Compositions and methods for inhibiting LECT2 gene expression

Abstract

The present invention provides a double-stranded ribonucleic acid (dsRNA) composition that targets the LECT2 gene, and a method for modifying (e.g., inhibiting) LECT2 expression using such a dsRNA composition. [Solution] The present invention provides compositions and methods that affect RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the LECT2 gene in cells or in subjects (e.g., mammals such as human subjects). The present invention also provides compositions and methods for treating disorders associated with LECT2 gene expression, such as LECT2 amyloidosis.

Description

[Technical Field]

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

[0002] Sequence List This application includes an electronically submitted sequence listing in ASCII format, the entirety of which is incorporated herein by reference. The ASCII copy, prepared on 27 August 2020, is named A2038-7232WO_SL.Txt and has a size of 221,086 bytes.

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

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

[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 hepatic complications (e.g., hepatitis, e.g., chronic hepatitis). It may be particularly prevalent in Mexican Americans and / or individuals who are homozygous for the G allele encoding valine at position 40 of the mature LECT2 protein (or position 58 of the unprocessed protein). Treatment for LECT2 amyloidosis is limited and new therapies are needed. [Overview of the project] [Problems that the invention aims to solve]

[0006] This disclosure describes methods and iRNA compositions for regulating LECT2 gene expression. In certain embodiments, LECT2 gene expression is reduced or inhibited using LECT2-specific iRNA. Such inhibition may be useful in treating disorders associated with LECT2 expression, such as amyloidosis, e.g., LECT2 amyloidosis (ALECT2). [Means for solving the problem]

[0007] Accordingly, the following compositions and methods are described herein that affect RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the 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 LECT2 gene expression, such as LECT2 amyloidosis.

[0008] In some embodiments, LECT2 amyloidosis is renal amyloidosis. In some embodiments, LECT2 amyloidosis includes amyloid deposition in the kidneys. In some embodiments, LECT2 amyloidosis is associated with kidney disease (e.g., nephrotic syndrome). In some embodiments, amyloidosis is associated with proteinuria. In some embodiments, proteinuria is absent. In some embodiments, LECT2 amyloidosis is hepatic amyloidosis. In some embodiments, LECT2 amyloidosis includes amyloid deposition in the liver. In some embodiments, LECT2 amyloidosis is associated with inflammation of the liver (e.g., hepatitis, e.g., chronic hepatitis). In some embodiments, the methods described herein are effective in inhibiting amyloid deposition or syndromes 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 deposition).

[0009] As used herein, the terms “iRNA,” “RNAi,” “iRNA agent,” “RNAi agent,” or “iRNA molecule” refer to an agent containing RNA as defined herein, which 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 cells or mammals.

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

[0012] In other embodiments, the iRNA includes a dsRNA having an RNA strand (antisense strand) having a region substantially complementary to a portion of the LECT2 mRNA. In one embodiment, the iRNA includes a dsRNA having an RNA strand (antisense strand) having a region substantially complementary to a portion of the LECT2 mRNA, for example, human LECT2 mRNA [e.g., provided in NM_002302.2 (SEQ ID NO: 1), or human LECT2 mRNA having an A-to-G substitution at nucleotide position 373 of SEQ ID NO: 1].

[0013] In one embodiment, an iRNA for inhibiting LECT2 gene expression comprises at least two sequences that are 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 that is substantially complementary to at least a portion of the mRNA encoding the LECT2 transcript, with the complementary region being 30 nucleotides or less and at least 15 nucleotides long. Generally, the iRNA is 19 to 24 nucleotides long.

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

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

[0016] In some embodiments, the iRNA is approximately 15 to 25 nucleotides long, and in other embodiments, the iRNA is approximately 25 to 30 nucleotides long. When the LECT2-targeting iRNA comes into contact with cells expressing LECT2, it inhibits the expression of the LECT2 gene (e.g., 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%).

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

[0018] In some embodiments, the 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, the 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, the 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, the iRNA (e.g., dsRNA) has the sense and / or antisense sequence of AD-454781.

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

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

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

[0022] In one embodiment, the iRNA described herein targets a variant of the wild-type LECT2 RNA transcript, and in another embodiment, the iRNA targets a mutant transcript (e.g., LECT2 RNA with an allele variant). For example, the iRNA 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) the valine-coding mRNA 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) the isoleucine-coding mRNA 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 the valine-coding mRNA and the isoleucine-coding mRNA 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 specific LECT2 transcript variants. In yet another embodiment, the iRNA agent targets multiple transcript variants.

[0025] In one embodiment, the iRNA featured in this disclosure targets a non-coding region of a LECT2 RNA transcript, such as 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 contribute as a targeting moiety and / or ligand, as described herein. In one embodiment, the conjugate is bound to the 3' end of the sense strand of the dsRNA. In some embodiments, the conjugate is bound via a linker, such as 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 an RNAi agent (e.g., dsRNA) to specific cells, e.g., hepatocytes. The GalNAc derivatives may be conjugated via linkers, e.g., divalent or trivalent branched linkers. In certain embodiments, the conjugate is

[0028] [ka] That is the case.

[0029] In some embodiments, the RNAi agent is bound to a carbohydrate conjugate via a linker, for example, 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 defined in Table 1 and is conjugated to L96 as 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., hepatocytes). 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 embodiment, provided herein are pharmaceutical compositions 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 medium. In one embodiment, the composition is used to treat a disorder related to LECT2 expression, such as amyloidosis, such as LECT2 amyloidosis.

[0036] In one embodiment, the iRNA provided herein is a double-stranded ribonucleic acid (dsRNA) for inhibiting LECT2 expression, comprising a sense strand and an antisense strand 15 to 30 base pairs long, wherein the antisense strand is complementary to at least 15 nucleotides of a double-stranded target sequence disclosed in Tables 2A to 2B, 3A to 3B, 4A to 4B, or 5 to 7, and is 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 the double-stranded 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 the double-stranded 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 nucleotides of the target sequence of the double-stranded 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 double-stranded 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 nucleotides of the target sequence of the double helix 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 double helix 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 the double-stranded 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 the double-stranded AD-454781. In some embodiments, the antisense strand is complementary to all nucleotides of the target sequence of the double-stranded 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 the 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 consecutive nucleotides of the target site 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 the antisense strand, wherein the antisense strand comprises a region complementary to the LECT2 RNA transcript, each strand has from about 14 to about 30 nucleotides, and the double-stranded RNAi agent has the 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) (wherein: i, j, k, and l are each independently 0 or 1; p, p’, q, and q’ are each independently from 0 to 6; Each N a and N a ’ represents an oligonucleotide sequence comprising from 0 to 25 nucleotides, either modified or unmodified, or a combination thereof, and each sequence comprises at least two different modified nucleotides; Each N b and N b ’ represents an oligonucleotide sequence comprising from 0 to 10 nucleotides, either modified or unmodified, or a combination thereof; Each n p , n p ’n q , and n q ’ represents an overhang 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 is different from the modification of Y, N b (The modification of ' is different from the modification of Y') It is a double-stranded RNAi (dsRNA) represented by [this symbol].

[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 arises from the 5' end at positions 11, 12, and 13 of the antisense chain.

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

[0051] In some embodiments, the double-stranded region is 15 to 30 nucleotide pairs long. In some embodiments, the double-stranded region is 17 to 23 nucleotide pairs long. In some embodiments, the double-stranded region is 19 to 21 nucleotide pairs long. In some embodiments, the double-stranded region is 21 to 23 nucleotide pairs long.

[0052] In some embodiments, nucleotide modifications are 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'-deoxy, 2'-hydroxyl, and any combination thereof.

[0053] In some embodiments, the nucleotide modification may be 2'-O-methyl, 2'-fluoro, or both, and may also be glycol nucleic acid (GNA).

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

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

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

[0057] In some embodiments, the ligand is

[0058] [ka] That is the case.

[0059] In some embodiments, the ligand and linker are expressed in formula XXIV:

[0060] [ka] As shown below.

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

[0062] In some embodiments, the dsRNA has (e.g., includes) 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 embodiment, the iRNA provided herein is a double-stranded ribonucleic acid (dsRNA) for inhibiting LECT2 expression, comprising a sense strand and an antisense strand comprising a region complementary to the LECT2 RNA transcript and containing at least 15 consecutive nucleotides that differ by three or fewer 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 chain includes at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides that differ by 3 nucleotides or less from one of the antisense sequences listed in any one of Tables 2A-2B, 3A-3B, 6, or 7. In some embodiments, the antisense chain includes at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides that differ by 2 nucleotides or less from one of the antisense sequences listed in any one of Tables 2A-2B, 3A-3B, 6, or 7. In some embodiments, the antisense chain includes at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides that differ by 1 nucleotide or less 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 includes at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides that differ by 3 nucleotides or less 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 includes at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides that differ by 2 nucleotides or less 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 includes at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides that differ by 1 nucleotide or less 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 double-stranded 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 double-stranded AD-454781, AD-133461, or AD-454746. In some embodiments, the sense and antisense sequences are the double-stranded AD-454781. In some embodiments, the sense and antisense sequences are the double-stranded sequences disclosed herein that suppress LECT2 mRNA expression by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, as evaluated using the assay disclosed in the examples provided herein.

[0067] In some embodiments, the dsRNA contains at least one modified nucleotide. In some embodiments, the first five nucleotides of the sense strand and the first five nucleotides of the antisense strand of the dsRNA are unmodified. In some embodiments, all nucleotides of the sense strand and all nucleotides of the antisense strand of the dsRNA contain modifications.

[0068] In some embodiments, 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 or dodecanoate bisdecylamide group linked to a cholesteryl derivative.

[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, debasalized nucleotides, glycol nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramides, and non-natural bases including nucleotides.

[0070] In some embodiments, the dsRNA may include a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or both, and may also be a glycol nucleotide.

[0071] In some embodiments, the dsRNA contains 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 contains 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 contains at least 1, 2, 3, 4 or more 2'-fluoromodified nucleotides in the sense strand. In some embodiments, the dsRNA contains 2'-fluoromodified nucleotides at positions 7, 9, 10, 11, or a combination thereof in the sense strand. In some embodiments, the dsRNA contains 2'-fluoromodified nucleotides at positions 7, 9, 10, and 11 in the sense strand. In some embodiments, the dsRNA contains at least 1, 2, 3, 4, 5, 6, 7 or more 2'-fluoromodified nucleotides in the antisense strand. In some embodiments, the dsRNA contains 2'-fluoromodified nucleotides at positions 2, 4, 6, 8, 9, 14, 16, or a combination thereof in the antisense strand. In some embodiments, the dsRNA contains 2'-fluoromodified nucleotides at positions 2, 14, and 16 in the antisense strand. In some embodiments, the dsRNA contains 2'-fluoromodified nucleotides at positions 2, 6, 8, 9, 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 located on the antisense strand. In some embodiments, the glycol nucleotide is located at position 7 of the antisense strand.

[0074] In some embodiments, the dsRNA contains phosphorothioate bonds between positions 1 and 2, between positions 2 and 3, or both, on the sense strand. In some embodiments, the dsRNA contains phosphorothioate bonds between positions 1 and 2 and between positions 2 and 3 on the sense strand.

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

[0076] In some embodiments, the complementary region is at least 17 nucleotides long. In some embodiments, the complementary region is between 19 and 23 nucleotides long. In some embodiments, the complementary region is 21 nucleotides long.

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

[0078] In some embodiments, at least one strand includes a 3' overhang of at least one nucleotide. In some embodiments, at least one strand includes a 3' overhang of at least two nucleotides. In some embodiments, the dsRNA includes a blunt end. In some embodiments, the dsRNA includes both 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 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 AD-454781, AD-133461, or AD-454746. In some embodiments, the complementary region consists of the antisense sequence of the double-stranded AD-454781. In some embodiments, the complementary region consists of an antisense sequence selected from the double-stranded sequences disclosed herein, which suppresses 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 containing or consisting of a sense strand sequence selected from Tables 2A-2B, 3A-3B, 6, or 7, and an antisense strand containing or consisting of an antisense sequence selected from Tables 2A-2B, 3A-3B, 6, or 7. In some embodiments, the dsRNA contains or consists of a corresponding sense-antisense sequence pair selected from the double-stranded sequences disclosed in Tables 2A-2B, 3A-3B, 4A-4B, or 5-7. In certain embodiments, the dsRNA contains or consists of a corresponding sense-antisense sequence pair selected from the double-stranded sequences disclosed in Table 2A, 3A, or 6. In certain embodiments, the dsRNA contains or consists of a corresponding sense-antisense sequence pair selected from the double-stranded sequences disclosed in Table 2B, 3B, or 7. In certain embodiments, the dsRNA contains or consists of a corresponding sense-antisense sequence pair selected from the double-stranded sequences disclosed in Table 4A or 4B. In certain embodiments, the dsRNA comprises or consists of a corresponding sense sequence and antisense sequence pair selected from the double-stranded ones disclosed in Table 5.

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

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

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

[0085] In one embodiment, the present disclosure provides cells containing at least one iRNA (e.g., dsRNA) disclosed herein. The cells are typically mammalian cells, such as human cells. In some embodiments, the cells are liver cells (e.g., hepatocytes).

[0086] In one embodiment, the Disclosure provides human cells (e.g., human cells described herein) in which levels of LECT2 mRNA or LECT2 protein are reduced compared to similar untreated cells, the levels of 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, human cells were produced by a process that included contacting human cells (e.g., human cells as described herein) with dsRNA, e.g., dsRNA as described herein.

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

[0089] In some embodiments of the pharmaceutical compositions described herein, iRNA (e.g., dsRNA) is administered in a non-buffered solution. In some embodiments, the non-buffered solution is physiological saline or water.

[0090] In some embodiments of the pharmaceutical compositions described herein, iRNA (e.g., dsRNA) is administered by a buffer solution. In some embodiments, the buffer solution comprises an 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, iRNA (e.g., dsRNA) is targeted to the liver (e.g., hepatocytes).

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

[0093] In some embodiments, the pharmaceutical composition includes an iRNA (e.g., dsRNA) containing a ligand (e.g., GalNAc ligand) that targets the iRNA (e.g., dsRNA) described herein to liver cells, for example, hepatocytes.

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

[0095] In certain embodiments, the pharmaceutical composition, for example, the composition described herein, comprises a lipid formulation. In some embodiments, the RNAi agent is an LNP formulation, for example, an MC3 formulation. In some embodiments, the LNP formulation targets the RNAi agent to specific cells, for example, liver cells (e.g., hepatocytes). In some embodiments, the lipid formulation is an LNP11 formulation. In some embodiments, the composition is administered intravenously.

[0096] In another embodiment, the pharmaceutical composition is formulated for administration according to the dosage regimens described herein, for example, once every four weeks or less, once every three weeks or less, once every two weeks or less, or once a week or less. In another embodiment, administration of the pharmaceutical composition may be maintained for one month or longer, for example, one, two, three, or six months, or one year or longer.

[0097] In another embodiment, a composition containing the iRNA featured in this 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 the iRNA-containing composition provided herein may be administered before, after, or concurrently 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 concurrently with the second treatment.

[0098] In some embodiments, the second treatment is a non-iRNA therapeutic agent effective in treating the disorder or its symptoms.

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

[0100] In some embodiments, the disorder treated by the compositions or methods disclosed herein is LECT2 amyloidosis, including 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 the removal of all or part of the organ(s) affected by amyloidosis (e.g., excision of all or part of the kidney or liver tissue affected by amyloidosis). The removal may be carried out in conjunction with the replacement of all or part of the removed organ(s) (e.g., in conjunction with kidney or liver transplantation).

[0102] In one embodiment, the foregoing provides a method for inhibiting LECT2 expression in cells, comprising (a) introducing an iRNA (e.g., dsRNA) described herein into cells, and (b) maintaining the cells from step (a) for a time sufficient to obtain degradation of the mRNA transcript of the LECT2 gene, thereby inhibiting the expression of the LECT2 gene in the cells.

[0103] In one embodiment, the foregoing provides a method for inhibiting LECT2 expression in cells (e.g., cells as described herein), comprising: (a) contacting, for example introducing, an iRNA (e.g., dsRNA) as described herein into cells; and (b) maintaining the cells from step (a) for a period of time sufficient to obtain a reduction in the levels of LECT2 mRNA, LECT2 protein, or both LECT2 mRNA and LECT2 protein, thereby inhibiting the expression of the LECT2 gene in the cells.

[0104] In one embodiment, provided herein is a method for reducing or inhibiting the expression of the LECT2 gene in cells (e.g., liver cells). The method comprises contacting cells with the dsRNA described herein, thereby inhibiting the expression of the LECT2 gene. As used herein, “contact” includes direct contact with cells as well as indirect contact with cells. For example, when a composition containing RNAi is administered to a subject (e.g., intravenously or subcutaneously), cells within the subject (e.g., liver cells) may be contacted.

[0105] In some embodiments, the method is: (a) Introducing into a cell a double-stranded ribonucleic acid (dsRNA) comprising at least two sequences that are complementary to each other, wherein the dsRNA has a sense strand having a first sequence and an antisense strand having a second sequence; the antisense strand has a complementary region that is substantially complementary to at least a portion of the mRNA encoding LECT2, the complementary region being 30 nucleotides or less in length, e.g., 15-30 nucleotides, and generally 19-24 nucleotides, and the dsRNA, upon contact with a cell expressing LECT2, inhibits the 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) Maintain the cells from step (a) for a sufficient time to obtain degradation of the mRNA transcript of the LECT2 gene, thereby reducing or inhibiting the expression of the LECT2 gene in the cells. Includes.

[0106] In some embodiments of the aforementioned methods for 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, cells are present in subjects that require treatment, prevention, and / or management of disorders related to LECT2 expression.

