175,770, filed May 5, 2009, and International Application No. PCT / US10 / 33777, filed May 5, 2010, which are incorporated herein by reference.

[0418] Synthesis of cationic lipids Any of the compounds used in the nucleic acid-lipid particles featured in this disclosure, such as cationic lipids, can be prepared by known organic synthesis techniques. All substituents, unless otherwise indicated, are as defined below.

[0419] "Alkyl" means a straight-chain or branched, acyclic or cyclic, saturated aliphatic hydrocarbon containing 1 to 24 carbon atoms. Representative saturated straight-chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

[0420] "Alkenyl" refers to an alkyl as defined above containing at least one double bond between adjacent carbon atoms. Alkenyl includes both cis and trans isomers. Representative straight-chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

[0421] "Alkynyl" means any alkyl or alkenyl as defined above further containing at least one triple bond between adjacent carbons. Representative straight-chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

[0422] "Acyl" means any alkyl, alkenyl, or alkynyl, as defined below, in which the carbon at the point of attachment is replaced with an oxo group. For example, -C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl are acyl groups.

[0423] "Heterocycle" means a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic ring that is either saturated, unsaturated, or aromatic and contains one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur, where the nitrogen and sulfur heteroatoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. Heterocyclic rings include bicyclic rings in which any of the above heterocycles are fused to a benzene ring. Heterocycles may be bonded via any heteroatom or carbon atom. Heterocycles include heteroaryls, as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

[0424] The terms "optionally substituted alkyl," "optionally substituted alkenyl," "optionally substituted alkynyl," "optionally substituted acyl," and "optionally substituted heterocycle," when substituted, mean that at least one hydrogen atom is replaced by a substituent. In the case of an oxo substituent (=O), two hydrogen atoms are replaced. In this regard, the substituent may be oxo, halogen, heterocycle, -CN, -OR x , -NR x R y , -NR x C(=O)R y , -NR x SO2R y , -C(=O)R x , -C(=O)OR x , -C(=O)NR x R y , -SO n R x , and -SO n NR x R y where n is 0, 1, or 2;x and R y are the same or different and independently hydrogen, alkyl, or heterocycle, and each of said alkyl and heterocycle substituents is selected from oxo, halogen, —OH, —CN, alkyl, —OR x , heterocycle, -NR x R y , -NR x C(=O)R y , -NR x SO2R y , -C(=O)R x , -C(=O)OR x , -C(=O)NR x R y , -SO n R x , and -SO n NR x R y may be further substituted by one or more of:

[0425] "Halogen" means fluoro, chloro, bromo, and iodo.

[0426] In some embodiments, the methods featured in the present disclosure may require the use of protecting groups. Protecting group methods are well known to those skilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, TW et al., Wiley-Interscience, New York City, 1999). Briefly, a protecting group in the context of the present disclosure is any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during a certain reaction and then removed to expose the original functional group. In some embodiments, an "alcohol protecting group" is used. An "alcohol protecting group" is any group that reduces or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added or removed using techniques well known in the art.

[0427] Synthesis of Formula A In one embodiment, the nucleic acid-lipid particles featured in this disclosure comprise a cationic lipid of Formula A:

[0428] [ka]

[0429] wherein R1 and R2 are independently alkyl, alkenyl, or alkynyl, each of which may be optionally substituted; R3 and R4 are independently lower alkyl; or R3 and R4 together may form an optionally substituted heterocyclic ring. In some embodiments, cationic lipid is XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane).Generally, the lipid of above formula A can be prepared by the following reaction scheme 1 or 2, wherein all substituents are as defined above unless otherwise indicated.

[0430] [ka]

[0431] Lipid A, in which R1 and R2 are independently alkyl, alkenyl, or alkynyl, each optionally substituted, and R3 and R4 are independently lower alkyl, or R3 and R4 together may form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those skilled in the art. Reaction of 1 and 2 produces ketal 3. Treatment of ketal 3 with amine 4 produces lipids of Formula A. Lipids of Formula A can be converted to the corresponding ammonium salts with organic salts of Formula 5, where X is an anionic counterion selected from halogen, hydroxide, phosphate, sulfate, etc.

[0432] [ka]

[0433] Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those skilled in the art. Reaction of 6 and 7 produces ketone 1. Conversion of ketone 1 to the corresponding lipid of formula A is as described in Scheme 1.

[0434] Synthesis of MC3 DLin-M-C3-DMA [i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate] was prepared as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred overnight at room temperature. The solution was washed with dilute hydrochloric acid, followed by dilute aqueous sodium bicarbonate. The organic fraction was dried over anhydrous magnesium sulfate, filtered, and the solvent removed on a rotary evaporator. The residue was passed through a silica gel column (20 g) using a 1-5% methanol / dichloromethane elution gradient. Fractions containing the purified product were combined, and the solvent was removed to give a colorless oil (0.54 g).

[0435] Synthesis of ALNY-100 The synthesis of ketal 519 [ALNY-100] was carried out using Scheme 3 below.

[0436] [ka]

[0437] Synthesis of 515: To a stirred suspension of LiAlH (3.74 g, 0.09852 mol) in 200 mL of anhydrous THF in a two-neck RBF (1 L) at 0 °C under nitrogen gas, a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF was slowly added. After complete addition, the reaction mixture was warmed to room temperature and then heated to reflux for 4 h. The reaction progress was monitored by TLC. After completion of the reaction (by TLC), the mixture was cooled to 0 °C and quenched by the careful addition of saturated NaSO solution. The reaction mixture was stirred at room temperature for 4 h and filtered. The residue was washed thoroughly with THF. The filtrate and washings were combined, diluted with 400 mL of dioxane and 26 mL of concentrated hydrochloric acid, and stirred at room temperature for 20 min. The volatiles were removed in vacuo to give the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400MHz): δ= 9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

[0438] Synthesis of 516: To a stirred solution of compound 515 in 100 mL of dry DCM in a 250 mL two-neck RBF, NEt (37.2 mL, 0.2669 mol) was added and cooled to 0 °C under nitrogen. After the slow addition of N-(benzyloxycarbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL of dry DCM, the reaction mixture was warmed to room temperature. After completion of the reaction (2-3 h by TLC), the mixture was washed successively with 1 N HCl solution (1 × 100 mL) and saturated NaHCO solution (1 × 50 mL). The organic layer was then dried over anhydrous NaSO, and the solvent was evaporated to give the crude material, which was purified by silica gel column chromatography to give 516 as a sticky mass. Yield: 11g (89%). 1H-NMR (CDCl3, 400MHz): δ = 7.36-7.27(m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60(m, 2H), 2.30-2.25(m, 2H). LC-MS [M+H] -232.3 (96.94%).

