Compositions and methods for inhibiting expression of the HAO1 (hydroxyacid oxidase 1 (glycolate oxidase)) gene

Abstract

Provided are RNAi agents, eg, double-stranded RNAi agents, that target the HAO1 gene, and methods of using such RNAi agents to inhibit expression of HAO1, and methods of treating a subject, eg, having PH1. [Solution] Provided are double-stranded RNAi agents that inhibit expression of the HAO1 gene in cells, e.g., cells within the body of a subject, e.g., a mammal, e.g., a human having an HAO1-associated disorder, and uses of such double-stranded RNAi agents. In certain aspects of the invention, substantially all of the nucleotides of the iRNA of the invention are modified.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application Nos. 62 / 062,751, filed October 10, 2014, 62 / 147,976, filed April 15, 2015, and 62 / 214,602, filed September 4, 2015, the entire disclosures of each of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on October 8, 2015, is entitled 30864PCT_CRF_sequencelisting.txt and is 735,705 bytes in size. [Background technology]

[0003] Background of the Invention Primary oxaluria type 1 (PH1) is an autosomal recessive disorder of glyoxylate metabolism. Hepatic glyoxylate detoxification is prevented by mutations in the AGXT gene, which encodes the hepatic peroxisomal alanine-glyoxylate aminotransferase (AGT) enzyme. AGT1 is the final enzyme in the metabolic degradation of hydroxyproline. Loss of AGT function, which converts the intermediate metabolite glyoxylate to glycine, leads to the accumulation of glyoxylate, which is then reduced to glycolate, which is oxidized to oxalate by the enzyme glycolate oxidase (GO), also known as hydroxyacid oxidase (HAO1).

[0004] Regulation of glyoxylate, a key precursor of oxalate, occurs at multiple intracellular sites, including mitochondria, peroxisomes, and the cytosol. Excess oxalate in PH1 patients cannot be adequately excreted by the kidneys, leading to the formation and accumulation of calcium oxalate crystals in the kidneys and urinary tract. Renal damage results from a combination of oxalate-induced tubular toxicity, nephrocalcinosis, and kidney obstruction by stones. More than 30% of patients progress to end-stage renal disease (ESRD).

[0005] The HAO1 gene encodes the enzyme hydroxyacid oxidase 1, also known as glycolate oxidase ("GO"). The HAO1 protein is a 2-hydroxyacid oxidase that is expressed primarily in the liver and is most active towards glycolate.

[0006] In a mouse model of PH1 in which the AGT1 gene is deleted, urinary oxalate levels are reduced when the HAO1 gene is deleted.

[0007] PH1, AGXT, and HAO1 are described in Non-Patent Documents 1-4. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Angel L. Pey, Armando Albert, and Eduardo Salido, “Protein Homeostasis Defects of Alanine-Glyoxylate Aminotransferase: New Therapeutic Strategies in Primary Hyperoxaluria Type I,” BioMed Research International, vol. 2013, Article ID 687658,15 pages,2013.doi:10.1155 / 2013 / 687658 [Non-patent document 2] Cochat and Rumsby (2013) NEJM 369:7 [Non-patent document 3] Salido et al (2006) PNAS 103:18249 [Non-patent document 4] Baker et al(2004)American Journal of Physiology - Heart and Circulatory Physiology Published 1 October 2004Vol.287no.4,H1771-H1779DOI:10.1152 / ajpheart.00234.2004 Summary of the Invention [Means for solving the problem]