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

[0109] In some embodiments, LECT2 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%, as determined by, for example, 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) is in the range of 0.0005 to 1 nM, for example, between 0.001 to 0.2 nM, between 0.002 to 0.1 nM, between 0.005 to 0.075 nM, or between 0.01 to 0.05 nM. 50 In some embodiments, the iRNA (e.g., dsRNA) has an IC of 0.02 nM or less, for example, 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 It has. In some embodiments, the iRNA (e.g., dsRNA) has an IC in the range of 0.01 to 1 nM. 50 It holds.

[0112] In some embodiments, the cells (e.g., hepatocytes) are mammalian cells (e.g., human, non-human primate, or rodent cells). 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 the LECT2 gene in the cells.

[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 deposition). The 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 treated with an untreated or non-targeted dsRNA (e.g., a dsRNA that does not target LECT2)].

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

[0115] In other embodiments, the Disclosure provides methods for treating pathogenesis processes (e.g., amyloid deposition) associated with LECT2 expression. In one embodiment, the method comprises administering an effective (e.g., therapeutically or prophylactically effective) amount of the dsRNA provided herein to a subject, e.g., a patient in need of such treatment.

[0116] In one embodiment, the foregoing provides a method for treating and / or preventing a disorder related to LECT2 expression (e.g., LECT2 amyloidosis), comprising administering to a subject requiring such treatment a therapeutically effective amount of the iRNA (e.g., dsRNA) described herein, or a composition comprising the iRNA (e.g., dsRNA) described herein.

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

[0119] In some embodiments, administration of LECT2-targeting iRNA 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, e.g., amyloidosis, e.g., LECT2 amyloidosis, includes the presence or level of some degree of amyloid deposition or LECT2 (e.g., 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 human.

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

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

[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 a subject. In some embodiments, measuring the level of LECT2 in a subject includes measuring the level of LECT2 gene, LECT2 protein, or LECT2 mRNA in a biological sample (e.g., tissue, blood, or serum sample) from the subject. In some embodiments, the methods described herein further include performing blood tests, imaging tests, or liver or kidney biopsies. In some embodiments, measuring the level of LECT2 (e.g., LECT2 gene, LECT2 mRNA, or LECT2 protein) in a subject is performed before treatment with a dsRNA agent or pharmaceutical composition. In some embodiments, if it is determined that the subject has a LECT2 (e.g., LECT2 gene, LECT2 mRNA, or LECT2 protein) level higher than a reference level, the 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 a subject is performed after treatment with a dsRNA agent or pharmaceutical composition.

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

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

[0127] In some embodiments, 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 body weight to 5 mg / kg body weight of the subject.

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

[0131] In some embodiments, the method 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 deposition). The 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 treated with an untreated or non-targeted dsRNA (e.g., a dsRNA that does not target LECT2)].

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

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

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

[0135] In some embodiments, iRNA (e.g., dsRNA) or a composition containing 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 embodiment, provided herein is a vector encoding at least one strand of iRNA (e.g., dsRNA) as described herein.

[0137] In one embodiment, the herein provides a vector encoding at least one strand of dsRNA, wherein the dsRNA comprises a region complementary to at least a portion of 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 complementary region is at least 15 nucleotides long. In some embodiments, the complementary region is 19 to 23 nucleotides long. In some embodiments, the complementary region is 21 to 23 nucleotides long.

[0139] In one embodiment, a vector is provided to inhibit 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 ligated 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 LECT2 iRNA.

[0140] In one embodiment, provided herein are cells containing a vector as described herein.

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

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

[0143] Details of various embodiments of this disclosure are described below. Other features, purposes, and advantages of this disclosure will be apparent from the description and drawings, as well as the claims.

[0144] A patent or application file shall contain at least one color drawing. A copy of the published patent or patent application, accompanied by the color drawing(s), shall be provided by the Secretariat upon request and payment of the required fees. [Brief explanation of the drawing]

[0145] [Figure 1] Figure 1 shows the human LECT2 mRNA transcript sequence (reference sequence NM_002302.2 GI:59806344, recorded on April 17, 2013; Sequence ID No. 1). [Figure 2] Figure 2 shows the sequences and chemistry of three exemplary LECT2 siRNAs designed to target regions of LECT2 mRNA in both human and cynomolgus monkeys: AD-454781, AD-133461, and AD-454746. For each siRNA, "F" in green indicates a "2' fluoro" modification, OMe in black indicates a methoxy group, GNA in purple indicates a glycol nucleic acid, and PS indicates a phosphonothioate bond. Figure 2 discloses sequence numbers 897–902, respectively, in the order in which they appear. [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 of circulating LECT2 protein in plasma (residual percentage) were quantified 14 days after treatment in the experimental (LECT2 siRNA) group and the control (PBS) group. [Figure 4] Figures 4A–4C show the dose-response of three exemplary LECT2 siRNAs in suppressing LECT2 in cynomolgus monkeys compared to a PBS control. Relative levels of plasma LECT2 (plasma LECT2 protein knockdown) were quantified by normalization to the pre-treatment protein levels of each individual monkey. [Figure 5] Figures 5A-5B show the relative levels (differences in expression ratios) 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) group or the control (PBS) group. [Figure 6]Figures 6A and 6B evaluate long-term LECT-2 knockdown in cynomolgus monkeys. In Figure 6A, the relative levels of LECT2 mRNA (difference in expression ratios) in the liver of monkeys 6 months after initial treatment were quantified in the experimental (siRNA AD-81725) group and the control (PBS) group. In Figure 6B, the circulating levels of plasma LECT2 protein (percentage of residual protein) were measured monthly before treatment and for 6 months after the first dose in both the experimental (siRNA AD-81725) and control (PBS) groups. [Modes for carrying out the invention]

[0146] iRNAs direct sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). This specification describes iRNAs and methods for modulating (e.g., inhibiting) the expression of the LECT2 gene using them. Also provided are compositions and methods for treating disorders associated with LECT2 expression, such as amyloidosis (e.g., LECT2 amyloidosis).

[0147] The iRNAs of the compositions characterized herein comprise an RNA chain (antisense chain) having a region of 30 nucleotides or less in length, i.e., 15–30 nucleotides, and generally 19–24 nucleotides, which is substantially complementary to at least a portion of the mRNA transcript of the LECT2 gene (also referred herein as "LECT2-specific iRNA"). The use of such iRNAs enables targeted degradation of mRNA of genes involved in impairments related to 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. LECT2-targeting iRNAs can specifically and efficiently mediate RNAi, resulting in significant inhibition of LECT2 gene expression, which can be evaluated, for example, in cell-based assays.

[0148] The following description discloses the preparation and use of compositions containing iRNA for modulating (e.g., inhibiting) the expression of the LECT2 gene, as well as compositions and methods for addressing disorders related to the expression of the LECT2 gene.

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

[0150] In some embodiments, the following are characterized herein: a pharmaceutical composition containing LECT2 iRNA and a pharmaceutically acceptable carrier; a method of using the composition to inhibit the expression of the LECT2 gene; and a method of using the pharmaceutical composition to treat a disorder related to the expression of the LECT2 gene (e.g., LECT2 amyloidosis).

[0151] I. Definition For convenience, the meanings of certain terms and phrases used in this specification, the examples, and the appended claims are provided below. In the event of any clear discrepancy between the use of a term in other parts of this specification and its definition provided in this section, the definition provided in this section shall prevail.

[0152] In this specification, “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 stimulator for chondrocytes and osteoblasts. The human LECT2 gene was mapped to chromosome 5q31.1–q32. See above.

[0153] The sequence of human LECT2 mRNA can be found in NM_002302.2 (SEQ ID NO: 1). The sequences 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 16kDa protein. It is secreted from the liver. It exhibits 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. (See above.)

[0155] LECT2 is expressed in various tissues, including the brain, stomach, and liver. Koshimizu, Y & Ohtomi, M. (2010) Brain Res. 1311:1-11. In studies using indirect immunoperoxidase staining to investigate LECT2 expression in normal and diseased human organs and tissues other than the liver, LECT2 was found to be generally expressed in blood vessels, endothelial and smooth muscle cells, adipocytes, brain nerve cells, apical squamous epithelial cells, parathyroid cells, sweat gland and sebaceous gland epithelium, Hassar's bodies, and some mononuclear cells in immunohematopoietic tissues. The protein was generally negative, but occasionally stained positive in osteoblasts, chondrocytes, cardiac and skeletal muscle cells, gastrointestinal smooth muscle cells, 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 from GTC to ATC) at nucleotide 172 in exon 3 of the gene has been identified, constituting the presence of either valine or 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. While we do not wish to be constrained by theory, it has been suggested that the substitution of the embedded isoleucine (A allele) side chain with valine (G allele) destabilizes the protein and may constitute the amyloid-forming tendency of this LECT2 mutant. Murphy, CL et al. (2010) Am J Kidney Dis, 56(6):1100-1107.

[0157] As used herein, “LECT2 amyloidosis” or “ALECT2” encompasses amyloidosis involving the deposition of amyloid or amyloid fibrils containing the LECT2 protein (e.g., any polymorphic variant of the LECT2 protein) or a portion of the LECT2 protein. The LECT2 protein may be a variant (e.g., a mutant) LECT2 protein. The amyloidosis may be systemic or focal. In some embodiments, LECT2 amyloidosis involves amyloid deposition in the kidneys and / or liver.

[0158] "G," "C," "A," "T," and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively. However, it is understood that the terms "ribonucleotide" or "nucleotide" may also refer to modified nucleotides or surrogate substitution sites, as further described below. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil may be substituted by other sites without substantially altering the base-pairing properties of oligonucleotides containing such substitution sites. For example, but not limited to, nucleotides containing inosine as a base may base-pair with nucleotides containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine may be substituted, for example, with inosine-containing nucleotides in the nucleotide sequences of dsRNAs featured in this disclosure. In another example, adenine and cytosine somewhere in an oligonucleotide can be substituted with guanine and uracil, respectively, to form GU fluctuation base pairs with target mRNA. Sequences containing such substitutional portions 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 agents containing RNA, as defined herein, that mediate targeted cleavage of RNA transcripts, for example, via the RNA-induced silencing complex (RISC) pathway. In one embodiment, the iRNA described herein results in inhibition of LECT2 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, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the ALECT2 gene, including mRNA, which 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, either in that portion or in its vicinity. For example, target sequences are generally 9–36 nucleotides long, e.g., 15–30 nucleotides long, and include all subranges between them. As a non-limiting example, target sequences include 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, and 19-26 nucleotides. Otide may consist of 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 “sequence-containing chain” refers to an oligonucleotide containing a chain of nucleotides described by a sequence referred to using standard nucleotide nomenclature.

[0161] As used herein, and unless otherwise indicated, the term “complementary” means, as understood by those skilled in the art, the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under certain conditions to form a double-stranded structure. Such conditions may be, for example, stringent conditions, which are 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, 50 o C or 70 o This may include washing C for 12–16 hours. Other conditions, such as physiologically relevant conditions that may be encountered in living organisms, can be applied. Those skilled in the art can determine the most appropriate set of conditions for testing the complementarity of the two sequences, depending on the final application of the hybridized nucleotides.

[0162] Complementary sequences within iRNA, for example, within dsRNA as described herein, include base pairing of one or both nucleotide sequences over the full length of an oligonucleotide or polynucleotide containing a first nucleotide sequence to an oligonucleotide or polynucleotide containing a second nucleotide sequence. Such sequences may be referred to herein as “fully complementary” to each other. However, where the first sequence is referred to herein as “substantially complementary” with respect to the second sequence, the two sequences may be fully complementary, or they may form one or more, but generally five, four, three, or two or fewer, mismatched base pairs during hybridization over a double helix of up to 30 base pairs, while retaining their ability to hybridize under conditions most relevant to their final application, e.g., inhibition of gene expression via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA containing one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length contains a 21-nucleotide sequence in which the longer oligonucleotide is fully complementary to the shorter oligonucleotide, and may be further referred to as “fully complementary” for the purposes described herein.

[0163] As used herein, “complementary” sequences may also include, or be formed entirely from, non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, provided that the above requirements regarding their hybridizing ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pair formations.

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

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

[0166] The term “double-stranded RNA” or “dsRNA,” as used herein, refers to an iRNA containing a hybridized double-stranded region comprising two antiparallel and substantially complementary nucleic acid strands, which are said to have “sense” and “antisense” orientations with respect to the target RNA. The double-stranded region can be of any length, but typically ranges from 9 to 36 base pairs, e.g., 15 to 30 base pairs, allowing for the specific degradation of the desired target RNA, for example, via the RISC pathway. Considering a double helix of 9–36 base pairs, the double helix is ​​defined as 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 in between, e.g., not limited to, 15–30 base pairs, 15–26 base pairs, 15–23 base pairs, 15–22 base pairs, 15–21 base pairs, 15–20 base pairs, 15–19 base pairs, 15–18 base pairs, 15–17 base pairs, 18–30 base pairs. dsRNA can be any length of 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 produced intracellularly by processing with Dicer and similar enzymes is generally 19-22 base pairs long. One strand of the double-stranded region of dsDNA contains a sequence that is substantially complementary to the region of the target RNA. The double-stranded structure may originate from a single RNA molecule having at least one self-complementary region, or it may be formed from two or more separate RNA molecules.When a double-stranded region is formed from two strands of a single molecule, the molecule may 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 the other strand, forming a double-stranded structure. The hairpin loop may contain at least one unpaired nucleotide; in some embodiments, the hairpin loop may contain 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 twenty, at least 23 or more unpaired nucleotides. When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules may be covalently linked, although this is not necessary. When the two strands are covalently linked by means other than a hairpin loop, the linking structure is referred to as a “linker”. The term “siRNA” is also used herein to refer to dsRNA as described above.

[0167] In another embodiment, the iRNA agent may be a "single-stranded siRNA" introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNA iRNA binds to the RISC endonuclease Argonaute 2 and then cleaves the target mRNA. Single-stranded siRNAs are typically 15-30 nucleotides long and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein (e.g., sequences provided in Tables 2A-2B, 3A-3B, 6, or 7) can be used as single-stranded siRNAs, either 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 terms “RNA molecule” or “ribonucleic acid molecule” include not only naturally expressed or found RNA molecules, but also analogs and derivatives of RNA containing one or more ribonucleotide / ribonucleoside analogs or derivatives described herein or known in the art. Strictly speaking, “ribonucleoside” contains a nucleoside base and a ribose sugar, and “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 its nucleic acid base structure, ribose structure, or ribose-phosphate backbone structure, for example, as described below herein. However, molecules containing ribonucleoside analogs or derivatives must retain the ability to form double helixes. As a non-limiting example, RNA molecules may also include, but are not limited to, 2'-O-methyl-modified nucleosides, nucleosides containing a 5'-phosphorothioate group, terminal nucleosides linked to a cholesterol derivative or dodecanoic acid bisdecylamide group, locked nucleosides, debased nucleosides, acyclic nucleosides, glycol nucleotides, 2'-deoxy-2'-fluoro-modified nucleosides, 2'-amino-modified nucleosides, 2'-alkyl-modified nucleosides, morpholino nucleosides, non-natural bases containing phosphoramides or nucleosides, or at least one modified ribonucleoside containing any combination thereof. Alternatively, the RNA molecule may 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, and may contain up to the full length of the dsRNA molecule. The modifications do not need to be identical for each of the multiple modified ribonucleosides in the RNA molecule.In one embodiment, the modified RNA intended for use in the methods and compositions described herein is a peptide nucleic acid (PNA) having the ability to form the required double-stranded structure, for example, enabling or mediating the specific degradation of target RNA via the RISC pathway.

[0169] In one embodiment, the modified ribonucleoside comprises a deoxyribonucleoside. In such an example, the iRNA agent may contain, for example, one or more deoxyribonucleosides including a deoxyribonucleoside overhang, or one or more deoxyribonucleosides within the double-stranded portion of the dsRNA. In certain embodiments, the RNA molecule contains, for example, a percentage of 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%) of deoxyribonucleosides in one or both strands. In other embodiments, the term “iRNA” does not include double-stranded DNA molecules (e.g., naturally occurring double-stranded DNA molecules or 100% deoxyribonucleoside-containing DNA molecules).

[0170] In one embodiment, the RNA interference agent contains a single-stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. While not wishing to be constrained by theory, the long double-stranded RNA introduced into the cell is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer is a ribonuclease III-like enzyme that treats the dsRNA into a short interference RNA of 19-23 base pairs with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double helix, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases within RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Therefore, in one embodiment, this disclosure relates to single-stranded RNA that promotes the formation of a RISC complex to result in the silencing of a target gene.

[0171] As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide protruding from the double-stranded structure of iRNA, for example, dsRNA. For example, a nucleotide overhang exists if the 3' end of one strand of dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide; or an overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may contain or consist of a nucleotide / nucleoside analog, including a deoxynucleotide / nucleoside. An overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of an overhang may be located at the 5' end, 3' end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0172] In one embodiment, the antisense strand of the dsRNA has 1 to 10 nucleotide overhangs at its 3' and / or 5' ends. In another embodiment, one or more nucleotides in the overhangs are substituted with nucleoside thiophosphates.

[0173] As used herein with respect to dsRNA, the terms “blunted” or “blunt-ended” mean that there is no unpaired nucleotide or nucleotide analog at a given end of the dsRNA, i.e., there is no nucleotide overhang. One or both ends of a dsRNA can be blunted. If both ends of a dsRNA are blunt, it is called a blunt-ended dsRNA. To clarify, a “blunt-ended” dsRNA is one that is blunt at both ends, i.e., has no nucleotide overhangs at either end of the molecule. In most cases, such a molecule is double-stranded along its entire length.

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

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

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

[0177] When referring to iRNA, "introduction into cells" means promoting or effecting uptake or absorption into cells, as understood by those skilled in the art. Absorption or uptake of iRNA can occur via passive or active cellular processes alone, or by means of adjunct agents or devices. The meaning of this term is not limited to cells in vitro; iRNA can also be "introduced into cells", where the cells are part of a living organism. In such cases, introduction into cells 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 by means of, for example, 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 hereby incorporated by reference in their entirety. In vitro introduction into cells 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 the expression of" refers to at least partial "inhibition" or partial "activation" of LECT2 gene expression in cells treated with an iRNA composition described herein as compared to LECT2 expression in control cells. Control cells include untreated cells or cells treated with a non-target control iRNA.