[0439] Synthesis of 517A and 517B: Cyclopentene 516 (5 g, 0.02164 mol) was dissolved in 220 mL of a 10:1 mixture of acetone and water in a single-neck 500 mL RBF and N-methylmorpholine-N-oxide (7.6 g, 0.06492 mol) was added, followed by 4.2 mL of a 7.6% solution of OsO in tert-butanol (0.275 g, 0.00108 mol) at room temperature. After completion of the reaction (approximately 3 h), the mixture was quenched by the addition of solid NaSO, and the resulting mixture was stirred at room temperature for 1.5 h. The reaction mixture was diluted with DCM (300 mL) and washed with water (2 × 100 mL), followed by saturated NaHCO (1 × 50 mL) solution, water (1 × 30 mL), and finally brine (1 × 50 mL). The organic phase was dried over NaSO, and the solvent was removed in vacuo. Silica gel column chromatography purification of the crude material gave a mixture of diastereomers which were separated by preparative HPLC. Yield: - 6 g crude 517A - Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400MHz): δ= 7.39-7.31(m, 5H), 5.04(s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47(d, 2H), 3.94-3.93(m, 2H), 2.71(s, 3H), 1.72- 1.67(m, 4H). LC-MS - [M+H]-266.3, [M+NH4 +]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

[0440] Synthesis of 518: Using a procedure similar to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400MHz): δ= 7.35-7.33(m, 4H), 7.30-7.27(m, 1H), 5.37-5.27(m, 8H), 5.12(s, 2H), 4.75(m,1H), 4.58-4.57(m,2H), 2.78-2.74(m,7H), 2.06-2.00(m,8H), 1.96-1.91(m, 2H), 1.62(m, 4H), 1.48(m, 2H), 1.37-1.25(br m, 36H), 0.87(m, 6H). HPLC-98.65%.

[0441] General procedure for the synthesis of compound 519: A solution of compound 518 (1 equivalent) in hexane (15 mL) was added dropwise to an ice-cold solution of LAH in THF (1 M, 2 equivalents). After complete addition, the mixture was heated at 40° C. for 0.5 h and then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous NaSO, then filtered through Celite and reduced to an oil. Column chromatography afforded pure 519 (1.3 g, 68%) obtained as a colorless oil. C NMR = 130.2, 130.1 (x2), 127.9 (x3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (x2), 29.7, 29.6 (x2), 29.5 (x3), 29.3 (x2), 27.2 (x3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): MW calculated for C44H80NO2 (M + H)+ 654.6, found 654.6.

[0442] Formulations prepared by either standard or extrusion-free methods can be characterized in a similar manner. For example, formulations are typically characterized visually. They should be a whitish, translucent solution without aggregates or precipitates. The particle size and size distribution of lipid nanoparticles can be measured by light scattering, for example, using a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be approximately 20-300 nm, e.g., 40-100 nm. The particle size distribution should be monomodal. The total dsRNA concentration in the formulation and captured fraction is estimated using a dye exclusion assay. Samples of formulated dsRNA may be incubated with an RNA-binding dye, e.g., Ribogreen (Molecular Probes), in the presence or absence of a formulation-disrupting detergent, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by comparing the signal from the detergent-containing sample to a standard curve. The captured fraction is determined by subtracting the "free" dsRNA content (measured by the signal in the absence of surfactant) from the total dsRNA content. The percentage of captured dsRNA is typically greater than 85%. For SNALP formulations, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The preferred range is typically at least about 50 nm to at least about 110 nm, at least about 60 nm to at least about 100 nm, or at least about 80 nm to at least about 90 nm.

[0443] Compositions and preparations for oral administration include powder or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or mini-tablets.Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.In some embodiments, oral preparations are those in which the dsRNAs featured in the present disclosure are administered with one or more penetration enhancers, surfactants and chelating agents.Suitable surfactants include fatty acids and / or their esters or salts, bile acids and / or their salts. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or their monoglycerides, diglycerides, or pharmaceutically acceptable salts (e.g., sodium). In some embodiments, a combination of penetration enhancers is used, for example, a fatty acid / salt combined with a bile acid / salt. One exemplary combination is the sodium salt of lauric acid, capric acid, and UDCA. Additional penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The dsRNA featured in this disclosure may be orally delivered in a granular form, including spray-dried particles, or complexed to form microparticles or nanoparticles.dsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulan, cellulose and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P (TDAE), polyaminostyrene (e.g., p-amino), poly(methyl cyanoacrylate), poly(ethyl cyanoacrylate), poly(butyl cyanoacrylate), poly(isobutyl cyanoacrylate), poly(isohexyl cyanoacrylate), DEAE-methacrylate, D These include EAE-hexyl acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethyl acrylate, polyhexyl acrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid) (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations for dsRNA and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. Publication No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

[0444] Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular, or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives, such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0445] Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids.

[0446] The pharmaceutical preparations featured in the present disclosure can be conveniently presented in unit dosage form, but can be prepared according to conventional techniques well known in the pharmaceutical industry.Such techniques include the step of combining active ingredient with pharmaceutical carrier(s) or excipient(s).Generally, preparations are prepared by uniformly and intimately combining active ingredient with liquid carrier or finely divided solid carrier or both, and then, if necessary, shaping the product.

[0447] The compositions featured in this disclosure can be formulated into any of many possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions can further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension can also contain stabilizers.

[0448] Additional preparations emulsion The compositions of the present disclosure may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of liquids dispersed within other liquids in the form of droplets, usually greater than 0.1 μm in diameter (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245). (See, e.g., "Emulsions of Water and Water," 1988, Marcel Dekker, Inc., New York, NY, volume 2, p. 335; Higuchi et al., in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 1985, p. 301"). Emulsions are often two-phase systems containing two immiscible liquid phases that are intimately mixed and dispersed within one another. Generally, emulsions can be either water-in-oil (w / o) or oil-in-water (o / w). When the aqueous phase is finely dispersed and dispersed as minute droplets within the bulk oil phase, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when the oil phase is finely dispersed and dispersed as minute droplets within the bulk aqueous phase, the resulting composition is called an oil-in-water (o / w) emulsion.Emulsions may contain additional ingredients in addition to the dispersed phase and the active drug, which may be present as a solution in either the aqueous or oily phase, or as a separate phase itself. Pharmaceutical excipients, such as emulsifiers, stabilizers, dyes, and antioxidants, may also be present in the emulsion as needed. Pharmaceutical emulsions may also be multiple emulsions, containing more than one phase, such as oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer certain advantages not offered by simple binary emulsions. Multiple emulsions, in which individual oil droplets of an o / w emulsion encapsulate small water droplets, constitute w / o / w emulsions. Similarly, oil droplet systems encapsulated in globules of water and stabilized within an oily continuous phase constitute o / w / o emulsions.