[0009] Summary of the Invention The invention provides compositions comprising RNAi agents, e.g., double-stranded iRNA agents, that target HAO1. The invention also provides methods of using the compositions of the invention to inhibit expression of HAO1 and to treat HAO1-associated diseases, e.g., PH1. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the nucleotide sequence of human (Homo sapiens) HAO1 mRNA (SEQ ID NO: 1). [Figure 2] FIG. 2 shows the nucleotide sequence of Mus musculus HAO1 mRNA (SEQ ID NO: 2). [Figure 3A] FIG. 3A is a graph showing the results of in vitro screening of GO(HAO)GalNac-siRNA conjugates in primary cynomolgus monkey hepatocytes. [Figure 3B] FIG. 3B is a graph showing the dose-response curve of GO(HAO)GalNac-siRNA conjugates in primary cynomolgus monkey hepatocytes. [Figure 4A]FIG. 4A is a graph showing the results of in vivo evaluation of GO(HAO)GalNac-siRNA conjugates in C57B6 mice after a single administration. [Figure 4B] FIG. 4B is a graph showing the results of in vivo evaluation of GO(HAO)GalNac-siRNA conjugates in C57B6 mice after a single administration. [Figure 5A] FIG. 5A is a graph showing urinary oxalate levels in AGXT knockout (KO) mice after treatment with GO(HAO)GalNac-siRNA conjugates. [Figure 5B] FIG. 5B is a graph showing urinary glycolate levels in AGXT KO mice after treatment with GO(HAO)GalNac-siRNA conjugates. [Figure 6A] FIG. 6A is a graph showing AGXT mRNA levels in a rat model of PH1 72 hours after a single administration of AGXT siRNA. [Figure 6B] FIG. 6B is a graph showing urinary oxalate levels in a rat model of PH1 72 hours after treatment with GO(HAO)GalNac-siRNA conjugates. [Figure 6C] FIG. 6C is a graph showing urinary oxalate levels in a rat model of PH1 after 49 days of continued weekly administration of both AF-011-63102 and AD-62994 on days 14 and 21 and 24-hour urine collection, as indicated. [Figure 6D] Figure 6D is a graph showing the duration of HAO1 knockdown in rats, with mRNA levels expressed relative to levels seen in PBS-treated rats 1 week or 4 weeks after the last of the four doses (corresponding to days 28 and 49 in Figure 6C). [Figure 7] FIG. 7 shows the reverse complement of the nucleotide sequence of human (Homo sapiens) HAO1 mRNA (SEQ ID NO: 3). [Figure 8]The reverse complement of the nucleotide sequence of Mus musculus HAO1 mRNA (SEQ ID NO: 4) is shown. [Figure 9] FIG. 9 shows the nucleotide sequence of cynomolgus monkey (Macaca fascicularis) HAO1 mRNA (SEQ ID NO: 5). [Figure 10] FIG. 10 shows the nucleotide sequence of the brown rat (Rattus norvegicus) HAO1 mRNA (SEQ ID NO: 6). [Figure 11] FIG. 11 shows the reverse complement of the nucleotide sequence of cynomolgus monkey (Macaca fascicularis) HAO1 mRNA (SEQ ID NO: 7). [Figure 12] FIG. 12 shows the reverse complement of the nucleotide sequence of Rattus norvegicus HAO1 mRNA (SEQ ID NO: 8). [Figure 13] FIG. 13 shows in vivo screening of GO GalNAc conjugates. [Figure 14] FIG. 14 is a graph showing in vivo evaluation of GO GalNAc conjugates in mice. [Figure 15] FIG. 15 is a graph showing dose-response assessment of GO GalNAc conjugates in mice. [Figure 16] FIG. 16 is a graph showing dose-response assessment of GO GalNAc conjugates in mice. [Figure 17] FIG. 17 is a graph showing dose-response assessment in mice. [Figure 18] FIG. 18 is two graphs showing the relationship between mRNA knockdown and serum glycolate levels in mice. [Figure 19] FIG. 19 is two graphs showing the relationship between mRNA knockdown and serum glycolate levels in rats. [Figure 20] FIG. 20 is a graph showing dose-dependent inhibition of HAO1 mRNA by ALN-65585 in primary cynomolgus monkey hepatocytes. [Figure 21] FIG. 21 is two graphs showing HAO1 mRNA and serum glycolate levels after a single dose treatment with ALN-GO1 in mice. [Figure 22] FIG. 22 is a graph showing the duration of HAO1 mRNA silencing after a single dose treatment with ALN-GO1 in mice. [Figure 23] FIG. 23 is a graph showing HAO1 mRNA and serum glycolate levels after a single dose treatment with ALN-GO1 in rats. [Figure 24] FIG. 24 shows two graphs showing urinary oxalate and glycolate levels in a mouse model of primary oxaluria type I after a single dose of ALN-GO1. [Figure 25A] FIG. 25A is a graph showing HAO1 mRNA levels in a rat model of primary oxaluria type I after a single administration of ALN-GO1. [Figure 25B] FIG. 25B is a graph showing urinary oxalate levels in a rat model of primary oxaluria type I after a single administration of ALN-GO1. [Figure 26] FIG. 26 is two graphs showing HAO1 mRNA and urinary oxalate levels in a rat model of primary oxaluria type I after repeated administration of ALN-GO1. [Figure 27] FIG. 27 is two graphs showing HAO1 mRNA and urinary glycolate levels after repeated administration in non-human primates. DETAILED DESCRIPTION OF THE INVENTION

[0011] Detailed Description of the Invention The present invention provides compositions comprising RNAi agents, e.g., double-stranded RNAi agents, that target HAO1. The present invention also provides methods of using the compositions of the present invention to inhibit the expression of HAO1 and to treat HAO1-related disorders.

[0012] I. Definition In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a numerical value or range of values ​​for a parameter is stated, intermediate values ​​and ranges to the stated values ​​are also intended to be part of the present invention.

[0013] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0014] The word "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0015] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless expressly stated to the contrary.

[0016] As used herein, "HAO1" refers to the gene encoding the enzyme hydroxyacid oxidase 1. Other gene names include GO, GOX, GOX1, and HAOX1. This protein is also known as glycolate oxidase and (S)-2-hydroxyacid oxidase. The GenBank accession numbers for human HAO1 mRNA are NM_017545.2; cynomolgus monkey (Macaca fascicularis) HAO1 mRNA is XM_005568381.1; house mouse (Mus musculus) HAO1 mRNA is NM_010403.2; and brown rat (Rattus norvegicus) HAO1 mRNA is XM_006235096.1.