[0179] The terms "activate", "enhance", "up-regulate the expression of", "increase the expression of", etc., insofar as they refer to the LECT2 gene, herein refer to at least partial activation of the expression of the LECT2 gene, as represented by an increase in the amount of LECT2 mRNA that can be isolated or detected from a first cell or cell group that has been treated such that the LECT2 gene is transcribed and the expression of the LECT2 gene is increased, as compared to a second cell or cell group that is substantially identical to the first cell or cell group (control cells) that has not been so treated.

[0180] In one embodiment, LECT2 gene expression is activated by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% upon administration of the iRNA described herein. In some embodiments, LECT2 gene is activated by at least about 60%, 70%, or 80% upon administration of the iRNA featured in this disclosure. In some embodiments, LECT2 gene expression is activated by at least about 85%, 90%, or 95% or more upon administration of the iRNA described herein. In some embodiments, LECT2 gene expression 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 500-fold, at least 1000-fold or more in cells treated with the iRNA described herein compared to expression in untreated cells. Activation of expression by low molecular weight dsRNA is described, for example, in Li et al., 2006 Proc. Natl. Acad. Sci. USA 103:17337-42, and in U.S. Patent Application Publications 2007 / 0111963 and 2005 / 226848, respectively, which are incorporated herein by reference.

[0181] The terms “silence,” “inhibit expression,” “downregulate expression,” and “suppress expression,” insofar as they refer to the LECT2 gene, hereby refer to at least partial suppression of LECT2 gene expression, assessed based on, for example, LECT2 mRNA expression, LECT2 protein expression, or another parameter functionally linked to LECT2 gene expression. For example, inhibition of LECT2 expression may be represented by a reduction in the amount of LECT2 mRNA that can be isolated or detected from a first group of cells or cells in which the LECT2 gene is transcribed and treated to inhibit LECT2 gene expression compared to a control. The control may be a second group of cells or cells that are substantially identical to the first group of cells or cells, except that the second group of cells or cells are not treated in the same way (control cells). The degree of inhibition is usually a percentage of the control level, for example,

[0182]

number

[0183] It is expressed as follows.

[0184] Alternatively, the degree of inhibition may be given in relation to a parameter functionally linked to LECT2 gene expression, such as a reduction in the amount of the protein encoded by the LECT2 gene. The reduction in 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 expressing LECT2 either constitutively or by genomic engineering, and by any appropriate assay.

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

[0186] In the context of this disclosure, the terms “to treat,” “treatment,” etc., mean to prevent, reduce or alleviate at least one symptom associated with a disorder related to LECT2 expression, or to slow or reverse the progression or predicted progression of such disorder. For example, when the methods featured herein are used to treat LECT2 amyloidosis, they can help inhibit amyloid deposition, reduce or prevent one or more symptoms of amyloidosis, or reduce the risk or severity of associated conditions (e.g., nephrotic syndrome or hepatitis). Therefore, unless the context clearly indicates otherwise, the terms “to treat,” “treatment,” etc., are intended to include prophylaxis, e.g., prevention of disorders and / or symptoms of disorders related to LECT2 expression.

[0187] In the context of disease markers or symptoms, “lower” means any reduction, for example, a statistically or clinically significant reduction at such levels. The reduction could 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%. This reduction can be lowered to a level that is considered within the normal range for individuals without such disorder.

[0188] As used herein, terms such as “therapeutic dose” and “preventive dose” refer to the amount that provides a therapeutic benefit in treating, preventing, or managing any disorder or pathological process associated with LECT2 expression. The specific amount that is therapeutically effective may 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 the disorder or pathological process, and the administration of other therapeutic agents.

[0189] As used herein, “pharmaceutical composition” comprises a pharmacologically effective amount of iRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutic effective amount,” or simply “effective amount” means that amount of iRNA effective to produce an intended pharmacological, therapeutic, or prophylactic outcome. For example, in a method for treating a disorder associated with LECT2 expression (e.g., LECT2 amyloidosis), the 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, the therapeutic effective amount of the drug for treating that disease or disorder is the amount required to obtain at least a 10% reduction in that parameter. For example, a therapeutically effective dose of LECT2-targeting iRNA can reduce the level of LECT2 mRNA or LECT2 protein 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 the administration of a therapeutic agent. Such carriers include, but are not limited to, physiological 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, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavorings, colorants, and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, as well as lactose, and corn starch and alginate are suitable disintegrants. Binders may include starch and gelatin, while lubricants, if present, are generally magnesium stearate, stearic acid, or talc. If desired, tablets may be coated with substances such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract. The drugs contained in the drug formulation are further described below in this specification.

[0191] When referring to a number or range of numbers, the term "approximately" means that the number or range being referred to is an approximation within experimental variability (or statistical experimental error), and therefore the number or range may vary, for example, between 1% and 15% of the stated number or range.

[0192] II. iRNA agents This specification describes iRNA agents that modulate (e.g., inhibit) the expression of the LECT2 gene.

[0193] In some embodiments, the iRNA agent activates the expression of the LECT2 gene in cells or mammals.

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

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

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

[0197] In some embodiments, the dsRNA is 15 to 20 nucleotides (including both ends) in length, and in other embodiments, the dsRNA is 25 to 30 nucleotides (including both ends) in length. As will be recognized by those skilled in the art, the targeted region of the RNA targeted for cleavage is often a part of a larger RNA molecule, often an mRNA molecule. When relevant, a "portion" of the mRNA target is a continuous sequence of the mRNA target that is long enough to serve as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). dsRNAs with double-strands as short as about 9 base pairs can mediate RNAi-directed RNA cleavage in certain situations. In most cases, the target is at least 15 nucleotides long, e.g., 15 to 30 nucleotides long.

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

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

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

[0201] In certain embodiments, the dsRNA includes 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 sense and antisense nucleotide sequences, wherein the sense strand is selected from sequences provided in Tables 2A-2B, 3A-3B, 6, or 7, and the corresponding antisense strand is selected from 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 the other sequence is substantially complementary to the mRNA sequence produced by the expression of the LECT2 gene. Thus, the dsRNA comprises two oligonucleotides, one of which is described as the sense strand and the second oligonucleotide as the corresponding antisense strand. As described elsewhere in this specification and known in the art, the complementary sequence of the dsRNA may be contained as a self-complementary region of a single nucleic acid molecule, as opposed to being located on separate oligonucleotides.

[0204] Those skilled in the art are well aware that dsRNAs with 20–23 base pairs, and especially 21 base pairs, in a double-stranded structure have been praised for their particularly effective ability to induce RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, some have found that shorter or longer RNA double-stranded structures may be equally effective.

[0205] In the embodiments described above, due to the properties of the oligonucleotide sequences provided in Tables 2A-2B, 3A-3B, 6, or 7, the dsRNAs described herein may include at least one strand with a length of at least 19 nucleotides. It can be reasonably predicted that shorter double helixes having one of the sequences in Tables 2A-2B, 3A-3B, 6, or 7 would be equally effective compared to the dsRNAs described above, even if only a few nucleotides are subtracted from one or both ends.

[0206] In some embodiments, the dsRNA has a partial sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive 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 containing at least 15, 16, 17, 18, or 19 consecutive nucleotides of the antisense sequence provided in Tables 2A-2B, 3A-3B, 6, or 7, and a sense sequence containing at least 15, 16, 17, 18, or 19 consecutive nucleotides of the corresponding sense sequence provided in Tables 2A-2B, 3A-3B, 6, or 7.

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

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

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

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

[0212] In some such embodiments, the dsRNA contains only a portion of the sequences provided in Tables 2A-2B, 3A-3B, 6, or 7, but is equally effective in inhibiting LECT2 expression levels as a dsRNA containing the full-length sequences provided in Tables 2A-2B, 3A-3B, 6, or 7. In some embodiments, the dsRNA differs in inhibiting LECT2 gene expression levels by 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or less compared to a dsRNA containing the full sequences 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 sensitive to RISC-mediated cleavage. Therefore, this disclosure further characterizes iRNAs that target one or fewer such sequences. As used herein, an iRNA is said to target a specific site within an RNA transcript if the iRNA facilitates cleavage of the transcript at any site within that particular site. Such iRNAs typically consist of at least 15 consecutive 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 adjacent to a selected sequence in the LECT2 gene.

[0214] Target sequences are typically 15–30 nucleotides long, but there is a wide variation in the suitability of specific sequences within this range for directing the cleavage of any given target RNA. Various software packages and the guidelines described herein provide guidance for identifying the optimal target sequence for any given gene target, but an empirical approach can also be taken, where a “window” or “mask” of a given size (as an unrestricted example, 21 nucleotides) is placed on the target RNA sequence, literally or graphically [including, e.g., in silico], to identify sequences within a size range that could serve as a target sequence. By progressively shifting the sequence “window” to one nucleotide upstream or downstream of the initial target sequence location, subsequent potential target sequences can be identified until a complete set of possible sequences for any given target size of selection has been identified. This process, combined with the systematic synthesis and testing of identified sequences (using assays described herein or known in the art), identifies sequences that can optimally identify RNA sequences that mediate the best inhibition of target gene expression when targeted with iRNA agents. For example, the sequences identified in Tables 2A-2B, 3A-3B, 6, or 7 represent effective target sequences, but further optimization of inhibition efficiency can be achieved by progressively "walking the window" by selecting a single nucleotide upstream or downstream of a given sequence, thereby identifying sequences with equivalent or better inhibitory properties.

[0215] Furthermore, it is intended that further optimization can be achieved by testing any identified sequence, e.g., in Tables 2A-2B, 3A-3B, 6, or 7, by either adding or removing nucleotides to generate longer or shorter sequences, and by walking up or down a longer or shorter size window of the target RNA from that point, thereby testing those sequences. Furthermore, this approach can be combined with testing the efficacy of iRNA based on the target sequence in inhibition assays known in the art or described herein to further improve the efficiency of inhibition. Moreover, such optimized sequences can be further modified by introducing modified nucleotides, adding or altering overhangs, or other modifications known in the art and / or considered herein, e.g., increased serum stability or circulating half-life, increased thermal stability, enhanced transmembrane delivery, targeting to specific locations or cell types, increased interaction with silencing pathway enzymes, increased release from endosomes, etc., e.g.

[0216] The iRNAs described herein may 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 the iRNA contains a mismatch 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 the iRNA contains a mismatch to the target sequence, it is preferable that the mismatch is confined 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 the LECT2 gene region, the RNA strand generally does not contain a mismatch in the central 13 nucleotides. Using the methods described herein or methods known in the art, it is possible to determine whether an iRNA containing a mismatch to the target sequence is effective in inhibiting LECT2 gene expression. Considering the effectiveness of iRNAs with mismatches in inhibiting LECT2 gene expression is particularly important when it is known that specific complementary regions in the LECT2 gene 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 nucleotides, typically 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang exhibit unexpectedly superior inhibitory properties compared to their blunt-end counterparts. In yet another embodiment, the RNA of the iRNA (e.g., the dsRNA) 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. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. Modifications include, for example, (a) terminal modifications, e.g., 5'-terminal modifications (phosphorylation, conjugation, reverse ligation, etc.), 3'-terminal modifications (conjugation, DNA nucleotide, reverse ligation, etc.), (b) base modifications, e.g., substitution with stabilizing bases, destabilizing bases, or bases that form base pairs with the extended repertoire of a partner, base removal (debastic nucleotide), or conjugated bases, (c) sugar modifications (e.g., 2' or 4' position, or acyclic sugars) or sugar substitutions, and (d) skeletal modifications, e.g., modification or substitution of phosphodiester linkages. Specific examples of RNA compounds useful in this disclosure include, but are not limited to, RNA containing a modified skeleton or RNA that does not contain natural internucleoside linkages. RNA containing a modified skeleton includes, among other things, RNA that does not have a phosphorus atom in its skeleton. For the purposes of this specification and as sometimes cited in the art, modified RNA that does not have a phosphorus atom in its internucleoside skeleton can also be considered an oligonucleoside. In certain embodiments, the modified RNA has a phosphorus atom in its internucleoside skeleton.

[0218] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and 3'-alkylene phosphonates, other alkyl phosphonates including chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates with the usual 3'-5' linkage, their 2'-5' linked analogues, as well as those with reverse polarity, where adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0219] Representative U.S. patents teaching the preparation of the phosphorus-containing linkage described above 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; and 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 This includes U.S. Patent Nos. 534,639; Nos. 6,608,035; Nos. 6,683,167; Nos. 6,858,715; Nos. 6,867,294; Nos. 6,878,805; Nos. 7,015,315; Nos. 7,041,816; Nos. 7,273,933; Nos. 7,321,029; and U.S. Patent RE39464, each incorporated herein by reference.

[0220] Modified RNA skeletons that do not contain phosphate atoms have skeletons formed by short alkyl or cycloalkyl nucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside linkages, or one or more short heteroatoms or heterocyclic nucleoside linkages. These include those having morpholino linkages (partially formed from the sugar portion of a nucleoside); siloxane skeletons; sulfide, sulfoxide, and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfonamide skeletons; amide skeletons; and others having mixed N, O, S, and CH2 components.

[0221] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides 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. This includes Nos. 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 mimetic compounds suitable for or intended for use with iRNA, both the sugar and nucleoside bonds of the nucleotide units, i.e., the backbone, are replaced by novel groups. The base units are maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, an RNA mimetic compound that has been shown to have excellent hybridizing properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and are directly or indirectly bound to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patents 5,539,082; 5,714,331; and 5,719,262, each incorporated herein by reference. Further teachings on 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 and heteroatom skeletons having a phosphorothioate backbone, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [also known as the methylene (methylimino) or MMI backbone], --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--], as well as oligonucleotides having an amide backbone as referenced in U.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have a morpholino backbone structure as referenced in U.S. Patent No. 5,034,506.

[0224] Modified RNA may also contain one or more substituted sugar moieties. iRNAs characterized herein, for example, dsRNAs, may contain at the 2' position one of the following: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 may be substituted or unsubstituted C1-C 10 Alkyl or C2-C 10 It can be an alkenyl or alkinyl. An example of a suitable modification is O[(CH2) 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 is included, and n and m are 1 to about 10. In other embodiments, dsRNA is below 2':C1~C 10The modifications include lower alkyl groups, substituted lower alkyl groups, alkali groups, aralkyl groups, O-alkaryl or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of iRNA, or groups for improving the pharmacodynamic properties of iRNA, and one of other substituents having similar properties. In some embodiments, the modifications include 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy groups. Other exemplary modifications include the O(CH2)2ON(CH3)2 group, also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2.

[0225] In other embodiments, the iRNA agent contains 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, contain fewer than 5 acyclic nucleotides per strand (e.g., 4, 3, 2, or 1 acyclic nucleotide per strand). 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, 3' end, or both ends of the sense or antisense strand of the iRNA agent. In one embodiment, one or more acyclic nucleotides are located at positions 1–8 of the sense strand or antisense strand, or both. In one embodiment, one or more acyclic nucleotides are found on the antisense strand from the 5' end to positions 4–10 (e.g., positions 6–8). In another embodiment, one or more acyclic nucleotides are found in the 3' end overhangs of one or both iRNA agents.

[0226] The terms “acyclic nucleotide” or “acyclic nucleoside,” as used herein, refer to any nucleotide or nucleoside having an acyclic sugar, e.g., acyclic ribose. Exemplary acyclic nucleotides or nucleosides may include nucleic acid bases, e.g., naturally occurring or modified nucleic acid bases (e.g., nucleic acid bases described herein). In certain embodiments, bonds between any of the ribose carbons (C1, C2, C3, C4, or C5), independently or in combination, are absent in the nucleotide. In one embodiment, the bond between the C2-C3 carbons of the ribose ring is absent, e.g., acyclic 2'-3'-seco-nucleotide monomers. In other embodiments, bonds between C1-C2, C3-C4, or C4-C5 are absent (e.g., 1'-2', 3'-4', or 4'-5'-seco-nucleotide monomers). Exemplary acyclic nucleotides are disclosed in U.S. Patent No. 8,314,227, which is incorporated herein by reference in its entirety. For example, an acyclic nucleotide may contain any of monomers D to J in Figures 1 to 2 of U.S. Patent No. 8,314,227. In one embodiment, the acyclic nucleotide includes the following monomers:

[0227] [ka]

[0228] [In the formula, the base is a nucleic acid base, for example, a naturally occurring or modified nucleic acid base (for example, the nucleic acid bases described herein)].

[0229] In certain embodiments, acyclic nucleotides can be modified or derivatized, in particular, by coupling them with another moiety, such as a ligand (e.g., GalNAc, a cholesterol ligand), an alkyl group, a polyamine, a sugar, or a polypeptide.

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

[0231] In other embodiments, the 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 the following: (i) reduced off-target effects; (ii) reduced involvement of the passenger strand in RNAi; (iii) increased specificity of the guide strand to 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'-OCH3), 2'-5-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also occur at other positions on the iRNA RNA, particularly at the 3' position of the sugar on the 3' terminal nucleotide, or at the 5' position of the 2'-5' ligated dsRNA and the 5' terminal nucleotide. iRNA may also have sugar mimetic molecules such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patents 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; and 5,576,42 This includes Nos. 7; Nos. 5,591,722; Nos. 5,597,909; Nos. 5,610,300; Nos. 5,627,053; Nos. 5,639,873; Nos. 5,646,265; Nos. 5,658,873; Nos. 5,670,633; and Nos. 5,700,920, some of which are commonly owned in this application, and each of these is incorporated herein by reference.