[0449] Emulsions are characterized by little or no thermodynamic stability. The dispersed or discontinuous phase of an emulsion is well dispersed within the external or continuous phase and is maintained in this form by means of emulsifiers or the viscosity of the formulation. Either of the emulsion phases can be semi-solid or solid, as in the case of emulsion-type ointment bases and creams. Another means of stabilizing emulsions involves the use of emulsifiers, which can be incorporated into either of the emulsion phases. Emulsifiers can be broadly classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0450] Synthetic surfactants, also known as surface active agents, have found wide application in the formulation of emulsions and have been reviewed in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p. 199).Surfactants are typically amphiphilic, comprising hydrophilic and hydrophobic moieties. The ratio of hydrophilicity and hydrophobicity of surfactant is called hydrophilic / lipophilic balance (HLB), which is a useful tool for classifying and selecting surfactant in the preparation of formulation.Surfactant can be classified into different classes based on the nature of hydrophilic group: nonionic, anionic, cationic and amphoteric (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285).

[0451] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phospholipids, lecithin, and acacia. Hydrophilic adsorbents, such as anhydrous lanolin and hydrophilic petrolatum, can absorb water and form water-in-oil emulsions, maintaining their semisolid consistency. Finely dispersed solids have also been used as excellent emulsifiers in viscous preparations, especially in combination with surfactants. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate.

[0452] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion, including fats, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0453] Hydrophilic colloids, or hydrocolloids, include naturally occurring gums and synthetic polymers, such as polysaccharides (e.g., acacia, agar, alginate, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomer, cellulose ethers, and carboxyvinyl polymers), which disperse in or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around dispersed phase droplets and by increasing the viscosity of the external phase.

[0454] Emulsions often contain several components, such as carbohydrates, proteins, sterols and phospholipids, which can easily support the growth of microorganisms, so these preparations often incorporate preservatives.The commonly used preservatives contained in emulsion preparations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.Antioxidants are also generally added to emulsion preparations to prevent the preparation from deteriorating.The antioxidants used can be free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0455] The application of emulsion formulations via dermal, oral, and parenteral routes, and methods for preparing them, have been reviewed in the literature (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Emulsion formulations for oral delivery are widely used due to ease of formulation and efficiency in terms of absorption and bioavailability (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutrients are materials that are commonly administered orally as o / w emulsions.

[0456] In one embodiment of the present disclosure, the iRNA and nucleic acid compositions are formulated as microemulsions. A microemulsion can be defined as a system of water, oil, and an amphiphile that is a single optically isotropic and thermodynamically stable solution (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Typically, microemulsions are prepared by first dispersing oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, usually a medium-chain alcohol, to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions are generally prepared through the combination of three to five components, including oil, water, surfactants, co-surfactants, and electrolytes.Whether a microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the properties of the oil and surfactant used, as well as the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecule (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0457] The phenomenological approach using phase diagrams has been extensively studied, providing those skilled in the art with comprehensive knowledge of microemulsion formulation (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of spontaneously formed, thermodynamically stable droplets.

[0458] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ether, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), and decaglycerol decaoleate (DAO750), either alone or in combination with cosurfactants. Cosurfactants, typically short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, serve to increase interfacial fluidity by penetrating the surfactant film, resulting in an irregular film due to the interstitial spaces between the surfactant molecules. However, microemulsions can be prepared without the use of cosurfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase can include, but is not limited to, materials such as Captex300, Captex355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0459] Microemulsions are particularly interesting in terms of drug solubilization and drug absorption enhancement.Lipid-based microemulsions (both o / w and w / o) have been proposed to enhance the oral bioavailability of drugs, including peptides (for example, U.S. Patent No. 6,191,105; U.S. Patent No. 7,063,860; U.S. Patent No. 7,070,802; U.S. Patent No. 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer advantages such as improved drug solubilization, drug protection from enzymatic hydrolysis, potential drug absorption enhancement due to surfactant-induced changes in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical efficacy, and reduced toxicity (see, e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Microemulsions can often form spontaneously when their components are combined at ambient temperature. This can be particularly advantageous when formulating heat-labile drugs, peptides, or iRNA. Microemulsions have also been effective for transdermal delivery of active ingredients in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present disclosure are expected to facilitate increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract, as well as improve localized cellular uptake of iRNA and nucleic acids.

[0460] The microemulsions of the present disclosure may also contain additional ingredients and additives, such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers, to improve formulation properties and enhance absorption of the iRNA and nucleic acids of the present disclosure. The penetration enhancers used in the microemulsions of the present disclosure can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

[0461] penetration enhancers In one embodiment, the present disclosure provides effective delivery of nucleic acids, particularly iRNA, to animal skin using various penetration enhancers. Most drugs exist in solution in both ionized and non-ionized forms. However, usually, only lipid-soluble or lipophilic drugs can easily pass through cell membranes. It has been discovered that even non-lipophilic drugs can pass through cell membranes when the membrane they pass through is treated with a penetration enhancer. In addition to helping the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also increase the permeability of lipophilic drugs.

[0462] Penetration enhancers can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above classes of penetration enhancers is described in more detail below.