[0233] iRNA may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include other synthetic and natural nucleic acid bases, 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 This includes 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-daazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0234] Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in this disclosure. These include 5-substituted pyrimidines containing 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines. 5-methylcytosine substitution has been shown to increase nucleic acid double-strand stability by only 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 is an exemplary base substitution, especially when combined with 2'-O-methoxyethyl sugar modification.

[0235] Representative U.S. patents teaching the above-mentioned modified nucleic acid bases and certain preparations of other modified nucleic acid bases, but not limited to, the above-mentioned U.S. Patents No. 4,845,205; No. 5,130,30; No. 5,134,066; No. 5,175,273; No. 5,367,066; No. 5,432,272; No. 5,457,187; No. 5,459,255; No. 5,484,908; No. 5,502,177; No. 5,525,711; No. 5,552,540; No. 5,587,469; No. This includes U.S. Patent 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. Patent No. 5,750,692, which is similarly incorporated herein by reference.

[0236] Furthermore, the RNA of the iRNA can 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, the locked nucleic acid is a nucleotide having a modified ribose moiety, the ribose moiety including, for example, an extra crosslink connecting the 2' and 4' carbons. This structure effectively “locks” the ribose into a 3'-end 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 U.S. patents teaching the preparation of locked nucleic acids include, but are not limited to, U.S. Patents 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 whole. Exemplary LNAs include, but are not limited to, 2',4'-C methylene bicyclonucleotides (see, for example, Wengel et al., International PCT Application, International Publication No. 00 / 66604 and International Publication No. 99 / 14226).

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

[0239] Potentially stabilizing modifications to the ends of RNA molecules 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-docosanoyluridine-3''-phosphate, and reverse base dT (idT). Disclosure of these modifications is described in PCT application International Publication No. 2011 / 005861.

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

[0241] In one embodiment, N a and / or N b This includes alternating modification patterns.

[0242] In one embodiment, the YYY motif occurs at or near a cleavage site on the sense strand. For example, if the RNAi agent has a double-stranded region of 17–23 nucleotides in length, the YYY motif may occur at or near a cleavage site on the sense strand (e.g., it may occur at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13), and the count may begin from the first nucleotide from the 5' end; or from the first paired nucleotide within 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 both i and j are 1. Therefore, the sense chain is given by the following equation: 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 represented by [this].

[0244] If the sense chain is represented by formula (Ib), then N bThis represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0245] If the sense chain is represented by equation (Ic), then N b This represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0246] If the sense chain is represented by the expression (Id), then each N b This independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Preferably, N b There are 0, 1, 2, 3, 4, 5, or 6 of each N. a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 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 chain is given by the formula: 5'n p -N a -YYY-N a -n q 3'(Ia) It can be represented by [this].

[0249] If the sense chain is represented by equation (Ia), then each N a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0250] In one embodiment, the antisense strand sequence of 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) and can be represented by [wherein k and l are each independently 0 or 1; p’ and q’ are each independently 0 to 6; each N a ’ represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, and each sequence contains at least 2 different modified nucleotides; each N b ’ represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p ’ and n q ’ each independently represent overhang nucleotides; 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 ’ contain modifications in an alternating pattern.

[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 double-stranded region of 17-23 nucleotides in length, the Y'Y'Y' motif may occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the first nucleotide from the 5' end; or from the first paired nucleotide within the double-stranded region from the 5' end. Preferably, the Y'Y'Y' motif occurs at positions 11, 12, 13.

[0253] In one embodiment, all Y'Y'Y' motifs are 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] Therefore, the antisense chain is given by 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 represented by [this].

[0256] If the antisense chain is represented by equation (IIb), then N b ' represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0257] If the antisense chain is expressed as equation (IId), then each N b ' independently represents oligonucleotide sequences containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N b It is 0, 1, 2, 3, 4, 5, or 6.

[0258] In other embodiments, k is 0 and l is 0, and the antisense chain is given by the following equation: 5'n p '-N a '-Y'Y'Y'-N a '-n q '3'(Ia) It can be represented by [this].

[0259] If the antisense chain is expressed as equation (IIa), then each N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

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

[0261] Each nucleotide in the sense and antisense strands 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 in the sense and antisense strands can be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' can, in particular, represent a 2'-O-methyl modification or a 2'-fluoro modification.

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

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

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

[0265] Therefore, the RNAi agent used in the method of this disclosure may include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi double helix is ​​given by 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) Represented by, [In the formula, i, j, k, and l are each independently either 0 or 1; p, p', q, and q' are each independently between 0 and 6; each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides. During the ceremony, each n p ',n p , n q ', and n q Each of these may or may not exist independently, and represents an overhang nucleotide; and XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single 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] An example combination of sense and antisense strands that form an RNAi double helix includes the following formula: 5'n p -N a-YYY-N a -n q 3' 3'n p '-N a '-Y'Y'Y'-N a 'n q '5' Xa) 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] If an RNAi agent is represented by formula (IIIa), then each N a These independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

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

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

[0271] When an RNAi agent is represented by formula (IIId), each N b , N b ' independently represents oligonucleotide sequences containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a , N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. a , N a ', N b and N b Each of these independently includes alternating modification patterns.

[0272] In equations (III), (IIIa), (IIIb), (IIIc), and (IIId), each of X, Y, and Z may be the same as or different from one another.

[0273] If an RNAi agent is represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), then at least one of the Y nucleotides may form a base pair with one of the Y' nucleotides. Alternatively, at least two of the Y nucleotides may form a base pair with the corresponding Y' nucleotide, or all three of the Y nucleotides may form a base pair with the corresponding Y' nucleotide.

[0274] If the RNAi agent is represented by formula (IIIb) or (IIId), then at least one of the Z nucleotides may form a base pair with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides may form a base pair with the corresponding Z' nucleotide, or all three of the Z nucleotides may form a base pair with the corresponding Z' nucleotide.

[0275] If the RNAi agent is represented by formula (IIIc) or (IIId), then at least one of the X nucleotides may form a base pair with one of the X' nucleotides. Alternatively, at least two of the X nucleotides may form a base pair with the corresponding X' nucleotide, or all three of the X nucleotides may form a base pair with the corresponding X' nucleotide.

[0276] In one embodiment, modifications on the Y nucleotide are different from modifications on the Y' nucleotide, modifications on the Z nucleotide are different from modifications on the Z' nucleotide, and / or modifications on the X nucleotide are different from modifications 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 2'-fluoro modification. In another embodiment, if the RNAi agent is represented by formula (IIId), then N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p' is linked to an adjacent nucleotide via phosphorothionate linkage. In yet another embodiment, if 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 p ' is linked to an adjacent nucleotide via phosphorothionate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker. In another embodiment, if 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 p The nucleotide is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand contains at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked 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 The nucleotide is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand contains at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0279] In one embodiment, the RNAi agent is a multimer comprising at least two double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), where the double helixes are linked by linkers. The linkers may be cleavable or incleavable. The multimer may further comprise ligands. Each double helix may target the same gene or two different genes; or each double helix may target the same gene at two different target sites.

[0280] In one embodiment, the RNAi agent is a multimer comprising three, four, five, six or more double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), where the double helixes are linked by linkers. The linkers may be cleavable or incleavable. The multimer may further contain ligands. Each double helix may target the same gene or two different genes; or each double helix may target the same gene at two different target sites.

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

[0282] iRNA conjugate The iRNA agents disclosed herein may be in the form of conjugates. The conjugate may be bound to any suitable position within the iRNA molecule, for example, to the 3' or 5' end of the sense or antisense strand. The conjugate may be bound via a linker.

[0283] In some embodiments, the iRNA agents described herein are chemically linked to one or more ligands, moieties, or conjugates that can confer functionality, for example, by influencing (e.g., enhancing) the activity, cell distribution, or cell uptake of the iRNA. These are not limited to these parts, but also include lipid parts, such as cholesterol (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, such as 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), and thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), aliphatic chains, e.g., 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, e.g., di-hexadecyl-rac-glycerol or triethylammonium 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 chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetate (Manoharan et al., Tetrahedron Lett.It contains either the palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or the octadecylamine or hexylaminocarbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0284] In one embodiment, a ligand alters the distribution, targeting, or lifespan of the iRNA agent it incorporates. In some embodiments, a ligand provides enhanced affinity to a selected target, e.g., a molecule, cell or cell type, compartment, e.g., a compartment in a cell or organ, tissue, organ, or region of the organism, compared, for example, to a species where such a ligand is absent. Typical ligands do not participate in double-strand pairing in double-stranded nucleic acids.

[0285] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); 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, such as 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 polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudo-peptide polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helix peptides.

[0286] Ligands may also include targeting groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids or proteins, or antibodies that bind to specific cell types, such as kidney cells. Targeting groups may be tyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, biotin, or RGD peptides or RGD peptide mimetic compounds.

[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). A further description of GalNAc ligands is provided in the section titled “Carbohydrate Conjugates”.

[0288] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (e.g., TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantane acetate, 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, This includes O3-(oleoyl)colenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphoric acid, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, 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 the tetraazamacrocycle), dinitrophenyl, HRP, or AP.

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

[0290] A ligand can be a substance, such as a drug, that can increase the uptake of an iRNA agent into a cell by, for example, disrupting the cytoskeleton of the cell, for example, by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell. A drug may be, for example, taxone, vincristine, vinblastine, cytochalasin, nocodazole, japlaquinolide, latruncrine A, phalloidin, swinolide A, indanosine, or myoserbine.

[0291] In some embodiments, the ligands that bind to iRNAs described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipid-soluble substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEGs, vitamins, and the like. Exemplary PK modulators, but not limited to, include cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. Oligonucleotides containing numerous phosphorothioate linkages are also known to bind to serum proteins; therefore, short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases containing numerous phosphorothioate linkages in the backbone, can also conform to this 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] The ligand-conjugate oligonucleotides of this disclosure can be synthesized by using oligonucleotides having pendant-reactive functional groups, such as those derived from the binding of linking molecules to oligonucleotides (described later). These reactive oligonucleotides can be directly reacted with commercially available ligands, ligands synthesized to have any of the various protecting groups, or ligands having a linked portion attached thereto.

[0293] The oligonucleotides used in the conjugates of this disclosure can be conveniently and routinely prepared by well-known solid-phase synthesis techniques. Apparatus for such synthesis is available from several vendors, including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art can be used additionally or alternatively. Similar techniques are also known to be used to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

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

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

[0296] Lipid conjugate In one embodiment, the ligand is a lipid or lipid-based molecule. Such lipids or lipid-based molecules can typically bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of the conjugate to target tissues. For example, the target tissue may be the liver, including the parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, nephroxine or aspirin can be used. Lipids 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 modulate binding to serum proteins, such as HSA.

[0297] Lipid-based ligands can be used to modulate, for example, control (e.g., inhibit) the binding of conjugates to target tissues. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidney and therefore less likely to be removed from the body. Lipids or lipid-based ligands that bind more weakly to HSA can be used to target conjugates to the kidney.

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

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

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

[0301] Cell permeabilizing agent In another embodiment, the ligand is a cell permeabilizer, such as a helical cell permeabilizer. In one embodiment, the drug is amphiphilic. Exemplary drugs are peptides such as tat or Antennapetia. If the drug is a peptide, it can be modified, including the use of peptidyl mimes, invertomers, non-peptide or pseudo-peptide linkers, and D-amino acids. Helix drugs are typically α-helix drugs and may have lipophilic and lipophilic phases.

[0302] The ligand may be a peptide or a peptide mimetic. Peptide mimes (also referred to herein as oligopeptide mimes) are molecules that can fold into a defined three-dimensional structure similar to a native peptide. Binding of peptides and peptide mimes to iRNA agents may affect the pharmacokinetic distribution of the iRNA, for example, by enhancing cell recognition and absorption. The peptide or peptide mimetic portion may be about 5 to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0303] Peptides or peptide mimes may be, for example, cell-permeable peptides, cationic peptides, amphiphilic peptides, or hydrophobic peptides (e.g., primarily composed of Tyr, Trp, or Phe). The peptide moiety may be a dendrimeric peptide, a constrained peptide, or a crosslinked peptide. Alternatively, the peptide moiety may contain 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 hydrophobic MTS [e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 904)] may also be targeting moieties. The peptide moiety may be a “delivery” peptide capable of carrying large polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. 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. Peptides or peptide mimetic compounds can be encoded by peptides identified from phage display libraries or by random sequences of DNA, such as in one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). Typically, peptides or peptide mimetic compounds anchored to dsRNA agents via incorporated monomer units are cell-targeting peptides such as arginine-glycine-aspartate (RGD) peptides or RGD mimetic compounds. The peptide moiety can range in length from approximately 5 to approximately 40 amino acids. The peptide moiety may have structural modifications that increase stability or direct conformational properties. Any of the structural modifications described below may be utilized.

[0304] The RGD peptides for use in the compositions and methods of this disclosure may be linear or cyclic and may be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissues. RGD-containing peptides and peptide mimetic drugs may contain D-amino acids, as well as synthetic RGD mimetic drugs. In addition to RGD, other parts that target integrin ligands may be used. Preferred conjugates of these ligands target PECAM-1 or VEGF.

[0305] The RGD peptide moiety can be used to target specific cell types, such as tumor cells like endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can facilitate the targeting of dsRNA agents to tumors in various other tissues, including the lungs, kidneys, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, RGD peptides facilitate the targeting of iRNA agents to the kidneys. 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] "Cell-permeable peptides" can permeate cells, such as microbial cells like bacteria or fungal cells, or mammalian cells like human cells. Microbial cell-permeable peptides may be, for example, α-helix linear peptides (e.g., LL-37 or seropine P1), disulfide bond-containing peptides (e.g., α-defensin, β-defensin, or bactenesin), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidine). Cell-permeable peptides may also contain nuclear localization signals (NLS). For example, cell-permeable peptides may be bifidopphienophilic peptides such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0307] Carbohydrate conjugate In some embodiments of the compositions and methods of this disclosure, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for compositions suitable for in vivo delivery of nucleic acids, as described herein, and for in vivo therapeutic use. As used herein, “carbohydrate” means a carbohydrate itself, which consists of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) having an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound which, as part thereof, has a carbohydrate moiety consisting of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) having an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Typical 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. Specific monosaccharides include sugars with C5 or more (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 entirety of which is incorporated herein by reference. In some embodiments, the GalNAc conjugate serves as a ligand for targeting iRNA to specific cells. In some embodiments, the GalNAc conjugate targets iRNA to liver cells, for example, by serving as a ligand for the asialocrycoprotein receptor in liver cells (e.g., hepatocytes).

[0309] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives may be conjugated via a linker, for example, a divalent or trivalent branched 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 (for example, 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 as follows:

[0311] [ka]

[0312] In some embodiments, the RNAi agent is bound to a 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 defined in Table 1 and conjugated to L96 as shown below.

[0315] [ka]

[0316] In some embodiments, L96 is as follows:

[0317] [ka]

[0318] In some embodiments, carbohydrate conjugates for use in the compositions and methods of this disclosure are selected from the group consisting of the following:

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

[0320] Other representative 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 of the above-described further ligands, including, but not limited to, PK modulators and / or cell-permeable peptides.

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

[0324] [ka] [ka]

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

[0326] The term "linker" or "linking group" refers to an organic part that connects two parts of a compound, for example, a covalent bond between two parts of a compound. Linkers are typically directly bonded to atoms, such as oxygen or sulfur, units, such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or chains of atoms, such as but not limited to substituted or unsubstituted alkyls, substituted or unsubstituted alkenyls, substituted or unsubstituted alkynyls, arylalkyls, arylalkenyls, arylalkynyls, heteroarylalkyls, heteroarylalkenyls, heteroarylalkynyls, heterocyclylalkyls, heterocyclylalkenyls, heterocyclylalkynyls, aryl, heteroaryl, heterocyclyl, cycloalkyls, cycloalkenyls, alkylarylalkyls, alkylarylalkenyls, alkylarylalkynyls, alkenylarylalkyls, alkenylarylalkenyls, alkenylarylalkynyls, alkenylarylalkynyls, alkynylarylalkyls, alkynylarylalkenyls, alkynylarylalkynyls, alkylheteroarylalkyls, alkylheteroarylalkenyls, alkylheteroarylalkenyls This includes loarylalkynyl, alkenyl heteroarylalkyl, alkenyl heteroarylalkenyl, alkenyl heteroarylalkynyl, alkenyl heteroarylalkynyl, alkenyl heteroarylalkynyl, alkenyl heteroarylalkenyl, alkenyl heteroarylalkynyl, alkylhererocyclylalkynyl, alkenyl heterocyclylalkynyl, alkenyl heterocyclylalkenyl, alkenyl heterocyclyl, alkenyl heterocyclic alkenyl, alkenyl heterocyclyl, alkynyl heterocyclic alkenyl, alkynyl heterocyclic alkenyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenyl heteroaryl, and alkynylhereroaryl, where one or more methylene groups can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclic, where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic.In one embodiment, the linker consists of approximately 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 this disclosure is conjugated to a bivalent or trivalent branched linker selected from the structural bases shown in any of formulas (XXXI) to (XXXIV):

[0328] [ka]

[0329] [In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently represent a number from 0 to 20 for each occurrence, and the repeating units may be identical 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 Each of these is independently, for each occurrence, nonexistent, CO, NH, O, S, OC(O), NHC(O), NHC(O), CH2, CH2NH, or CH2O; Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5CThese are, independently, for each occurrence, non-existent alkylenes, substituted alkylenecholines, and one or more methylenes, O, S, S(O), SO2, N(R) N ), C(R')=C(R''), C≡C, or C(O) can interrupt or halt one or more of these; R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C Each of these is independent, and for each occurrence, they do not exist: 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 heterocycline];

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

[0333] [ka]

[0334] [In the formula, L 5A , L 5B and L 5C This represents monosaccharides such as GalNAc derivatives.