[0463] Surfactants: In the context of the present disclosure, surfactants (or "surface-active agents") are chemicals that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in enhanced absorption of iRNA through mucous membranes. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0464] Fatty acids: Various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, and their C 1ー20These include alkyl esters (e.g., methyl, isopropyl, and t-butyl), and their mono- and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see, for example, Touitou, E., et al., Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0465] Bile salts: The physiological role of bile includes promoting the dispersion and absorption of lipids and fat-soluble vitamins (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts and their synthetic derivatives act as penetration enhancers. Therefore, the term "bile salts" includes any of the naturally occurring components of bile and any of their synthetic derivatives.Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glycolic acid (sodium glycolate), glycolic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), taurosodium-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE) (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; see Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0466] Chelating Agent: As used in the context of the present disclosure, a chelating agent may be defined as a compound that removes metal ions from solution by forming a complex with them, resulting in enhanced absorption of iRNA through mucous membranes. With regard to their use as penetration enhancers in the present disclosure, chelating agents have the added advantage of also serving as DNase inhibitors, since most characterized DNA nucleases require divalent metal ions for catalysis and are therefore inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetic acid (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylic acid, and homovanilate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of β-diketones (enamines) (see, e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0467] Non-chelating non-surfactant: As used herein, a non-chelating non-surfactant penetration enhancer may be defined as a compound that demonstrates insignificant activity as a chelator or surfactant, but nonetheless enhances the absorption of iRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancer includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacycloalkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal anti-inflammatory agents such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0468] The agent that enhances iRNA uptake at cellular level can also be added to the pharmaceutical and other compositions of the present disclosure.For example, cationic lipids such as lipofectin (Junichi et al., US Patent No. 5,705,188), cationic glycerol derivatives and polycationic molecules such as polylysine (Lollo et al., International Publication No. WO 97 / 30731) are also known to enhance the cellular uptake of dsRNA.Examples of commercially available transfection reagents include Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000, among others. CD (Invitrogen; Carlsbad, CA), Lipofectamine™ (Invitrogen; Carlsbad, CA), RNAiMAX (Invitrogen; Carlsbad, CA), Oligofectamine™ (Invitrogen; Carlsbad, CA), Optifect™ (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 transfection reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal transfection reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal transfection reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® reagent (Promega; Madison, WI), TransFast™ transfection reagent (Promega; Madison, WI), Tfx™-20 reagent (Promega; Madison, WI), Tfx™-50 reagent (Promega; Madison, WI), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass. aD1 transfection reagent (NEW England Biolabs; Ipswich, MA, USA), LyoVec™ / LipoGen™ (Invivogen; San Diego, CA, USA), PerFectin transfection reagent (Genlantis; San Diego, CA, USA), NeuroPORTER transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin transfection reagent (Genlantis; San Diego, CA, USA), BaculoPORTER transfection reagent (Genlantis; San Diego, CA, USA), TroganPORTER™ transfection reagent (Genlantis; San Examples of suitable fusion proteins include RiboFect (Bioline; Taunton, MA, USA), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), or HiFect™ (B-Bridge International, Mountain View, CA, USA).

[0469] Other agents can be utilized to enhance penetration of the administered nucleic acid, including glycols such as ethylene glycol and propylene glycol, pyrroles such as 2-pyrrole, azone, and terpenes such as limonene and menthone.

[0470] Carrier Certain compositions of the present disclosure also incorporate a carrier compound in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid or analog thereof that is inert (i.e., has no biological activity itself) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of biologically active nucleic acids, for example, by degrading the biologically active nucleic acid or facilitating its removal from the circulation. Co-administration of a nucleic acid and a carrier compound, typically in excess of the latter substance, can result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidney, or other extracirculatory reservoir, likely due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of partial phosphorothioate dsRNA in liver tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic acid, or 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0471] excipients In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert vehicle for delivering one or more nucleic acids to an animal. Excipients may be liquid or solid and are selected based on the proposed mode of administration so as to provide the desired bulk, consistency, etc., when combined with the nucleic acids and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (such as pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (such as starch, sodium starch glycolate, etc.); and wetting agents (such as sodium lauryl sulfate, etc.).

[0472] The compositions of the present disclosure can also be formulated using pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not adversely react with nucleic acids.Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0473] Preparations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or solutions of nucleic acids in liquid or solid oil bases.The solutions can also contain buffers, diluents, and other suitable additives.Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not adversely react with nucleic acids can be used.

[0474] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0475] Other ingredients The compositions of the present disclosure may further contain other auxiliary ingredients conventionally found in pharmaceutical compositions, at their utilization levels established in the art.Thus, for example, the compositions may contain additional compatible pharmaceutically active materials, such as antipruritic agents, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful for physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers.However, when added, such materials should not excessively interfere with the biological activity of the components of the compositions of the present disclosure.The preparations can be sterilized, and if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances, which do not adversely interact with the nucleic acid(s) of the preparation.

[0476] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers.

[0477] In some embodiments, the pharmaceutical compositions featured in this disclosure comprise (a) one or more iRNA compounds and (b) one or more biological agents that function via a non-RNAi mechanism. Examples of such biological agents include agents that interfere with the interaction of LECT2 and at least one LECT2 binding partner.

[0478] The toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are typical.

[0479] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of the compositions featured in this disclosure typically lies within a range of circulating concentrations that includes the ED50 with little or no toxicity. Dosages can vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods featured in this disclosure, the therapeutically effective amount can be initially estimated from cell culture assays. Dosages can also be formulated in animal models to achieve a circulating plasma concentration range of the compound, or, if appropriate, the polypeptide product of the target sequence, that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture (e.g., to achieve a reduction in polypeptide concentration). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0480] In addition to the administration described above, the iRNAs featured in this disclosure can be administered in combination with other known drugs that are effective in treating diseases or disorders associated with LECT2 expression.In any case, the administering physician can adjust the amount and timing of iRNA administration based on the results observed using standard measures of effectiveness known in the art or as described herein.

[0481] Methods for treating disorders associated with expression of the LECT2 gene The present disclosure relates to the use of iRNAs that target LECT2 to inhibit LECT2 expression and / or to treat diseases, disorders, or pathological processes associated with LECT2 expression.

[0482] In one aspect, a method for treating disorders associated with LECT2 expression is provided, comprising administering iRNA (e.g., dsRNA) disclosed herein to a subject in need thereof.In some embodiments, the iRNA inhibits (reduces) LECT2 expression.In some embodiments, the iRNA increases LECT2 expression.

[0483] As used herein, "disorders associated with LECT2 expression," "diseases associated with LECT2 expression," "pathological processes associated with LECT2 expression," and the like, include any condition, disorder, or disease in which LECT2 expression is altered (e.g., decreased or increased compared to normal levels). In some embodiments, LECT2 expression is decreased. In some embodiments, LECT2 expression is increased. In some embodiments, the decrease or increase in LECT2 expression is detectable in the blood (e.g., plasma) of a subject. In some embodiments, the decrease or increase in LECT2 expression is detectable in a tissue sample from the subject (e.g., kidney or liver sample). The decrease or increase may be assessed relative to levels observed in the same individual before the onset of the disorder or compared to other individual(s) without the disorder. The decrease or increase may be limited to a specific organ, tissue, or region of the body (e.g., kidney or liver).

[0484] As used herein, a "subject" to be treated according to the methods described herein includes a human or non-human animal, e.g., a mammal. A mammal may be, for example, a rodent (e.g., a rat or a mouse) or a primate (e.g., a monkey). In some embodiments, the subject is a human.

[0485] A "subject in need thereof" includes a subject who has, is suspected of having, or is at risk of developing a disorder associated with LECT2 expression. In some embodiments, the subject has or is suspected of having a disorder associated with LECT2 expression. In some embodiments, the subject is at risk of developing a disorder associated with LECT2 expression.

[0486] In some embodiments, the subject is an animal that serves as a model for a disorder associated with LECT2 expression, eg, LECT2 amyloidosis.