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

[0336] The cleavable linking group is sufficiently stable outside the cell but is cleaved upon entry into the target cell, releasing the two parts that the linker holds together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10, 20, 30, 40, 50, 60, 60, 70, 80, 90 or more, or at least about 100 times faster in the target cell or under a first reference condition (which may be selected to mimic or represent intracellular conditions) or under a second reference condition (which may be selected to mimic or represent conditions found in blood or serum).

[0337] Scleavable linkers are sensitive to cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleavage agents are more common or found at higher levels or activity within cells than in serum or blood. Examples of such degraders include redox agents that are selected for a particular substrate or do not have substrate specificity, such as oxidases or reductases or reducing agents such as mercaptans present in cells that can degrade redox cleavable linkers by reduction; esterases; endosomes or agents that can create an acidic environment, such as those with a pH of 5 or less; and enzymes that can hydrolyze or degrade acid-cleavable linkers by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0338] Scleavable linking groups, 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 in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH at approximately 5.0. Some linkers have scleavable linking groups that are cleaved at a favorable pH, thereby releasing cationic lipids from intracellular ligands or into desired compartments within the cell.

[0339] Linkers can contain cleavable linking groups that can be cleaved by specific enzymes. The type of cleavable linking group incorporated into a linker may depend on the target cell. For example, liver-targeting ligands can be linked to cationic lipids via linkers containing ester groups. Liver cells are rich in esterases, and therefore linkers are cleaved more efficiently in liver cells than in cells that are not rich in esterases. Other cell types that are rich in esterases include lung, renal cortex, and testicular cells.

[0340] Linkers containing peptide bonds can be used to target peptidase-rich cell types such as liver cells and synovial cells.

[0341] Generally, the suitability of a candidate cleavable linker can be evaluated by testing the ability (or conditions) of a degrading agent to cleave the candidate linker. It is also desirable to test the candidate cleavable linker for its resistance to cleavage in blood or in contact with other non-target tissues. Therefore, the relative sensitivity to cleavage between a first and second condition can be determined, with the first condition selected to demonstrate cleavage in target cells and the second condition selected to demonstrate cleavage in other tissues or bodily fluids, such as blood or serum. Evaluations can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, 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 intracellularly (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 group In one embodiment, the cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). Methods described herein can be found to determine whether a candidate cleavable linking group is a suitable “reductively cleavable linking group” or, for example, suitable for use with a particular iRNA moiety and a particular targeting agent. For example, a 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. Candidates may also be evaluated under conditions selected to mimic blood or serum conditions. For example, a candidate compound is cleaved by only about 10% in blood. 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 intracellularly (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 candidate compounds can be determined using standard enzyme kinetics assays under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

[0343] Phosphate-based cleavable linking groups In another embodiment, the cleavable linker includes a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by a substance that decomposes or hydrolyzes the phosphate group. Examples of substances that cleave phosphate groups in cells include enzymes such as intracellular phosphatases. Examples of linking groups based on phosphoric acid 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-, and -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 includes an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group 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 cells, specific low-pH organelles such as endosomes and lysosomes can provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable linking groups, but not limited to, include hydrazones, esters, and amino acid esters. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon bonded 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 linking groups In another embodiment, the cleavable linker includes an ester-based cleavable linking group. The ester-based cleavable linking group is cleaved in cells by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. The ester-based cleavable linking group has 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 linking groups In yet another embodiment, the cleavable linker includes a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases within the cell. The peptide-based cleavable linking group is a peptide bond formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not contain an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene, or alkylene. A peptide bond is a special type of amide bond formed between amino acids to produce peptides and proteins. The peptide-based cleavable group is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that produce peptides and proteins, and does not include the entire amide functional group. The peptide-based cleavable linking group has the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.Representative U.S. patents teaching the preparation of RNA conjugates include, but are not limited to, U.S. Patents 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. This includes Nos. 5,574,142; No. 5,585,481; No. 5,587,371; No. 5,595,726; No. 5,597,696; No. 5,599,923; No. 5,599,928; and Nos. 5,688,941; No. 6,294,664; No. 6,320,017; No. 6,576,752; No. 6,783,931; No. 6,900,297; No. 7,037,646; and No. 8,106,022, the entire contents of each of these are incorporated herein by reference.

[0347] Not all positions in a given compound need to be uniformly modified; in fact, more than one of the modifications described above may be incorporated into a single compound, or even a single nucleoside within an iRNA. This disclosure also includes iRNA compounds that are chimeric compounds.

[0348] In the context of this disclosure, a “chimeric” iRNA compound, or “chimeric,” is an iRNA compound, e.g., a dsRNA, that contains two or more chemically distinct regions, each composed of at least one monomeric 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 to the iRNA increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to a target nucleic acid. Further regions of the iRNA can serve as substrates for enzymes that can cleave RNA:DNA or RNA:RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA double helix. Thus, activation of RNase H results in cleavage of the RNA target, thereby significantly enhancing the iRNA inhibition efficiency of gene expression. As a result, when using chimeric dsRNA, comparable results are often obtained with shorter iRNAs compared to phosphorothionate deoxy dsRNAs that hybridize to the same target region. Cleavage of RNA targets can be routinely detected by gel electrophoresis and, if necessary, by relevant nucleic acid hybridization techniques known in the art.

[0349] In certain cases, 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, cell distribution, or cell uptake, and procedures for such conjugations are available in the scientific literature. Such non-ligand portions include lipid portions, 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. Let., 1993, 3:2765), and thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains, e.g., 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, e.g., 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 acetate (Manoharan et al.These include the , 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 having aminolinkers at one or more positions in its sequence. The amino group is then reacted with the molecule to be conjugated using a suitable coupling or activating reagent. The conjugation reaction can be performed using RNA still bound to a solid support, or in solution phase after RNA cleavage. Purification of RNA conjugates by HPLC typically yields a pure conjugate.

[0350] iRNA delivery The delivery of iRNA to targets requiring it can be achieved in numerous different ways. In vivo delivery can be carried out directly by administering a composition containing iRNA, such as dsRNA, to the target. Alternatively, delivery can be carried out indirectly by administering one or more vectors that encode the iRNA and direct its expression. These alternatives will be further discussed below.

[0351] Direct delivery Generally, any method of delivering nucleic acid molecules can be adapted for use with iRNA (see, for example, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and International Publication No. 94 / 02595, which is incorporated herein by reference in whole). 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, e.g., direct injection or transplantation into tissue (in non-limiting examples, tumors), or local administration of preparations. Local administration to the treatment site maximizes the local concentration of the drug, limits exposure of the active ingredient to systemic tissues that may be harmed by the drug or otherwise degrade the drug, and allows for a lower total dose of the iRNA molecule administered. Several studies have shown that local administration of iRNA successfully knocks down gene products. For example, intravitreal injection of VEGF dsRNA 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) have both been shown to prevent angiogenesis in experimental models of age-related macular degeneration. Furthermore, direct intratumoral injection of dsRNA into mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and extends the 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 can also be delivered locally to the CNS by 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 delivered locally to the lungs by intranasal administration (Howard, KA., et al (2006) Mol. Ther. Success has also been demonstrated in 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For systemic administration of iRNA for disease treatment, 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] Modification of RNA or a pharmaceutical carrier also enables the targeting of iRNA compositions to target tissues and avoids undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to other groups, such as lipid or carbohydrate groups as described herein. Such conjugates can be used to target iRNA to specific cells, such as liver cells. For example, galNAc conjugates or lipid (e.g., LNP) formulations can be used to target iRNA to specific cells, such as liver cells.

[0353] Lipophilic groups such as cholesterol enhance cellular uptake and prevent degradation. For example, systemic injection of iRNA directed to 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 aptamers 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 facilitate the binding of negatively charged iRNA molecules and enhance interactions with negatively charged cell membranes, enabling efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can be induced to bind to iRNA or form vesicles or micelles that enclose iRNA (see, e.g., Kim SH., et al (2008) Journal of Controlled Release 129(2):107-116). Vesicle or micelle formation further prevents iRNA degradation when administered systemically. Methods for preparing and administering cationic RNA complexes are well within the capabilities of those skilled in the art (see, for example, 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 entirety).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, DR., et al (2003), op. cit.; Verma, UN., et al (2003), op. cit.), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS., et al (2006) Nature 441:111-114), Cardiolipin (Chien, PY., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), and polyethyleneimine (Bonnet ME., et al (2008) Pharm. Res. Aug 16 Epub ahead). This includes, 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, DA., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, the iRNA forms a complex with cyclodextrin for systemic administration. Methods of administering iRNA and cyclodextrin and pharmaceutical compositions are described in U.S. Patent No. 7,427,60, which is incorporated herein by reference in whole.

[0354] iRNA-encoding vector In another embodiment, iRNAs targeting the LECT2 gene may be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. 00 / 22113, Conrad, International PCT Publication No. 00 / 22114, and Conrad, U.S. Patent No. 6,054,299). Expression may be transient (on the order of hours to weeks) or persistent (weeks to months or more), depending on the specific construct used and the target tissue or cell type. These transgenes may be introduced as linear constructs, circular plasmids, or viral vectors, and may be embedded or non-embedded 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 or multiple strands of iRNA can be transcribed from a promoter on an expression vector. For example, if two separate strands are to be expressed to generate dsRNA, two separate expression vectors can be simultaneously introduced into target cells (e.g., by transfection or infection). Alternatively, individual strands of dsRNA can both be transcribed by promoters located on the same expression plasmid. In one embodiment, dsRNA is expressed as reverse repeats linked by a linker polynucleotide sequence, such that the dsRNA has a stem-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, e.g., 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 desired nucleic acid segments. Delivery of iRNA expression vectors can be systemic, for example, by intravenous or intramuscular administration to target cells explanted from a patient, followed by reintroduction into the patient, or by any other means enabling introduction into desired target cells.

[0357] iRNA expression plasmids can be transfected into target cells as a complex with a cationic lipid carrier (e.g., oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdown targeting different regions of the target RNA over a period of one week or longer are also contemplated in this disclosure. The success of vector introduction 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 such as green fluorescent protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that impart resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance, to the transfected cells.

[0358] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, e.g., lentivirus vectors, Moloney's mouse leukemia virus, etc.; (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 vector or avipox, e.g., canarypox or fowlpox; and (j) helper-dependent or gutless adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors may be taken up or not taken up into the cell genome. Constructs may, if desired, contain viral sequences for transfection. Alternatively, constructs may be incorporated into vectors that enable episomal replication, e.g., EPV and EBV vectors. Constructs for the recombinant expression of iRNA generally require regulatory elements, such as promoters and enhancers, to ensure iRNA expression in target cells. Other aspects of vectors and constructs to consider are further described below.

[0359] A vector useful for iRNA delivery contains sufficient regulatory elements (promoter, enhancer, etc.) to express the iRNA in the desired target cells or tissues. These regulatory elements may be selected to provide either constitutive or regulatory / inducible expression.

[0360] iRNA expression can be precisely controlled, for example, by using inducible regulatory sequences that are 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, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG). Those skilled in the art can select an appropriate regulatory / promoter sequence based on the intended use of the iRNA transgene.

[0361] In certain embodiments, viral vectors containing a nucleic acid sequence encoding iRNA 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 the correct packaging of the viral genome and its integration into host cell DNA. The nucleic acid sequence encoding iRNA is cloned into one or more vectors to facilitate the delivery of the nucleic acid to the patient. More details on 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 intended for use include, for example, HIV-based vectors described in U.S. Patents 6,143,520; 5,665,557; and 5,981,276, which are incorporated herein by reference.

[0362] Adenoviruses are also intended for use in iRNA delivery. Adenoviruses are particularly attractive as a medium for delivering genes, for example, to respiratory epithelium. Adenoviruses spontaneously infect respiratory epithelium, causing mild illness. Other targets for adenovirus-based delivery systems include 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) present 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 rhesus monkey respiratory epithelium. Other examples of the use of adenoviruses 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 the 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 intended (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); US Pat. No. 5,436,146). In one embodiment, the iRNA may be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, a U6 or H1 RNA promoter, or a cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNAs characterized 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] Other typical viral vectors are poxviruses such as vaccinia virus, including attenuated vaccinia such as modified virus Ankara (MVA) or NYVAC, and avipox such as fowlpox or canarypox.

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

[0366] A vector-based pharmaceutical preparation may contain the vector in an acceptable diluent, or it may contain a sustained-release matrix in which the gene delivery vehicle is embedded. Alternatively, if a complete gene delivery vector can be produced intact from recombinant cells, such as a retroviral vector, the pharmaceutical preparation may contain one or more cells that produce the gene delivery system.

[0367] III. Pharmaceutical composition containing iRNA In one embodiment, this disclosure provides a pharmaceutical composition comprising an iRNA described herein and a pharmaceutically acceptable carrier. A pharmaceutical composition containing iRNA is useful for treating diseases or disorders related to 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, a composition can be formulated for parenteral delivery, such as systemic administration by 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 containing GalNAc conjugates) are formulated for subcutaneous delivery.

[0368] The pharmaceutical compositions described herein are administered in doses sufficient to inhibit the expression of the LECT2 gene. Generally, appropriate doses of iRNA are in the range of 0.01 to 200.0 mg / kg of recipient body weight / day, and generally in the range of 1 to 50 mg / kg of body weight / day. For example, dsRNA can be administered in single doses of 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. The pharmaceutical compositions can be administered once daily, or iRNA can be administered two, three, or more lower doses at appropriate intervals throughout the day, and delivery via continuous infusion or controlled-release formulations may also be used. In that case, the amount of iRNA contained in each lower dose must be correspondingly small in order to achieve the total daily dose. The dosing unit may also be formulated for delivery over several days, for example, using conventional sustained-release formulations that provide sustained release of iRNA over several days. Sustained-release formulations are well known in the art and are particularly useful for drug delivery at specific sites, such as those that can be used with the drugs of this disclosure. In this embodiment, the dosing unit comprises a multiple of the corresponding daily dose.

[0369] The effect of a single dose at LECT2 level can be long-lasting, and subsequent doses should be administered at intervals of 3, 4, or 5 days or less, or at intervals of 1, 2, 3, or 4 weeks or less.

[0370] Those skilled in the art will understand, but will not be limited, that certain factors, including the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other pre-existing conditions, may influence the dosage and timing required to effectively treat the subject. Furthermore, treatment of a subject with a therapeutically effective dose of the composition may include a single treatment or a series of treatments. Estimations of the effective dosage and in vivo half-life of individual iRNAs included in the disclosure can be made using conventional methods or based on in vivo studies using appropriate animal models.

[0371] Using an appropriate animal model, such as a mouse containing a transgene expressing human LECT2, the therapeutically effective dose and / or effective dosage regimen of LECT2 siRNA can be determined.

[0372] This disclosure also includes pharmaceutical compositions and formulations comprising the iRNA compounds characterized herein. The pharmaceutical compositions of this disclosure can be administered in a number of ways, depending on whether topical or systemic treatment is desired and the area to be treated. Administration may be topical (e.g., by a transdermal patch), inhalation or inhalation of powder or aerosol including in the lung, e.g., by a nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration may include intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous, e.g., via an implantable device; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular administration.

[0373] iRNAs can be delivered in a way that targets specific tissues, such as tissues that produce red blood cells. For example, iRNAs can be delivered to the bone marrow, liver (e.g., hepatocytes of the liver), lymph nodes, spleen, lungs (e.g., pleura of the lungs), or spine. In one embodiment, iRNAs are 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 bases, powders, or oily bases, thickeners, etc., may be required or desirable. Coated condoms, gloves, etc., may also be useful. Suitable topical formulations include those in which the iRNAs characterized 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 with them, 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, dicaplate, tricaplate, monoolein, dilaurin, glyceryl 1-monocaplate, 1-dodecyl azacycloheptan-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] Liposome formulations Beyond microemulsions, which have been studied and used for drug formulation, many other organized surfactant structures exist. These include monolayers, micelles, bilayers, and vesicles. Vesicles, such as liposomes, have attracted considerable 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” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or multiple bilayers.

[0376] Liposomes are monolayer or multilayer vesicles having 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 efficiently fuse with the cell wall, but are taken up by macrophages in vivo.

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

[0378] Further advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can encapsulate 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). Key considerations in the preparation of liposomal formulations are lipid surface charge, vesicle size, and the aqueous capacity of the liposomes.

[0379] Liposomes are useful for the movement and delivery of active ingredients to the site of action. Because the liposome membrane is structurally similar to biological membranes, when liposomes are applied to tissue, they begin to fuse with the cell membrane, and as the fusion of liposomes and cells progresses, the contents of the liposome become empty within the cell where the activator can act.

[0380] Liposome formulations have become the focus of extensive research as a mode of drug delivery for many drugs. Regarding topical administration, there is growing evidence that liposomes offer several advantages over other formulations. Such 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 detail the ability of liposomes to deliver drugs containing high molecular weight DNA to the skin. Analgesics, antibodies, hormones, and compounds containing high molecular weight DNA have been administered to the skin. The majority of applications have involved 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 complex binds to the negatively charged cell surface and is 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 engulf DNA rather than complex it with it. Since both DNA and lipids are similarly charged, repulsion occurs rather than complex formation. Nevertheless, some DNA is incorporated into the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding thymidine kinase genes to cell monolayers in culture. Exogenous gene expression was detected in 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 derived phosphatidylcholine. Neutral liposome compositions can be formed from, for example, dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic fusion liposomes are mainly formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soy PC and egg PC. Yet 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 guinea pig skin resulted in a reduction of 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 efficacy of interferon administered as part of a liposomal formulation compared to administration using an aqueous system, concluding that the liposomal formulation is superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0386] Nonionic liposome systems have also been tested to determine their efficacy in drug delivery to the skin, particularly in systems containing nonionic surfactants and cholesterol. Cyclosporine A was delivered to the dermis of mouse skin using nonionic liposome formulations containing Novasome® I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome® II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether). The results showed that such nonionic liposome systems are effective in promoting the deposition of cyclosporine-A to different layers of the skin (Hu et al. STP Pharma. Sci., 1994, 4, 6, 466).

[0387] Liposomes also include “stereostabilized” liposomes, which, as used herein, refer to liposomes containing one or more specific lipids that, when incorporated into liposomes, enhance their circulating lifespan compared to liposomes lacking such specific lipids. An example of a stereostabilized liposome is liposome (A) in which a portion of the vesicle-forming lipid portion contains monosialoganglioside G M1(B) These include one or more glycolipids, or are derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While we do not wish to be bound by any particular theory, in the art, for sterically stabilized liposomes containing at least gangliosides, sphingomyelin, or PEG-derivativeized lipids, the enhancement of the circulating half-life of these sterically stabilized liposomes is thought to be due to a reduction in uptake by the reticuloendothelial system (RES) into cells (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) described monosialoganglioside G M1 The ability of galactocerebroside sulfate and phosphatidylinositol to improve the half-life of liposomes in the blood was reported. These findings were described by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). Both U.S. Patent No. 4,837,028 and International Publication No. 88 / 04924 of Allen et al. describe (1) sphingomyelin and (2) ganglioside G M1 Alternatively, liposomes containing galactocerebroside sulfate are disclosed. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimiristoylphosphatidylcholine are disclosed in International Publication No. 97 / 13499 (Lim et al.).

[0389] Many liposomes containing lipids derivatized with one or more hydrophilic polymers, and methods for preparing them, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described a nonionic surfactant containing a PEG moiety, 2C 1215GLiposomes containing [the specified substance] were described. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymer glycols resulted in a significant enhancement of the blood half-life. Synthetic phospholipids modified by the bonding of carboxyl groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Patents 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 significant increase in the circulating half-life. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivative phospholipids formed from combinations of distearoylphosphatidylethanolamine (DSPE) and PEG, e.g., DSPE-PEG. Liposomes having a covalently bound PEG moiety on their outer surface are described in Fisher's European Patent No. 0445131B1 and International Publication No. 90 / 04384. Liposome compositions containing 1 to 20 mol percent of PEG-derivative PE, and methods of use thereof, are described by Woodle et al. (U.S. Patents No. 5,013,556 and 5,356,633) and Martin et al. (U.S. Patent No. 5,213,804 and European Patent No. 0496813B1). Liposomes containing numerous other lipid-polymer conjugates are disclosed in International Publication No. 91 / 05545 and U.S. Patent No. 5,225,212 (both by 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 using a functional moiety on the surface.

[0390] Numerous liposomes containing nucleic acids are known in the art. International Publication 96 / 40062 by Thierry et al. discloses a method for encapsulating high molecular weight nucleic acids in liposomes. U.S. Patent No. 5,264,221 by Tagawa et al. discloses protein-bound liposomes and claims that the contents of such liposomes may contain dsRNA. U.S. Patent No. 5,665,710 by Rahman et al. describes a specific method for encapsulating oligodeoxynucleotides in liposomes. International Publication 97 / 04787 by Love et al. discloses liposomes containing dsRNA targeted to the 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 permeate pores smaller than 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 the target without fragmentation, and are often self-loading. To construct transfersomes, surface edge activators, usually surfactants, can be added to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Delivery of serum albumin via transfersomes has been shown to be as effective as subcutaneous injection of a serum albumin-containing solution.

[0392] Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common method for classifying and ranking the properties of many different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The properties of the hydrophilic group (also known as the "head") provide 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] When a surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have found broad applications in pharmaceuticals and cosmetics and can be used across a wide range of pH values. Generally, their HLB values ​​range from 2 to about 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 ethoxylated products, propoxylated alcohols, and ethoxylated / propoxylated block polymers also fall into this class. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0394] Surfactants are classified as anionic if they carry a negative charge when dissolved or dispersed in water. 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 alkylbenzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0395] Surfactants are classified as cationic if they carry a positive charge when dissolved or dispersed in water. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used compounds in this class.

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

[0397] The use of surfactants in drug products, formulations, and emulsions is outlined (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 to form, for example, SPLPs, pSPLPs, SNALPs, or other nucleic acid-lipid particles. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle containing an SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle containing plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALPs and SPLPs exhibit a long circulating lifetime 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 “pSPLPs” containing an encapsulating condensant-nucleic acid complex as described in PCT International Publication No. 00 / 03683. The particles of this 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 nontoxic. Furthermore, when present in the nucleic acid-lipid particles of this disclosure, the nucleic acids are resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for preparing them are disclosed, for example, in U.S. Patents 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT International Publication No. 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 approximately 1:1 to approximately 50:1, approximately 1:1 to approximately 25:1, approximately 3:1 to approximately 15:1, approximately 4:1 to approximately 10:1, approximately 5:1 to approximately 9:1, or approximately 6:1 to approximately 9:1.

[0400] Cationic lipids include, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleyloxy)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-dilinolelenyloxy-N,N-dimethylaminopropane (DLenDMA), and 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 (DLinDAP), 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).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-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or similar compounds. The compounds 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)piperazine-1-yl)ethylazanejyl)didodecane-2-ol (Tech G1), or mixtures thereof. Cationic lipids can constitute approximately 20 mol% to 50 mol% or 40 mol% of the total lipids present in the particles.

[0401] In another embodiment, lipid-siRNA nanoparticles can be prepared using the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane. 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 contain 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] Noncationic lipids are anionic or neutral lipids, but are not limited to distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyloleoyl phosphatidylethanolamine (POPE), and dioleoyl phosphatidylethanolamine (DOPE). This includes oleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. Noncationic lipids may constitute about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% if cholesterol is included, of the total lipids present in the particles.

[0404] The conjugated lipids that inhibit particle aggregation may be, but are not limited to, polyethylene glycol (PEG) lipids, and include PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. PEG-DAA conjugates may be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimiristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C)8. The amount of conjugated lipids that inhibit particle aggregation may be 0 mol% to about 20 mol% or about 2 mol% of the total lipids present in the particles.

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

[0406] In some embodiments, the iRNA is formulated in lipid nanoparticles (LNPs).

[0407] LNP01 In one embodiment, lipid-dsRNA nanoparticles (e.g., LNP01 particles) can be prepared using 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). Each stock solution 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 molar ratio of 42:48:10. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such 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 hot barrel extruder, such as a 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, approximately pH 7, e.g., approximately pH 6.9, approximately pH 7.0, approximately pH 7.1, approximately pH 7.2, approximately pH 7.3, or approximately pH 7.4.

[0408] [ka]

[0409] The LNP01 formulation is described, for example, in International Patent Application Publication No. 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-Didimyristoylglycerol (C14-PEG or PEG-C14) (PEG with an average molecular weight of 2000) PEG-DSG: PEG-distylylglycerol (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 International Publication No. 2009 / 127060, filed on April 15, 2009, and are incorporated herein by reference.

[0414] Formulations containing XTC are described, for example, in U.S. Provisional Application No. 61 / 148,366 filed on 29 January 2009; U.S. Provisional Application No. 61 / 156,851 filed on 2 March 2009; U.S. Provisional Application No. 61 / 185,712 filed on 10 June 2009; U.S. Provisional Application No. 61 / 228,373 filed on 24 July 2009; U.S. Provisional Application No. 61 / 239,686 filed on 3 September 2009; and International Application No. PCT / US2010 / 022614 filed on 29 January 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 on 22 September 2009 and International Application No. PCT / US10 / 28224 filed on 10 June 2010, which are incorporated herein by reference.

[0416] Formulations containing ALNY-100 are described, for example, in International Patent Application PCT / US09 / 63933, filed on November 10, 2009, which is incorporated herein by reference.

[0417] C12-200, including the formulation, is described in U.S. Provisional Application No. 61 / 175,770, filed on 5 May 2009, and International Application No. PCT / US10 / 33777, filed on 5 May 2010, and is incorporated herein by reference.

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

[0419] "Alkyl" refers to a linear or branched, acyclic or cyclic, saturated aliphatic hydrocarbon containing 1 to 24 carbon atoms. Typical saturated linear alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl; on the other hand, saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, and isopentyl. Typical saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl; on the other hand, unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl.

[0420] "Alkenyl" means an alkyl group as defined above, which contains at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Typical linear and branched alkenyls include ethyleneyl, propyrenyl, 1-butenyl, 2-butenyl, isobutyrenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, and 2,3-dimethyl-2-butenyl.

[0421] "Alkynyl" means any alkyl or alkenyl as defined above, further containing at least one triple bond between adjacent carbon atoms. Typical linear and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-butynyl.

[0422] "Acyl" refers to any alkyl, alkenyl, or alkynyl group in which the carbon at the bonding site is substituted with an oxo group, as defined below. For example, -C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl are acyl groups.

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

[0424] The terms "appropriately substituted alkyl," "appropriately substituted alkenyl," "appropriately substituted alkynyl," "appropriately substituted acyl," and "appropriately substituted heterocyclic" mean that, if substituted, at least one hydrogen atom is substituted by the substituent. In the case of an oxo substituent (=O), two hydrogen atoms are substituted. In this regard, substituents are oxo, halogen, heterocyclic, -CN, and -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 This includes, where n is 0, 1, or 2, and Rx and R y These are the same or different, independently of hydrogen, alkyl, or heterocyclic, and each of the alkyl and heterocyclic substituents is oxo, halogen, -OH, -CN, alkyl, -OR x , complex algebra, -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 It may be further replaced by one or more of the following.

[0425] "Halogen" refers to fluoro, chloro, bromo, and iodine.

[0426] In some embodiments, the methods characterized in this 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 this disclosure is any group that reduces or removes unwanted reactivity of a functional group. Protecting groups can be added to a functional group to mask its reactivity during a particular reaction and then removed to expose the original functional group. In some embodiments, "alcohol protecting groups" are used. "Alcohol protecting groups" are any groups that reduce or remove unwanted reactivity of an alcohol functional group. Protecting groups can be added or removed using techniques well known in the art.

[0427] Synthesis of Equation A In one embodiment, the nucleic acid-lipid particles characterized in this disclosure are cationic lipids of formula A:

[0428] [ka]

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

[0430] [ka]

[0431] Lipid A can be prepared according to Scheme 1, where R1 and R2 are independently alkyl, alkenyl, or alkynyl, each of which may be optionally substituted, and R3 and R4 are independently lower alkyls, or R3 and R4 together may form an optionally substituted heterocyclic ring. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those skilled in the art. The reaction of 1 and 2 produces ketal 3. Treatment of ketal 3 with amine 4 produces lipid of formula A. Lipid of formula A can be converted to the corresponding ammonium salt by the organic salt of formula 5, where X is an anion counterion selected from halogens, hydroxides, phosphates, sulfates, and others.

[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. The reaction of 6 and 7 produces ketone 1. The conversion of ketone 1 to the lipid of the corresponding formula A is as described in Scheme 1.

[0434] MC3 synthesis The preparation of DLin-M-C3-DMA [i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate] was carried out as follows: A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyrate (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 on anhydrous magnesium sulfate, filtered, and the solvent was removed using a rotary evaporator. The residue was passed through a silica gel column (20 g) using a 1-5% methanol / dichloromethane elution gradient. The fractions containing the purified product were mixed, the solvent was removed, and a colorless oil (0.54 g) was obtained.

[0435] Synthesis of ALNY-100 Ketal 519 [ALNY-100] was synthesized using the following scheme 3.

[0436] [ka]

[0437] Synthesis of 515: At 0°C under nitrogen gas, a stirring suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml of anhydrous THF in 1 L of 2-neck RBF was slowly added to a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF. After complete addition, the reaction mixture was warmed to room temperature and then heated under reflux for 4 hours. The progress of the reaction was monitored by TLC. After the completion of the reaction (by TLC), the mixture was cooled to 0°C and inhibited by careful addition of saturated Na2SO4 solution. The reaction mixture was stirred at room temperature for 4 hours and filtered. The residue was thoroughly washed with THF. The filtrate and washings were mixed, diluted with 400 mL of dioxane and 26 mL of concentrated hydrochloric acid, and stirred at room temperature for 20 minutes. Volatile substances were removed under vacuum to obtain the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400MHz): δ= 9.34 (wide, 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 250 mL of two-neck RBF, NEt3 (37.2 mL, 0.2669 mol) was added and the mixture was cooled to 0°C under nitrogen gas. After the slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL of dry DCM, the reaction mixture was warmed to room temperature. After the reaction was complete (by TLC for 2-3 hours), the mixture was successively washed with 1N HCl solution (1 x 100 mL) and saturated NaHCO3 solution (1 x 50 mL). The organic layer was then dried on anhydrous Na2SO4, and the solvent was evaporated to obtain the crude product, which was purified by silica gel column chromatography to obtain 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] Combination of 517A and 517B: Cyclopentene 516 (5 g, 0.02164 mol) was dissolved in 220 mL of acetone and water (10:1) in a 500 mL single-neck RBF. N-methylmorpholine N-oxide (7.6 g, 0.06492 mol), followed by 4.2 mL of a 7.6% solution of OsO4 in tert-butanol (0.275 g, 0.00108 mol), was added at room temperature. After the reaction was complete (approximately 3 hours), the mixture was inhibited by the addition of solid Na2SO3, and the resulting mixture was stirred at room temperature for 1.5 hours. The reaction mixture was diluted with DCM (300 mL) and washed with water (2 x 100 mL), followed by saturated NaHCO3 (1 x 50 mL) solution, water (1 x 30 mL), and finally with saline solution (1 x 50 mL). The organic phase was dried over Na2SO4, and the solvent was removed under vacuum. A mixture of diastereomers was obtained by silica gel column chromatography of the crude material, and this mixture was separated by preparative HPLC. Yield: - 6 g crude product 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-rays.

[0440] Synthesis of 518: Compound 518 (1.2 g, 41%) was obtained as a colorless oil using a procedure similar to that described for the synthesis of compound 505. 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 hours, and then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous solution Na2SO4, and then filtered through Celite to reduce it to an oil. Column chromatography yielded pure 519 (1.3 g, 68%) as a colorless oil. 13C 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): Molecular weight of C44H80NO2 (M + H)+: Calculated value 654.6, Measured value 654.6.

[0442] Formulations prepared by either the standard method or the extrusion-free method can be characterized in a similar manner. For example, formulations are typically characterized visually. They should be whitish, translucent solutions free of aggregates or precipitates. The particle size and particle size distribution of lipid nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). The particles should be approximately 20–300 nm in size, e.g., 40–100 nm. The particle size distribution should be monomorphic. The total dsRNA concentration in the formulation and captured fractions 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 or out of the presence of a formulation-destroying surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the surfactant-containing sample compared to a standard curve. The captured fraction is determined by subtracting the "free" dsRNA content (measured by signal in the absence of surfactant) from the total dsRNA content. The percentage of captured dsRNA is typically over 85%. For SNALP formulations, particle sizes are 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. Preferred ranges are 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 formulations for oral administration include powders or granules, fine particles, nanoparticles, suspensions or solutions in water or a non-aqueous medium, capsules, gel capsules, sachets, tablets, or small tablets. Thickeners, flavorings, diluents, emulsifiers, dispersion aids, or binders may be desirable. In some embodiments, the oral formulation is an oral formulation in which the dsRNA characterized in this disclosure is administered together with one or more osmotherapists, 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, glutolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydrofusidate, 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-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or their monoglycerides, diglycerides, or pharmaceutically acceptable salts (e.g., sodium). In some embodiments, combinations of osmotic enhancers, e.g., fatty acid / salt combinations with bile acids / salts, are used. One exemplary combination is lauric acid, capric acid, and the sodium salt of UDCA. Further osmotic enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The dsRNAs featured in this disclosure may be delivered orally in the form of spray-dried particles or in granular forms complexed to form microparticles or nanoparticles.dsRNA complex-forming agents include poly-amino acids; polyimines; polyacrylates; polyalkyl acrylates, polyoxethane, polyalkylcyanoacrylates; cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG), and starch; polyalkylcyanoacrylates; DEAE-derivativeized polyimines, pullulan, cellulose, and starch. Suitable complex-forming agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene p (TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcyanoacrylate), DEAE-methacrylate, and D The oral formulations for dsRNA and their preparations are described in detail in U.S. Patent No. 6,887,906, U.S. Publication No. 20030027780, and U.S. Patent 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 a sterile aqueous solution containing a buffer, diluent, and other suitable additives, such as, not limited to, osmotic enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0445] The pharmaceutical compositions of this disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of components, including, but not limited to, ready-made liquids, self-emulsifying solids, and self-emulsifying semi-solids.

[0446] The pharmaceutical formulations featured in this disclosure can be conveniently presented in unit dosage forms, but can also be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques involve associating an active ingredient with a pharmaceutical carrier(s) or excipient(s). Generally, formulations are prepared by uniformly and closely associating an active ingredient with a liquid carrier, a subdivided solid carrier, or both, and then forming a product, if necessary.

[0447] The compositions featured in this disclosure may, without limitation, be formulated into any of many possible dosage forms, such as tablets, capsules, gel capsules, liquid syrups, softgels, suppositories, and enemas. The compositions may also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspensions may also contain stabilizers.

[0448] Additional formulations Emulsion The compositions of this disclosure may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of liquids dispersed in the form of separate droplets, usually larger 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.)). (See 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 densely mixed and dispersed from 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 in a bulk oil phase as tiny droplets, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when the oil phase is finely dispersed in a bulk aqueous phase as tiny droplets, the resulting composition is called an oil-in-water (o / w) emulsion.Emulsions may contain additional components in addition to the active drug, which may exist as a dispersed phase and a solution in either an aqueous or oil phase, or as separate phases themselves. Pharmaceutical excipients, such as emulsifiers, stabilizers, dyes, and antioxidants, may also be present in the emulsion as needed. Pharmaceutical emulsions can also be multiple emulsions, for example, oil-in-water (o / w / o) and water-in-oil (w / o / w) emulsions, which contain two or more phases. Such complex formulations often offer specific advantages that simple two-component emulsions do not. Among these, a multiple emulsion in which individual oil droplets of an o / w emulsion encapsulate smaller water droplets constitutes a w / o / w emulsion. Similarly, an oil droplet system encapsulated in small water droplets and stabilized in an oily continuous phase provides an o / w / o emulsion.