[0487] LECT2 amyloidosis In some embodiments, the disorder associated with LECT2 expression is amyloidosis, for example, LECT2 amyloidosis. LECT2 amyloidosis has been described in several clinical studies. For example, see Benson, MD et al. (2008) Kidney International, 74: 218-222; Murphy, CL et al. (2010) Am J Kidney Dis, 56(6):1100-1107; Larsen, CP et al. (2010) Kidney Int., 77(9):816-819; Holanda, DG et al. (20011) Nephrol. Dial. Transplant., 26 (1): 373-376; and Sethi, S. et al. (2012) Kidney International 82, 226-234 (hereinafter referred to as Sethi et al.).

[0488] The clinical and pathological features of LECT2 amyloidosis resemble those of amyloid light chain (AL) amyloidosis. These symptoms include, for example, symptoms of kidney disease and renal failure, such as fluid retention, swelling, and shortness of breath. Amyloidosis can affect the heart, peripheral nervous system, gastrointestinal tract, blood, lungs, and skin. Cardiac complications include, for example, heart failure and arrhythmia. Other symptoms include, for example, stroke, gastrointestinal disorders, hepatomegaly, decreased spleen function, decreased function of the adrenal glands and other endocrine glands, skin discoloration or growths, lung problems, bleeding and bruising problems, fatigue, and weight loss. In some embodiments, the methods described herein are associated with improvement of one or more symptoms described herein.

[0489] Methods for the diagnosis of amyloidosis, e.g., LECT2 amyloidosis, are described, for example, in Leung, N. et al. (2010) Blood, published online September 4, 2012; DOI 10.1182 / blood-2012-03-413682; Shiller, SM et al. (2011). Laboratory Methods for the Diagnosis of Hereditary Amyloidoses, Amyloidosis - Mechanisms and Prospects for Therapy, Dr. Svetlana Sarantseva (Ed.), ISBN: 978-953-307-253-1; Sethi et al. (see above), and in U.S. Patent Application Publication No. 20100323381.

[0490] Based on the results provided by Sethi et al., LECT2 amyloidosis accounts for a significant proportion of cases of renal amyloidosis. See Table 1 of Sethi et al., which shows that 26 of 127 cases of renal amyloidosis studied by laser microdissection and mass spectrometry of renal biopsy and / or nephrectomy specimens were determined to be LECT2 amyloid-type renal amyloidosis. Sethi et al. further report that apolipoprotein E protein and serum amyloid P component (SAP) were present in all cases of LECT2 amyloidosis.

[0491] In some embodiments, the amyloidosis, e.g., LECT2 amyloidosis, is involved in systemic amyloid deposition. In some embodiments, the amyloidosis, e.g., LECT2 amyloidosis, is completely or predominantly localized to a particular tissue or organ (e.g., the kidney or liver).

[0492] In some embodiments, the amyloidosis, eg, LECT2 amyloidosis, is hereditary.

[0493] In some embodiments, LECT2 amyloidosis is diagnosed using analysis of a sample (e.g., a biopsy sample) from a subject. In some embodiments, the biopsy sample is a kidney biopsy. In some embodiments, the sample is a nephrectomy sample. In some embodiments, the sample is a sample from a liver biopsy or other resected liver tissue. In some embodiments, the sample is analyzed using a method selected from one or more of immunohistochemistry, LECT2 immunoassay, electron microscopy, laser microdissection, and mass spectrometry. In some embodiments, LECT2 amyloidosis is diagnosed using laser microdissection and mass spectrometry.

[0494] In some embodiments, amyloidosis, for example, LECT2 amyloidosis, affects the kidney, for example, involves amyloid deposition in the kidney. In some embodiments, kidney function is impaired as a result of amyloidosis. In some embodiments, the subject suffers from one or more of fluid retention, swelling, and shortness of breath. In some embodiments, the subject has nephrotic syndrome. In some embodiments, the subject suffers from proteinuria. In some embodiments, the subject has renal failure.

[0495] In some embodiments, amyloidosis, for example, LECT2 amyloidosis, affects the liver, for example, is involved in amyloid deposition in the liver.In some embodiments, liver function is impaired as a result of amyloidosis.In some embodiments, the subject has hepatitis, for example, chronic hepatitis.In some embodiments, the hepatitis is viral hepatitis.

[0496] LECT2 amyloidosis has been found to be particularly prevalent in Mexican Americans and has been associated with homozygosity for the G allele of the LECT2 gene, which encodes valine at position 40 of the mature protein (amino acid 58 of the unprocessed protein). See, e.g., Benson, MD et al. (2008) Kidney International, 74: 218-222; Murphy, CL et al. (2010) Am J Kidney Dis, 56(6):1100-1107.

[0497] In some embodiments, the subject is of Mexican descent. In some embodiments, the subject is Mexican American.

[0498] In some embodiments, the subject has a G allele of the LECT2 gene, which encodes a valine at position 40 of the mature protein (amino acid 58 of the unprocessed protein). In some embodiments, the subject is homozygous for the G allele (G / G genotype). In some embodiments, the LECT2 protein expressed in the subject has a valine at position 40 of the mature protein (or amino acid 58 of the unprocessed protein).

[0499] In some embodiments, the method reduces LECT2 expression. In some embodiments, the reduction in LECT2 expression is assessed by comparing the level of the same individual before treatment. In some embodiments, the method is shown to reduce LECT2 expression by comparing the LECT2 expression level of a treated subject (or group of subjects) with the level of a control subject (or group of subjects), for example, an untreated subject (or group of subjects) or a subject (or group of subjects) treated with a control treatment (e.g., an iRNA (e.g., dsRNA) that does not target LECT2).

[0500] In some embodiments, the method reduces amyloid deposits, for example, amyloid deposits comprising a LECT2 protein or a portion thereof. In some embodiments, the protein is a wild-type protein. In some embodiments, the protein is a human LECT2 protein or a portion thereof that includes a valine at position 40 (as described herein, at position 40 of the mature secreted protein or at amino acid 58 of the unprocessed protein). In some embodiments, the method reduces the size, number, and / or extent of amyloid deposits.

[0501] In some embodiments, the method reduces one or more symptoms associated with amyloid deposits.

[0502] In some embodiments, the dsRNA is administered in a form that targets the dsRNA to a particular organ or tissue to inhibit amyloid deposition in the organ or tissue.

[0503] In some embodiments, dsRNA is targeted to the liver.In some embodiments, dsRNA is conjugated with a ligand, such as GalNac ligand (for example, GalNac ligand described herein), that targets dsRNA to the liver (for example, to hepatocytes).

[0504] Also provided herein is a method for reducing amyloid deposition, comprising administering the dsRNA disclosed herein to a subject in need thereof (for example, a subject who has, is suspected of having, or is at risk of developing LECT2 amyloidosis).In some embodiments, the method reduces (for example, prevents or reduces) the size, number, and / or extent of amyloid deposition.The size, number, and / or extent of amyloid deposits can be evaluated using any method known in the art (for example, immunoassay, immunohistochemistry, mass spectrometry).The reduction of amyloid deposition can involve at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more reduction in amyloid deposition (for example, the size, number, and / or extent of amyloid deposits).

[0505] In the methods provided herein, iRNA (e.g., dsRNA) and compositions thereof are administered in therapeutically effective amounts. The therapeutic effect of administering LECT2 siRNA can be established, for example, by comparison with an appropriate control. For example, the inhibition of amyloid deposition can be established, for example, in a group of patients with amyloidosis (e.g., LECT2 amyloidosis), by comparing any suitable parameter (e.g., a parameter that evaluates the size, number, or extent of amyloid deposits) with the same parameter in an appropriate control group. The control group (e.g., a similar group or the same group in a crossover design) can include, for example, an untreated group, a group treated with conventional treatment; a group treated with placebo or non-targeting iRNA; etc.

[0506] Rheumatoid arthritis Rheumatoid arthritis is also a disorder associated with LECT2 expression. In particular, it has been found that in the Japanese population, possessing one A allele of the LECT2 gene, which encodes isoleucine at position 40 of the mature protein (or amino acid 58 of the unprocessed protein), increases the overall risk of developing rheumatoid arthritis. Possessing two A alleles is strongly associated with disease severity. See Kameoka, Y. et al. (2000) Arth Rheum, 43(6):1419-20.

[0507] In one embodiment of the method provided herein, the disorder related to LECT2 expression is rheumatoid arthritis.In one embodiment, dsRNA inhibits LECT2 expression in the subject with rheumatoid arthritis.In some such embodiments, dsRNA inhibits LECT2 expression in synovial tissue and / or synovial fluid-derived cells (for example, mononuclear cells and fibroblasts).In some embodiments, dsRNA targets the mRNA that codes for isoleucine at position 40 of mature protein (amino acid 58 of unprocessed protein).

[0508] liver damage LECT2 expression can be increased during acute liver injury.

[0509] In one embodiment of the method provided herein, the disorder associated with LECT2 expression is acute liver injury.In some embodiments, iRNA (e.g., dsRNA) modulates (e.g., increases or decreases) LECT2 expression.In some embodiments, iRNA modulates LECT2 expression in the liver.In some embodiments, iRNA reduces LECT2 expression in the liver.In some embodiments, iRNA increases LECT2 expression in the liver.

[0510] Combination therapy In some embodiments, the iRNA (e.g., dsRNA) disclosed herein is administered in combination with a second therapy (e.g., one or more additional therapies) known to be effective in treating a disorder associated with LECT2 expression (e.g., LECT2 amyloidosis) or a symptom of such a disorder. The iRNA can be administered before, after, or simultaneously with the second therapy. In some embodiments, the iRNA is administered before the second therapy. In some embodiments, the iRNA is administered after the second therapy. In some embodiments, the iRNA is administered simultaneously with the second therapy.

[0511] The second treatment can be an additional therapeutic agent. The iRNA and the additional therapeutic agent can be administered in combination in the same composition, or the additional therapeutic agent can be administered as part of a separate composition.

[0512] In some embodiments, the second therapy is a non-iRNA therapeutic effective to treat a disorder or a symptom of a disorder.

[0513] In some embodiments, the disorder treated by the compositions or methods disclosed herein is LECT2 amyloidosis, which affects kidney function, e.g., by amyloid deposition in the kidney. In some such embodiments, the iRNA is administered in conjunction with a therapy to support kidney function (e.g., dialysis, diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), or dialysis).

[0514] In some embodiments, the disorder treated by the compositions or methods disclosed herein is LECT2 amyloidosis, which involves amyloid deposition in the liver. In some such embodiments, the iRNA is administered in conjunction with a treatment that supports liver function.

[0515] In some embodiments, the disorder treated by the compositions or methods disclosed herein is LECT2 amyloidosis, and the iRNA is administered in conjunction with removal of all or part of an organ(s) affected by amyloidosis (e.g., resection of all or part of kidney or liver tissue affected by amyloidosis). Removal may also be performed in conjunction with replacement of all or part of the removed organ (e.g., in conjunction with kidney or liver transplantation).

[0516] Dosage, route, and timing A subject (e.g., a human subject, e.g., a patient) can be administered a therapeutic amount of iRNA. A therapeutic amount can be, for example, 0.05 to 50 mg / kg. For example, a therapeutic amount can be 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg / kg of dsRNA.

[0517] In some embodiments, the iRNA is formulated for delivery to a target organ, for example, the liver.

[0518] In some embodiments, the iRNA is formulated as a lipid formulation, e.g., an LNP formulation as described herein. In some such embodiments, the therapeutic amount is 0.05-5 mg / kg, e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg / kg of dsRNA. In some embodiments, the lipid formulation, e.g., an LNP formulation, is administered intravenously. In some embodiments, the iRNA (e.g., dsRNA) is formulated as an LNP formulation and administered (e.g., intravenously) at a dose of 0.1-0.5 mg / kg.

[0519] In some embodiments, the iRNA is administered by intravenous infusion over a period of time, e.g., 5, 10, 15, 20, or 25 minutes. In some embodiments, the iRNA is in the form of a GalNAc conjugate described herein. In some such embodiments, the therapeutic amount is 0.5-50 mg, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg / kg of dsRNA. In some embodiments, the GalNAc conjugate is administered subcutaneously. In some embodiments, the iRNA (e.g., dsRNA) is in the form of a GalNAc conjugate and is administered (e.g., subcutaneously) at a dose of 1-10 mg / kg.

[0520] In some embodiments, administration is, for example, repeated periodically, for example, every day, every other week (i.e., every two weeks) for one, two, three, four or more months. After the initial treatment regimen, treatment can be administered less frequently. For example, after administration every other week for three months, administration can be repeated once per month, six months, or one year or more.

[0521] In some embodiments, the iRNA agent is administered in two or more doses, in some embodiments, the number or amount of subsequent doses depends on achieving a desired effect, e.g., inhibiting amyloid deposition, or achieving a therapeutic or prophylactic effect, e.g., reducing or preventing one or more symptoms associated with a disorder.

[0522] In some embodiments, iRNA agent is administered according to a schedule.For example, iRNA agent can be administered once a week, twice a week, three times a week, four times a week, or five times a week.In some embodiments, schedule comprises administration at regular intervals, for example, once every hour, every four hours, every six hours, every eight hours, every 12 hours, every day, every two days, every three days, every four days, every five days, every week, every two weeks, or every month.In some embodiments, iRNA agent is administered as frequently as needed to achieve desired effect.