[0449] Emulsions are characterized by having little to no thermodynamic stability. The dispersed or discontinuous phases of an emulsion are well dispersed externally or within the continuous phase and are maintained in this form by means of emulsifiers or the viscosity of the formulation. In the case of emulsion-type ointment bases and creams, any of the emulsion phases may be semi-solid or solid. Other means of stabilizing an emulsion involve the use of emulsifiers, which may be incorporated into any of the emulsion phases. Emulsifiers can be broadly classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorbent bases, and finely dispersed solids (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; 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 surfactants, have found a wide range of applications in emulsion formulations and are outlined 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 and contain both hydrophilic and hydrophobic moieties. The ratio of hydrophilicity to hydrophobicity in a surfactant is called the hydrophilic / lipophilic balance (HLB), and it is a useful means of classifying and selecting surfactants in the preparation of pharmaceutical formulations. Surfactants can be classified into different classes based on the properties of their hydrophilic groups: 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; and 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. Adsorbents with hydrophilic properties that can absorb water and form w / o emulsions, such as anhydrous lanolin and hydrophilic petrolatum, maintain their semi-solid viscosity. Finely dispersed solids are also used as excellent emulsifiers in viscous preparations, particularly in combination with surfactants. These include polar inorganic solids such as heavy metal hydroxides, non-expanding clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloids of aluminum silicate and 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. These include 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] Examples of hydrophilic colloids include naturally occurring gums and synthetic polymers, such as polysaccharides (e.g., acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carbomer, cellulose ether, and carboxyvinyl polymer). These disperse in water or swell in water to form colloidal solutions that stabilize emulsions by creating a strong interfacial film around the dispersed phase droplets and by increasing the viscosity of the outer phase.

[0454] Emulsions often contain several components, such as carbohydrates, proteins, sterols, and phospholipids, that can easily support microbial growth; therefore, preservatives are frequently incorporated into these formulations. Commonly used preservatives in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, p-hydroxybenzoic acid esters, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may include free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, as well as antioxidant co-agents such as citric acid, tartaric acid, and lecithin.

[0455] The application of emulsion formulations via skin, oral, and non-enteral routes, and methods for manufacturing them, are outlined 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; 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 their ease of formulation and efficiency in terms of absorption and bioavailability (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; 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, fat-soluble vitamins, and high-fat nutritional supplements are commonly administered orally as o / w emulsions.

[0456] In one embodiment of the present disclosure, the iRNA and nucleic acid composition is formulated as a microemulsion. A microemulsion can be defined as a system of water, oil, and an amphiphilic substance 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, a microemulsion is a system prepared by first dispersing the oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, usually an alcohol of intermediate chain length, to form a clear system. Therefore, microemulsions are also described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surfactant 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 via a combination of 3 to 5 components, including oil, water, surfactant, 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 filling of the polar head and hydrocarbon tail of the surfactant molecule (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0457] Phenomenological approaches utilizing phase diagrams have been extensively studied, providing those skilled in the art with comprehensive knowledge of microemulsion formulation methods (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 naturally formed, thermodynamically stable droplet formulation.

[0458] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ethers, 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 co-surfactants. Co-surfactants, typically short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, work to increase interfacial fluidity by penetrating the surfactant film, resulting in the creation of an irregular film due to gaps between surfactant molecules. However, microemulsions are prepared without the use of co-surfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but not limited to, water, aqueous solutions of drugs, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and derivatives of ethylene glycol. The oil phase may include, but not limited to, materials such as Captex300, Captex355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono, di, and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolated glycerides, saturated polyglycolated C8-C10 glycerides, vegetable oils, and silicone oils.

[0459] Microemulsions are of particular interest from the standpoint of enhancing drug solubilization and drug absorption. Lipid-based microemulsions (both o / w and w / o) containing peptides have been proposed to increase the oral bioavailability of drugs (e.g., U.S. Patent Nos. 6191105; 7063860; 7070802; 7157099; 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, protection of drugs from enzymatic hydrolysis, enhanced expected drug absorption due to surfactant-induced changes in membrane fluidity and permeability, ease of preparation, ease of oral administration compared to solid dosage forms, improved clinical efficacy, and reduced toxicity (see, e.g., U.S. Patent No. 6191105; No. 7063860; No. 7070802; No. 7157099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Microemulsions often form spontaneously when their components are combined at ambient temperature. This can be particularly advantageous when formulating heat-unstable drugs, peptides, or iRNAs. Microemulsions have also been effective for transdermal delivery of active ingredients in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of this disclosure are expected to promote increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as to improve local intracellular uptake of iRNAs and nucleic acids.

[0460] The microemulsions of this disclosure may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and osmotic enhancers to improve the formulation properties and enhance the absorption of iRNAs and nucleic acids of this disclosure. Osmotic enhancers used in the microemulsions of this disclosure can be classified into 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 is discussed above.

[0461] Penetration enhancer In one embodiment, the present disclosure describes how various penetration enhancers can be used to facilitate the effective delivery of nucleic acids, particularly iRNAs, to animal skin. Most drugs exist in solution in both ionized and non-ionized forms. However, typically only lipid-soluble or lipophilic drugs readily cross cell membranes. It has been found that even non-lipophilic drugs can cross cell membranes when the membrane being crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also increase the permeability of lipophilic drugs.

[0462] Penetration enhancers can be classified into one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, for example, 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 relation to this disclosure, surfactants (or “surfactants”) are chemical substances 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, thereby enhancing the 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 (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; see 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-monoleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, and its C 1ー20Examples include alkyl esters (e.g., methyl, isopropyl, and t-butyl), as well as their mono- and di-glycerides (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 naturally occurring bile salts, and their synthetic derivatives, act as osmotic enhancers. Therefore, the term “bile salt” includes any of the naturally occurring components of bile, as well as 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), glucoseic acid (sodium glucose), 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-dihydrofusidate sodium (STDHF), glycodihydrofusidate sodium, and polyoxyethylene-9-lauryl ether (POE) (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 agents: When used in connection with the present disclosure, chelating agents can be defined as compounds that remove metal ions from solution by forming complexes with them, thereby enhancing the absorption of iRNA through mucosa. 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 catalytic activity and are therefore inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (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 (enamine) (see, for example, 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-chelated non-surfactant compounds: As used herein, non-chelated non-surfactant osmotic enhancers can be defined as compounds that demonstrate activity that is not superior as a chelating agent or surfactant, but nevertheless enhance the absorption of iRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Examples of osmotic enhancers in this class include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenyl azacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal anti-inflammatory drugs such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0468] Agents that enhance iRNA uptake at the cellular level may also be added to the pharmaceuticals and other compositions of this disclosure. For example, cationic lipids such as lipofectin (Junichi et al., U.S. Patent No. 5705188), cationic glycerol derivatives, and polycationic molecules such as polylysine (Lollo et al., International Publication No. 97 / 30731) are known to enhance dsRNA uptake within cells.Examples of commercially available transfection reagents include, for example, Lipofectamine(trademark) (Invitrogen; Carlsbad, CA), Lipofectamine 2000(trademark) (Invitrogen; Carlsbad, CA), 293fectin(trademark) (Invitrogen; Carlsbad, CA), Cellfectin(trademark) (Invitrogen; Carlsbad, CA), DMRIE-C(trademark) (Invitrogen; Carlsbad, CA), FreeStyle(trademark)MAX(Invitrogen; Carlsbad, CA), and Lipofectamine(trademark) 2000. CD (Invitrogen; Carlsbad, CA), Lipofectamine (trademark) (Invitrogen; Carlsbad, CA), RNAiMAX (Invitrogen; Carlsbad, CA), Oligofectamine (trademark) (Invitrogen; Carlsbad, CA), Optifect (trademark) (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 include 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 (trademark) (B-Bridge International, Mountain View, CA, USA).

[0469] Other agents, including glycols such as ethylene glycol and propylene glycol, pyrroles such as 2-pyrrole, azon, and terpenes such as limonene and menthone, may be used to enhance the penetration of administered nucleic acids.

[0470] Carrier Certain compositions of this disclosure also incorporate a carrier compound in the formulation. As used herein, “carrier compound” or “carrier” may refer to a nucleic acid or its analogue that is inert (i.e., not biologically active itself) but is recognized as a nucleic acid by an in vivo process that reduces the bioavailability of biologically active nucleic acids, for example, by degrading biologically active nucleic acids or facilitating their removal from the circulation. Co-administration of nucleic acids and carrier compounds, typically in excess of the latter, may result in a substantial reduction in the amount of nucleic acid recovered by the liver, kidneys or other extracirculatory storage organs, possibly due to competition between the carrier compound and nucleic acid for common receptors. For example, the recovery of partial phosphorothioate dsRNAs in liver tissue may be reduced when administered co-administered with polyinosinic acid, dextran sulfate, polycytidic acid, or 4-acetamido-4'-isothiocyanostilbene-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 carrier compounds, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspension agent, or any other pharmaceutically inert medium for delivering one or more nucleic acids to an animal. Excipients may be liquid or solid and are selected based on the planned mode of administration so as to provide the desired bulk, viscosity, etc., when combined with the nucleic acid and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica, silicon dioxide colloids, stearic acid, metal stearate salts, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate); disintegrants (e.g., starch, sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

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

[0473] Formulations for topical administration of nucleic acids may 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 may also contain buffers, diluents, and other suitable additives. pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not react adversely with nucleic acids may be used.

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

[0475] Other ingredients The compositions of this disclosure may further contain other auxiliary components conventionally found in pharmaceutical compositions, at levels of use established in the art. For example, a composition may contain pharmaceutically active materials of further suitability, such as antipruritics, astringents, topical anesthetics, or anti-inflammatory agents, or further materials useful for physically formulating various dosage forms of the compositions of this disclosure, such as dyes, flavorings, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, such materials, when added, should not excessively interfere with the biological activity of the components of the compositions of this disclosure. The formulations may be sterilized and, if desired, mixed with auxiliary agents that do not adversely interact with the nucleic acids of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, colorants, flavorings, and / or aromatics.

[0476] The aqueous suspension may contain substances that increase the viscosity of the suspension, such as 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 bioagents that function by a non-RNAi mechanism. Examples of such bioagents include agents that interfere with the interaction between LECT2 and at least one LECT2-binding partner.

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

[0479] Data obtained from cell culture assays and animal experiments may be used to formulate dosage ranges for use in humans. Doses of the compositions featured in this disclosure are typically within the range of circulating concentrations, including an ED50 that is of little to no toxicity. Doses may 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 dose may first be estimated from a cell culture assay. Doses may be formulated in animal models to achieve the circulating plasma concentration range of the compound, or, where appropriate, the polypeptide product of the target sequence, including the IC50 (i.e., the concentration of the test compound that achieves maximum half-dose inhibition of the symptoms) determined in cell culture (e.g., achieving a reduction in polypeptide concentration). Using such information, useful doses in humans can be more accurately determined. Plasma levels may be measured, for example, by high-performance liquid chromatography.

[0480] The iRNAs featured in this disclosure may be administered in combination with other known agents effective in treating diseases or disorders related to LECT2 expression, in addition to the administrations discussed above. In any case, the administering physician may adjust the amount and timing of iRNA administration based on the results observed using standard efficacy measures known in the art or described herein.

[0481] Methods to address disorders related to LECT2 gene expression This disclosure relates to the use of LECT2-targeting iRNAs to inhibit LECT2 expression and / or to treat diseases, disorders, or pathological processes associated with LECT2 expression.

[0482] In one embodiment, a method is provided for treating a disorder related to LECT2 expression, comprising administering an 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, “LECT2 expression-related disorder,” “LECT2 expression-related disease,” “LECT2 expression-related pathological process,” and others 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 of the subject (e.g., in plasma). In some embodiments, the decrease or increase in LECT2 expression is detectable in a tissue sample from the subject (e.g., in a kidney or liver sample). The decrease or increase may be evaluated in comparison to levels observed in the same individual prior to the onset of the disorder, or in comparison to other individuals without the disorder. The decrease or increase may be limited to a specific organ, tissue, or region of the body (e.g., the kidney or liver).

[0484] When used herein, the “subject” to be treated according to the methods described herein includes humans or non-human animals, such as mammals. Mammals may be, for example, rodents (e.g., rats or mice) or primates (e.g., monkeys). In some embodiments, the subject is human.

[0485] "Subjects requiring it" include subjects who have, are suspected of having, or are at risk of developing a disorder related to LECT2 expression. In some embodiments, subjects have, or are suspected of having, a disorder related to LECT2 expression. In some embodiments, subjects are at risk of developing a disorder related to LECT2 expression.

[0486] In some embodiments, the subjects are animals that serve as models for disorders related to LECT2 expression, such as LECT2 amyloidosis.

[0487] LECT2 amyloidosis In some embodiments, disorders associated with LECT2 expression are amyloidosis, e.g., LECT2 amyloidosis. LECT2 amyloidosis has been described in several clinical studies. See, for example, 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; Hollanda, DG et al. (2011) Nephrol. Dial. Transplant., 26(1): 373-376; and Sethi, S. et al. (2012) Kidney International 82, 226-234 (hereinafter Sethi et al.).

[0488] The clinical and pathological features of LECT2 amyloidosis are similar to those of amyloid light chain (AL) amyloidosis. These symptoms include, for example, symptoms of kidney disease and renal failure, such as fluid retention, bloating, 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 arrhythmias. Other symptoms include, for example, stroke, gastrointestinal disorders, hepatomegaly, decreased splenic function, decreased adrenal and other endocrine gland function, skin discoloration or proliferation, lung problems, bleeding and bruising problems, fatigue, and weight loss. In some embodiments, the methods described herein are associated with the improvement of one or more of the symptoms described herein.

[0489] Methods for diagnosing amyloidosis, such as 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 by Sethi et al., which shows that 26 out of 127 cases of renal amyloidosis studied by laser microanalysis and mass spectrometry of renal biopsy and / or nephrectomy specimens were determined to be LECT2 amyloid-type renal amyloidosis. Sethi et al. also further report that apolipoprotein E protein and serum amyloid P component (SAP) were present in all cases of LECT2 amyloidosis.

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

[0492] In some embodiments, amyloidosis, such as LECT2 amyloidosis, is hereditary.

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

[0494] In some embodiments, amyloidosis, such as LECT2 amyloidosis, affects the kidneys, for example, and is involved in amyloid deposition in the kidneys. In some embodiments, kidney function is impaired as a result of amyloidosis. In some embodiments, the subject suffers from one or more of the following: fluid retention, bloating, 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, such as 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 is known to be particularly prevalent among Mexican Americans and has been linked to homozygosity of the G allele of the LECT2 gene, which codes for valine at position 40 of the mature protein (amino acid 58 of the unprocessed protein). See, for example, 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 of Mexican American descent.

[0498] In some embodiments, the subject possesses a G allele of the LECT2 gene that encodes 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 contains valine at position 40 of the mature protein (or at 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 evaluated by comparing it to 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 the treated subject (or control group) to the level of a control subject (or control group), e.g., an untreated subject (or control group) or a subject (or control group) treated with a control treatment [e.g., iRNA that does not target LECT2 (e.g., dsRNA)].

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

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

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

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

[0504] Furthermore, methods for reducing amyloid deposition are provided herein, comprising administering the dsRNA disclosed herein to a subject in need thereof (e.g., a subject having, suspected of having, or at risk of developing LECT2 amyloidosis). In some embodiments, the methods reduce (e.g., prevent or reduce) the size, number, and / or extent of amyloid deposition. The size, number, and / or extent of amyloid deposition can be assessed using any method known in the art (e.g., immunoassay, immunohistochemistry, mass spectrometry). Reduction of amyloid deposition may involve a reduction of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more of amyloid deposition (e.g., size, number, and / or extent of amyloid deposition).

[0505] In the methods provided herein, iRNA (e.g., dsRNA) and its composition are administered in a therapeutically effective dose. The therapeutic effect of LECT2 siRNA administration can be established, for example, by comparison with a suitable control. For example, inhibition of amyloid deposition can be established, for example, by comparing any suitable parameter (e.g., a parameter for evaluating the size, number, or extent of amyloid deposition) in a group of patients with amyloidosis (e.g., LECT2 amyloidosis) with the same parameter in a suitable control group. The control group (e.g., a similar or identical population in a cross-design) may include, for example, an untreated population, a population treated with conventional treatment; a population treated with placebo or untargeted iRNA; etc.

[0506] Rheumatoid arthritis Furthermore, rheumatoid arthritis is a disorder associated with LECT2 expression. In particular, in the Japanese population, it was found that 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), increased the overall risk of developing rheumatoid arthritis. Possessing two A alleles was 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 associated with LECT2 expression is rheumatoid arthritis. In one embodiment, dsRNA inhibits LECT2 expression in subjects having rheumatoid arthritis. In some such embodiments, dsRNA inhibits LECT2 expression in synovial tissue and / or synovial fluid-derived cells (e.g., mononuclear cells and fibroblasts). In some embodiments, dsRNA targets mRNA encoding isoleucine at position 40 of the mature protein (amino acid 58 of the unprocessed protein).

[0508] liver damage LECT2 expression may increase during acute liver injury.

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

[0510] Combination therapy In some embodiments, the iRNAs disclosed herein (e.g., dsRNAs) are administered in combination with a second treatment (e.g., one or more additional treatments) known to be effective in treating disorders associated with LECT2 expression (e.g., LECT2 amyloidosis) or symptoms of such disorders. The iRNAs may be administered before, after, or concurrently with the second treatment. In some embodiments, the iRNAs are administered before the second treatment. In some embodiments, the iRNAs are administered after the second treatment. In some embodiments, the iRNAs are administered concurrently with the second treatment.

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

[0512] In some embodiments, the second treatment is a non-iRNA therapeutic agent effective in treating the disorder or its symptoms.

[0513] In some embodiments, the disorder treated by the compositions or methods disclosed herein is LECT2 amyloidosis, which affects renal function due to amyloid deposition in the kidneys, for example. In some such embodiments, the iRNA is administered in conjunction with a treatment that supports renal 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, including 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 the removal of all or part of the organ(s) affected by amyloidosis (e.g., excision of all or part of the kidney or liver tissue affected by amyloidosis). The removal may be carried out in conjunction with the replacement of all or part of the removed organ(s) (e.g., in conjunction with kidney or liver transplantation).

[0516] Dosage, route, and timing The subject (e.g., human subject, e.g., patient) may be administered a therapeutic dose of iRNA. The therapeutic dose may be, for example, 0.05 to 50 mg / kg. For example, the therapeutic dose may 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, or 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, such as the liver.

[0518] In some embodiments, the iRNA is formulated as a lipid formulation, for example, an LNP formulation as described herein. In some such embodiments, the therapeutic dose is 0.05–5 mg / kg, for example, 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, for example, an LNP formulation, is administered intravenously. 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 (e.g., intravenously).

[0519] In some embodiments, the iRNA is administered by intravenous infusion over a set period of time, for example, 5, 10, 15, 20, or 25 minutes. In some embodiments, the iRNA is in the form of a GalNAc conjugate as described herein. In some such embodiments, the therapeutic dose is 0.5 to 50 mg of dsRNA, for example, 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. 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 at a dose of 1 to 10 mg / kg (e.g., subcutaneously).

[0520] In some embodiments, administration is repeated, for example, regularly, daily or every other week (i.e., every two weeks) for a period of one, two, three, four months or longer. After the initial treatment regimen, treatment may be administered less frequently. For example, after administration every three months, administration may be repeated once every one month, six months, or one year or longer.

[0521] In some embodiments, the iRNA agent is administered in two or more doses. In some embodiments, the number or amount of the following doses depends on achieving the desired effect, e.g., inhibition of amyloid deposition, or a therapeutic or preventive effect, e.g., reduction or prevention of one or more symptoms associated with the disorder.

[0522] In some embodiments, the iRNA agent is administered according to a schedule. For example, the iRNA agent may be administered once, twice, three times, four times, or five times per week. In some embodiments, the schedule includes regularly spaced administration, such as once every hour, every four hours, every six hours, every eight hours, every twelve hours, daily, every two days, every three days, every four days, every five days, weekly, every other week, or monthly. In some embodiments, the iRNA agent is administered at the frequency required to achieve the desired effect.

[0523] In some embodiments, the schedule includes a period of no drug administration after a period of no drug administration. For example, the schedule may include an initial set of doses administered at relatively short intervals (e.g., every 6 hours, every 12 hours, every 24 hours, every 48 hours, or every 72 hours) followed by a period of no iRNA administration (e.g., every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks). In one embodiment, the iRNA is initially administered every hour, and then at longer intervals (e.g., daily, weekly, bi-weekly, or monthly). In another embodiment, the iRNA is initially administered daily, and then at longer intervals (e.g., weekly, bi-weekly, or monthly). In certain embodiments, the longer intervals increase over time or are determined based on the achievement of the desired effect.

[0524] Before administering the full dose of iRNA, the patient may be given a small dose, such as a 5% infusion dose, and adverse effects, such as allergic reactions or elevated lipid levels or blood pressure, may be monitored. In another case, the patient may be monitored for undesirable effects.

[0525] Method for modulating LECT2 gene expression In yet another embodiment, the Disclosure provides methods for modulating (e.g., inhibiting or activating) the expression of the LECT2 gene, for example, in cells or in a subject. In some embodiments, the cells are ex vivo, in vitro, or in vivo. In some embodiments, the cells are located in the liver (e.g., hepatocytes). In some embodiments, the cells are located in a subject (e.g., a mammal such as a human). In some embodiments, the subject (e.g., a human) is diagnosed as being at risk of or having a disorder associated with LECT2 expression as described herein.

[0526] In one embodiment, the method involves contacting cells with the iRNA described herein in an amount effective in reducing the expression of the LECT2 gene in the cells. As used herein, “contact” includes direct contact with cells as well as indirect contact with cells. For example, when a composition containing the iRNA is administered to a subject (e.g., intravenously or subcutaneously), cells within the subject may be contacted.

[0527] LECT2 gene expression can be assessed based on the level of LECT2 mRNA, LECT2 protein expression, or the level of another parameter functionally related to the level of LECT2 gene expression. 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 is IC in the range of 0.001-0.01nM, 0.001-0.10nM, 0.001-1.0nM, 0.001-10nM, 0.01-0.05nM, 0.01-0.50nM, 0.02-0.60nM, 0.01-1.0nM, 0.01-1.5nM, and 0.01-10nM. 50 It has an IC. 50 The value should be an appropriate control value, such as the IC of untargeted iRNA. 50 It can be normalized to this.

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

[0529] In one embodiment, the method involves administering a composition described herein, for example, a composition comprising an iRNA that targets LECT2, to a mammal so as to reduce the expression of a target LECT2 gene for an extended period, for example, at least two, three, four days or more, for example, one, two, three, or four weeks or more. In some embodiments, the reduction in LECT2 expression is detectable within one, two, four, eight, twelve, or 24 hours of the first dose.

[0530] In another embodiment, the method involves administering a composition described herein to a mammal such that the expression of a target LECT2 gene is increased by at least 10% compared to, for example, an untreated animal. In some embodiments, LECT2 activation occurs over an extended period, for example, at least two, three, or four days or longer, for example, one, two, three, or four weeks or longer. While we do not wish 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 for the methods and the compositions featured in this disclosure specifically target the RNA (primary or processed) of the LECT2 gene. Compositions and methods for inhibiting LECT2 gene expression using iRNAs may be prepared and carried out as otherwise described herein.

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

[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 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 commonly understood by traders in the art to which this disclosure belongs. Similar or equivalent methods and materials to those described herein may be used in carrying out or testing the iRNAs and methods characterized herein, but preferred methods and materials are described below. All papers, patent applications, patents, and other references described herein are incorporated in their entirety by reference. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative and not intended to limit the scope of this disclosure.

[0536] Specific Embodiments 1. A double-stranded ribonucleic acid (dsRNA) for inhibiting LECT2 expression, comprising a sense strand and an antisense strand comprising a region complementary to the LECT2 RNA transcript and containing at least 15 consecutive nucleotides that differ by 3 nucleotides or less 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 LECT2 expression, comprising a sense strand 15 to 30 nucleotides long and an antisense strand 15 to 30 nucleotides long 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 according to Embodiment 1 or 2, comprising at least one modified nucleotide. 4. The dsRNA agent according to Embodiment 3, wherein the sense strand has 5 nucleotides or less and the antisense strand has 5 nucleotides or less that are unmodified nucleotides. 5. The dsRNA agent according to Embodiment 3, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand are modified. 6. A dsRNA according to any of the embodiments, comprising a double-stranded region having a length of 6.15 to 30 base pairs. 7. The dsRNA according to Embodiment 6, wherein the double-stranded region is 17 to 25 base pairs long. 8. The dsRNA according to Embodiment 6 or 7, wherein the double-stranded region is 19 to 22 base pairs long. 9. A dsRNA according to any one of embodiments 6 to 8, wherein the double-stranded region is 21 base pairs long. 10. The dsRNA according to any of the embodiments, wherein the antisense strand comprises a nucleotide sequence 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, and comprising at least 15 consecutive nucleotides. 11. A dsRNA agent according to any of the embodiments, wherein the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0, 1, 2, or 3 mismatches from the sense sequences listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, and 6B, which correspond to the antisense sequences. 12. The dsRNA according to any of the embodiments, wherein the antisense strand comprises a nucleotide sequence having at least 17 consecutive 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. 13. A dsRNA agent according to any of the embodiments, wherein the sense strand comprises a nucleotide sequence having 0, 1, 2, or 3 mismatches from a sense sequence corresponding to an antisense sequence, which is listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, and 6B, and which comprises at least 17 consecutive nucleotides. 14. The dsRNA according to any of the embodiments, wherein the antisense strand comprises a nucleotide sequence having at least 19 consecutive 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. 15. A dsRNA agent according to any of the embodiments, wherein the sense strand comprises a nucleotide sequence comprising at least 19 consecutive nucleotides having 0, 1, 2, or 3 mismatches from sense sequences listed in any one of Tables 2A, 2B, 4A, 4B, 5A, 5B, 6A, and 6B, which correspond to the antisense sequence. 16. The dsRNA according to any of the embodiments, wherein the antisense strand comprises a nucleotide sequence having at least 21 consecutive 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. 17. A dsRNA agent according to any of the emb...

Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting LECT2 expression, comprising a sense strand and an antisense strand comprising a region complementary to the LECT2 RNA transcript and containing at least 15 consecutive nucleotides that differ by three or fewer 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 LECT2 expression, comprising a sense strand 15 to 30 nucleotides long and an antisense strand 15 to 30 nucleotides long, 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 according to claim 1 or 2, comprising at least one modified nucleotide.

4. The dsRNA according to any one of the claims, comprising a double-stranded region having a length of 15 to 30 base pairs.

5. The dsRNA according to claim 4, wherein the double-stranded region is 17 to 25 base pairs long.

6. The dsRNA according to claim 4 or 5, wherein the double-stranded region is 19 to 22 base pairs long.

7. The dsRNA according to any one of claims 4 to 6, wherein the double-stranded region is 21 base pairs long.

8. The dsRNA according to any one of claims 1 or 3 to 7, wherein the complementary region is at least 17 nucleotides long.

9. The dsRNA according to any one of claims 1 or 3 to 8, wherein the complementary region is between 21 and 25 nucleotides in length.

10. The dsRNA according to any one of claims 1 or 3 to 9, wherein the complementary region is 23 nucleotides long.

11. The dsRNA according to any one of the claims, wherein at least one strand comprises a 3' overhang of at least one or two nucleotides.

12. The dsRNA according to any one of the claims, comprising a blunt end.

13. The dsRNA according to any one of claims 3 to 12, wherein at least one of the modified nucleotides 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.

14. The dsRNA according to any one of claims 3 to 12, wherein at least one of the modified nucleotides is selected from the group consisting of: 2'-deoxy-2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleic acids (LNAs), acyclic nucleotides, debasic nucleotides, glycol nucleotides (GNAs), 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural bases including nucleotides.

15. The dsRNA according to any one of claims 3 to 12, wherein the nucleotide modification 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'-O-methyl, 2'-deoxy, 2'-hydroxy, and combinations thereof.

16. The dsRNA according to any one of claims 3 to 12, wherein the nucleotide modification may be 2'-O-methyl, 2'-fluoro, or both, and GNA.

17. The dsRNA according to any one of the claims, wherein the sense strand is conjugated to at least one ligand.

18. The dsRNA according to claim 17, wherein the ligand is bound to the 3' end of the sense strand.

19. The dsRNA according to claim 17 or 18, wherein the ligand contains a carbohydrate.

20. The dsRNA according to any one of claims 17 to 19, wherein the ligand is a GalNAc ligand.

21. Ligand 【Chemistry 1】 The dsRNA according to any one of claims 17 to 20.

22. The dsRNA according to any one of claims 17 to 21, wherein the ligand is bound via a linker.

23. The dsRNA according to claim 22, wherein the linker is a divalent or trivalent branched linker.

24. The ligand and linker are of formula XXIV: 【Chemistry 2】 The dsRNA according to claim 22, as shown below.

25. The dsRNA according to any one of claims 17 to 24, wherein the ligand targets the dsRNA to hepatocytes.

26. The dsRNA according to any one of the claims, wherein the complementary region consists of an antisense sequence selected from the antisense sequences disclosed in Tables 2A-2B, 3A-3B, 6, or 7.

27. The dsRNA according to any one of the claims, wherein the dsRNA comprises a sense strand consisting of a sense sequence selected from the sense sequences disclosed in Tables 2A-2B, 3A-3B, 6, or 7, and an antisense strand consisting of an antisense sequence selected from the antisense sequences disclosed in Tables 2A-2B, 3A-3B, 6, or 7.

28. A double-stranded ribonucleic acid (dsRNA) for inhibiting LECT2 expression, comprising a sense strand and an antisense strand containing a region complementary to the LECT2 RNA transcript, wherein the sense strand comprises the sequence gsgsucagAfuCfUfUfcaaaaaauaaaaL96 (SEQ ID NO: 143) and all modifications, and the antisense strand comprises the sequence asUfsusuuaUfuUfUfgaagAfuCfugaccsgsg (SEQ ID NO: 144) and all modifications.

29. A double-stranded ribonucleic acid (dsRNA) for inhibiting LECT2 expression, comprising a sense strand and an antisense strand containing a region complementary to the LECT2 RNA transcript, wherein the complementary region is substantially complementary to nucleotides 669-691 of Sequence ID No.

1.

30. A cell containing dsRNA as described in any of the above claims.

31. A pharmaceutical composition for inhibiting the expression of the LECT2 gene, comprising the dsRNA described in any one of claims 1 to 29.

32. The pharmaceutical composition according to claim 31, wherein dsRNA is administered in a non-buffered solution.

33. The pharmaceutical composition according to claim 32, wherein the non-buffered solution is physiological saline or water.

34. The pharmaceutical composition according to claim 31, wherein the dsRNA is administered by a buffer solution.

35. The pharmaceutical composition according to claim 34, wherein the buffer solution comprises an acetate, citrate, prolamin, carbonate, phosphate, or any combination thereof.

36. The pharmaceutical composition according to claim 34 or 35, wherein the buffer solution is phosphate-buffered saline (PBS).

37. A pharmaceutical composition according to any one of claims 31 to 36, comprising a lipid preparation.

38. The pharmaceutical composition according to claim 37, wherein the lipid preparation is an LNP preparation.

39. The pharmaceutical composition according to claim 37 or 38, wherein the lipid preparation is an LNP11 preparation.

40. The pharmaceutical composition according to any one of claims 31 to 39, wherein dsRNA is targeted to liver cells or hepatocytes.

41. A pharmaceutical composition according to any one of claims 31 to 40, which is administered intravenously.

42. A pharmaceutical composition according to any one of claims 31 to 40, which is administered subcutaneously.

43. The pharmaceutical composition according to claim 41, comprising a lipid preparation and administered intravenously.

44. A pharmaceutical composition according to any one of claims 31 to 43, comprising a dsRNA conjugated to a ligand selected from a carbohydrate ligand or a GalNAc ligand.

45. A method for inhibiting LECT2 expression in cells, (a) bringing the dsRNA described in any of claims 1 to 29 into contact with a cell, for example, introducing it, and (b) Maintain the cells from step (a) for a sufficient time to obtain degradation of the mRNA transcript of the LECT2 gene, thereby inhibiting the expression of the LECT2 gene in the cells. Methods that include...

46. The method according to claim 45, wherein the cells are treated ex vivo, in vitro, or in vivo.

47. The method according to claim 45 or 46, wherein the cells are present in a subject requiring treatment, prevention, and / or management of a disorder related to LECT2 expression.

48. The method according to claim 47, wherein the disorder is amyloidosis.

49. The method according to claim 48, wherein the amyloidosis is LECT2 amyloidosis.

50. The method according to any one of claims 45 to 49, wherein the cells are liver cells or hepatocytes.

51. The method according to any one of claims 45 to 50, wherein LECT2 expression is inhibited by at least 20%.

52. The method according to any one of claims 45 to 51, wherein LECT2 expression is inhibited by at least 90%.

53. A method for treating disorders related to LECT2 expression, wherein a therapeutically effective amount (i) dsRNA according to any one of claims 1 to 29, or (ii) Pharmaceutical composition according to any one of claims 31 to 44 A method comprising administering to a subject requiring such treatment.

54. A method for treating LECT2 amyloidosis, wherein the therapeutically effective amount (i) dsRNA according to any one of claims 1 to 29, or (ii) Pharmaceutical composition according to any one of claims 31 to 44 A method comprising administering to a subject requiring such treatment.

55. The method according to claim 52 or 53, wherein the subject has amyloidosis or is at risk of developing amyloidosis.

56. The method according to any one of claims 53 to 55, wherein the amyloidosis is LECT2 amyloidosis.

57. The method according to any one of claims 45 to 56, wherein dsRNA or a composition containing dsRNA is administered according to a dosage regimen.

58. The method according to claim 57, wherein the administration regimen is weekly, bi-weekly, or monthly.

59. The method according to any one of claims 45 to 58, which reduces LECT2 amyloid deposition.

60. A method for reducing LECT2 amyloid deposition in subjects with LECT2 amyloidosis, (i) dsRNA according to any one of claims 1 to 29, or (ii) Pharmaceutical composition according to any one of claims 31 to 44 A method including administering to a target.

61. The method according to any one of claims 53 to 60, wherein dsRNA is administered at a dose of 0.05 to 50 mg / kg.

62. The method according to any one of claims 53 to 61, wherein dsRNA is administered at a concentration of 0.01 mg / kg body weight to 5 mg / kg body weight.

63. The method according to any one of claims 53 to 62, wherein dsRNA is formulated as an LNP preparation and administered at a dose of 0.1 mg / kg to 0.5 mg / kg.

64. The method according to any one of claims 53 to 63, wherein dsRNA is conjugated to a GalNAc ligand.

65. The method according to any one of claims 53 to 64, wherein dsRNA is conjugated to a GalNAc ligand and administered at a dose of 1 mg / kg to 10 mg / kg, which may be 1 mg / kg or 3 mg / kg.

66. A vector encoding at least one strand of dsRNA according to any one of claims 1 to 29.

67. A cell containing the vector according to claim 66.

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