[0523] In some embodiments, the schedule includes closely spaced administrations followed by an extended period during which the agent is not administered. For example, the schedule can include an initial set of doses administered at relatively short intervals (e.g., about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, or about every 72 hours), followed by an extended period during which the iRNA agent is not administered (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks). In one embodiment, the iRNA agent is initially administered hourly and later administered at longer intervals (e.g., daily, weekly, biweekly, or monthly). In another embodiment, the iRNA agent is initially administered daily and later administered at longer intervals (e.g., weekly, biweekly, or monthly). In certain embodiments, the longer intervals increase over time or are determined based on achieving a desired effect.

[0524] Prior to administration of the full dose of iRNA, the patient may receive a smaller dose, for example, a 5% infusion dose, and be monitored for adverse effects, such as allergic reactions, or increases in lipid levels or blood pressure. In another example, the patient may be monitored for undesirable effects.

[0525] Methods for modulating LECT2 gene expression In yet another aspect, the present disclosure provides a method for modulating (e.g., inhibiting or activating) the expression of the LECT2 gene, e.g., in a cell or in a subject. In some embodiments, the cell is ex vivo, in vitro, or in vivo. In some embodiments, the cell is in the liver (e.g., a hepatocyte). In some embodiments, the cell is in a subject (e.g., a mammal, e.g., a human). In some embodiments, the subject (e.g., a human) is at risk for or diagnosed with a disorder associated with LECT2 expression described herein.

[0526] In one embodiment, the method comprises contacting cells with the iRNA described herein in an amount effective to reduce the expression of LECT2 gene in the cells.As used herein, "contacting" includes direct contacting of cells and indirect contacting of cells.For example, when a composition comprising iRNA is administered to a subject (for example, intravenously or subcutaneously), the cells in the subject can be contacted.

[0527] Expression of the LECT2 gene can be evaluated based on the level of expression of LECT2 mRNA, LECT2 protein, or another parameter functionally related to the level of expression of the LECT2 gene. In some embodiments, LECT2 expression is inhibited by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, the iRNA has an IC in the range of 0.001-0.01 nM, 0.001-0.10 nM, 0.001-1.0 nM, 0.001-10 nM, 0.01-0.05 nM, 0.01-0.50 nM, 0.02-0.60 nM, 0.01-1.0 nM, 0.01-1.5 nM, or 0.01-10 nM. 50 IC 50 The value should be taken relative to an appropriate control value, e.g., the IC of a non-targeting iRNA. 50 can be normalized to

[0528] In some embodiments, the method includes introducing an iRNA described herein into a cell and maintaining the cell for a time sufficient to result in degradation of the mRNA transcript of the LECT2 gene, thereby inhibiting expression of the LECT2 gene in the cell.

[0529] In one embodiment, the method includes administering to a mammal a composition described herein, e.g., a composition comprising an iRNA targeting LECT2, to reduce expression of the target LECT2 gene for an extended period of time, e.g., at least 2, 3, 4, or more days, e.g., 1, 2, 3, or 4 weeks or more. In some embodiments, the reduction in LECT2 expression is detectable within 1, 2, 4, 8, 12, or 24 hours after the first administration.

[0530] In another embodiment, the method comprises administering a composition described herein to a mammal so that expression of the target LECT2 gene is increased, for example, by at least 10% compared to an untreated animal. In some embodiments, activation of LECT2 occurs over an extended period, for example, at least 2, 3, 4 days or more, for example, 1 week, 2 weeks, 3 weeks, 4 weeks or more. Without wishing to be bound by theory, iRNAs can activate LECT2 expression by stabilizing LECT2 mRNA transcripts, interacting with the genomic promoter, and / or inhibiting inhibitors of LECT2 expression.

[0531] The iRNAs useful in the methods and compositions featured in this disclosure specifically target the RNA (primary or processed) of the LECT2 gene. Compositions and methods for inhibiting the expression of the LECT2 gene using iRNAs can be prepared and performed as described elsewhere herein.

[0532] In one embodiment, the method includes administering a composition containing an iRNA, wherein the iRNA comprises a nucleotide sequence complementary to at least a portion of an RNA transcript of the LECT2 gene of a subject, e.g., a mammal, e.g., a human, to be treated. The composition can be administered by any suitable means known to those skilled in the art, including, but not limited to, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, intradermal, respiratory (aerosol), intranasal, rectal, and topical (buccal and sublingual) administration.

[0533] In certain embodiments, the composition is administered by intravenous infusion or injection. In some such embodiments, the composition comprises a lipid-formulated siRNA (e.g., an LNP formulation, such as an LNP11 formulation) for intravenous infusion.

[0534] In other embodiments, the composition is administered subcutaneously. In some such embodiments, the composition comprises an iRNA conjugated to a GalNAc ligand. In some such embodiments, the ligand targets the iRNA to the liver (e.g., hepatocytes).

[0535] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure belongs.Methods and materials similar or equivalent to those described herein can be used in the implementation or testing of the iRNA and methods featured in this disclosure, and suitable methods and materials are described below.All papers, patent applications, patents and other references described herein are incorporated by reference in their entirety.In the case of conflict, the present specification, including definitions, shall prevail.In addition, materials, methods and examples are only illustrative and are not intended to be limiting.

[0536] Specific Embodiments 1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of LECT2, comprising a sense strand and an antisense strand comprising a region complementary to a LECT2 RNA transcript and comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from one of the antisense sequences listed in Tables 2A-2B, 3A-3B, 6 or 7, or a pharmaceutically acceptable salt thereof. 2. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of LECT2, comprising a sense strand that is 15 to 30 nucleotides in length, and an antisense strand that is 15 to 30 nucleotides in length and complementary to at least 15 consecutive nucleotides of a target sequence listed in Table 2A, 2B, 3A, 3B, 6, or 7, or a pharmaceutically acceptable salt thereof. 3. The dsRNA of embodiment 1 or 2, comprising at least one modified nucleotide. 4. The dsRNA agent of embodiment 3, wherein no more than 5 nucleotides of the sense strand and no more than 5 nucleotides of the antisense strand are unmodified nucleotides. 5. The dsRNA agent of embodiment 3, wherein every nucleotide of the sense strand and every nucleotide of the antisense strand comprises a modification. 6. The dsRNA of any of the preceding embodiments, comprising a duplex region that is 15 to 30 base pairs in length. 7. The dsRNA of embodiment 6, wherein the duplex region is 17 to 25 base pairs in length. 8. The dsRNA of embodiment 6 or 7, wherein the duplex region is 19 to 22 base pairs in length. 9. The dsRNA of any of embodiments 6 to 8, wherein the duplex region is 21 base pairs in length. 10. The dsRNA of any of the previous embodiments, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides having 0, 1, 2, or 3 mismatches from one of the antisense sequences listed in any one of Tables 2A, 2B, 3A, 3B, 6, or 7. 11. The dsRNA agent of any preceding embodiment, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides having 0, 1, 2, or 3 mismatches from a sense sequence listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, and 6B corresponding to the antisense sequence. 12. The dsRNA of any of the previous embodiments, wherein the antisense strand comprises a nucleotide sequence com...

Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of LECT2, the dsRNA comprising a sense strand and an antisense strand, the antisense strand comprising a region complementary to a LECT2 RNA transcript, the antisense strand being 21 to 23 nucleotides in length and comprising at least 15 consecutive nucleotides derived from the nucleotide sequence 5'-AUUUAUUUUGAAGAUCUGACCGG-3' (SEQ ID NO: 371), or a pharmaceutically acceptable salt thereof.

2. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of LECT2, wherein the dsRNA comprises a sense strand and an antisense strand, the antisense strand is 21 to 23 nucleotides in length, and the sequence of the antisense strand is complementary to nucleotides 669 to 691 of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.

3. 3. The dsRNA of claim 1 or 2, comprising at least one modified nucleotide.

4. (a) the dsRNA comprises a duplex region that is 21 base pairs in length; (b) the complementary region is 21 or 23 nucleotides in length; (c) at least one strand comprises a 3' overhang of at least 1 or 2 nucleotides; (d) the dsRNA comprises a blunt end; (e) the complementary region consists of the antisense strand nucleotide sequence 5'-AUUUAUUUUGAAGAUCUGACCGG-3' (SEQ ID NO: 371); (f) the dsRNA comprises a sense strand consisting of the sense strand sequence set forth in SEQ ID NO: 370 and an antisense strand consisting of the antisense strand sequence set forth in SEQ ID NO: 371; and / or (g) the sense strand is conjugated to at least one ligand; The dsRNA according to any one of claims 1 to 3.

5. at least one modified nucleotide; and (a) the at least one modified nucleotide is selected from the group consisting of a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group; (b) the at least one modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleic acids (LNAs), acyclic nucleotides, abasic nucleotides, glycol nucleotides (GNAs), 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural nucleotide bases; (c) the at least one modified nucleotide is selected from the group consisting of locked nucleic acids (LNA), acyclic nucleotides, hexitol or hexose nucleic acids (HNA), cyclohexene nucleic acids (CeNA), glycol nucleic acids (GNA), 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, and combinations thereof; and / or (d) the at least one modified nucleotide is 2'-O-methyl, 2'-fluoro, and / or GNA; The dsRNA of claim 3 or 4.

6. (a) a ligand is attached to the 3′ end of the sense strand; (b) the ligand comprises a carbohydrate; (c) the ligand is a GalNAc ligand; (d) the ligand 【Chemistry 1】 is; (e) the ligand is attached via a linker; (f) the ligand is attached via a bivalent or trivalent branched linker; (g) The ligand is attached via a linker, wherein the ligand and linker are represented by Formula XXIV: 【Chemistry 2】 and / or (h) the ligand targets the dsRNA to hepatocytes; The dsRNA of claim 4.

7. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of LECT2, comprising a sense strand and an antisense strand comprising a region complementary to a LECT2 RNA transcript; (a) the sense strand is 21 nucleotides in length and contains the sequence of gsgsucagAfuCfUfUfcaaaauaaaauL96 (SEQ ID NO: 143) and all modifications, and the antisense strand is 23 nucleotides in length and contains the sequence of asUfsuuaUfuUfUfgaagAfuCfugaccsgsg (SEQ ID NO: 144) and all modifications; wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively, s is a phosphorothioate linkage, Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively, and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol-Hyp-(GalNAc-alkyl)3; or (b) the complementary region is substantially complementary to nucleotides 669 to 691 of SEQ ID NO:1; dsRNA.

8. A cell comprising the dsRNA of any one of claims 1 to 7.

9. A pharmaceutical composition for inhibiting the expression of the LECT2 gene, comprising the dsRNA of any one of claims 1 to 7.

10. 10. The pharmaceutical composition of claim 9, comprising a lipid formulation or an LNP11 formulation.

11. the dsRNA is conjugated to a carbohydrate ligand or a GalNAc ligand; The dsRNA is administered in an unbuffered solution, saline, water, a buffered solution, phosphate buffered saline (PBS), or a buffer containing acetate, citrate, prolamin, carbonate, phosphate, or any combination thereof; the composition is administered intravenously or subcutaneously; and / or The dsRNA is targeted to liver cells or hepatocytes. The pharmaceutical composition according to claim 9 or 10.

12. 8. The dsRNA according to any one of claims 1 to 7, which is used to inhibit LECT2 expression in a cell, wherein the inhibition is (a) introducing the dsRNA into a cell; and (b) maintaining the cells of step (a) for a time sufficient to obtain degradation of the mRNA transcripts of the LECT2 gene, thereby inhibiting expression of the LECT2 gene in the cells. Including, wherein the dsRNA is introduced into the cells ex vivo, in vitro, or in vivo; dsRNA.

13. 13. The dsRNA of claim 12, wherein the cell is present in a subject in need of treatment, prevention and / or management of a disorder associated with LECT2 expression or a subject with amyloidosis.

14. (a) the cell is a liver cell or a hepatocyte; and / or (b) the expression of LECT2 is inhibited by at least 20%; 14. The dsRNA of claim 12 or 13.

15. A pharmaceutical composition comprising a therapeutically effective amount of the dsRNA of any one of claims 1 to 7 for treating a disorder associated with LECT2 expression, for treating LECT2 amyloidosis, and / or for reducing LECT2 amyloid deposition in a subject with LECT2 amyloidosis.

16. (a) the subject has amyloidosis or is at risk of developing amyloidosis or LECT2 amyloidosis; (b) a pharmaceutical composition comprising the dsRNA is administered according to a dosing regimen; (c) the pharmaceutical composition comprising the dsRNA is administered according to a weekly, biweekly, or monthly dosing regimen; (d) the treatment reduces LECT2 amyloid deposition; (e) the dsRNA is administered at a dose of 0.05 to 50 mg / kg of subject body weight; (f) the dsRNA is conjugated to a GalNAc ligand; (g) the dsRNA is conjugated to a GalNAc ligand and administered at a dose of 1 mg / kg to 10 mg / kg; and / or (h) the dsRNA is conjugated to a GalNAc ligand and administered at a dose of 1 mg / kg or 3 mg / kg; 16. The pharmaceutical composition of claim 15.

17. A vector encoding at least one strand of the dsRNA of any one of claims 1 to 7.

18. A cell comprising the vector of claim 17.

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