[0017] As used herein, the term "HAO1" also refers to naturally occurring DNA sequence variants of the HAO1 gene, such as single nucleotide polymorphisms (SNPs) in the HAO1 gene. Exemplary SNPs can be found in the NCBI dbSNP Short Genetic Variations database, available at www.ncbi.nlm.nih.gov / projects / SNP.

[0018] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed upon transcription of the HAO1 gene, including mRNA that is the product of RNA processing of the primary transcript.

[0019] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising the strand of nucleotides represented by this sequence, referenced using standard nucleotide nomenclature.

[0020] "G," "C," "A," and "U" are generally abbreviations for nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. "T" and "dT" are used interchangeably herein to refer to a deoxyribonucleotide whose nucleobase is thymidine, e.g., deoxyribothymine, 2'-deoxythymidine, or thymidine. However, it should be understood that the terms "ribonucleotide," "nucleotide," or "deoxyribonucleotide" can also refer to modified nucleotides, or surrogate replacement moieties, as described in more detail below. Those skilled in the art will appreciate that guanine, cytosine, adenine, and uracil can be substituted with other moieties without substantially altering the base pairing properties of oligonucleotides containing nucleotides with such replacement moieties. For example, but not limited to, a nucleotide containing inosine as a base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine can be substituted in the nucleotide sequences of the invention by nucleotides containing, for example, inosine, and sequences containing such substituted moieties are embodiments of the invention.

[0021] The terms " iRNA ", " RNAi agent ", " iRNA agent ", " RNA interference agent " are used interchangeably herein and refer to an agent that comprises RNA as defined herein and mediates the target cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. iRNA induces the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates, for example, inhibits, the expression of HAO1 in cells, for example, target cells, for example, mammalian target cells.

[0022] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, for example, an HAO1 target mRNA sequence, to induce cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into short interfering RNAs of 19 to 23 base pairs, characterized by a 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 duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases in RISC cleave the target and induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect, the present invention relates to a single-stranded RNA (siRNA) that is produced in cells and promotes the formation of a RISC complex to silence a target gene, for example, the HAO1 gene. Thus, the term "siRNA" is also used herein to refer to the above-mentioned RNAi.

[0023] In another embodiment, the RNAi agent may be a single-stranded siRNA introduced into a cell or organ to inhibit the target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides long and chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, each of which is incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as single-stranded siRNAs as described herein or chemically modified by the methods described in Lima et al., (2012) Cell 150;:883-894.

[0024] In yet another embodiment, the present invention provides a single-stranded antisense oligonucleotide molecule targeting HAO1. A "single-stranded antisense oligonucleotide molecule" is complementary to a sequence within the target mRNA (i.e., HAO1). The single-stranded antisense oligonucleotide molecule can stoichiometrically inhibit translation by base pairing to the mRNA and physically interfering with the translation machinery. See Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. Alternatively, the single-stranded antisense oligonucleotide molecule inhibits the target mRNA by hybridizing to the target and cleaving the target via an RNase H cleavage event. The single-stranded antisense oligonucleotide molecule can be about 10 to about 30 nucleotides in length and has a sequence complementary to the target sequence. For example, a single-stranded antisense oligonucleotide molecule can comprise any one of the antisense nucleotide sequences described herein, e.g., a sequence that is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more consecutive nucleotides of a sequence set forth in any one of Tables 1 or 2, or can bind to any target site described herein. A single-stranded antisense oligonucleotide molecule can comprise modified RNA, DNA, or a combination thereof.

[0025] In another embodiment, the "iRNA" used in the compositions, uses, and methods of the present invention is double-stranded RNA, and is referred to herein as a "double-stranded RNAi agent," a "double-stranded RNA (dsRNA) molecule," a "dsRNA agent," or a "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands, which are oriented in "sense" and "antisense" directions relative to the target RNA, i.e., the HAO1 gene. In some embodiments of the present invention, the double-stranded RNA (dsRNA) causes degradation of the target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0026] Generally, the majority of the nucleotides in each strand of dsRNA molecule are ribonucleotides; however, as described in detail herein, each strand or both strands can also comprise one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides.In addition, " RNAi agent " as used herein can comprise ribonucleotides with chemical modification; RNAi agent can comprise substantial modification of multiple nucleotides.This modification can comprise any type of modification disclosed herein or known in the art.Any of these modifications used in siRNA type molecules are included in " RNAi agent " in this specification and claims.

[0027] The two strands forming the duplex structure may be different parts of a single larger RNA molecule, or they may be separate RNA molecules. When the two strands are part of a single larger molecule and are connected by a continuous chain of nucleotides between the 3' end of one strand and the 5' end of the other strand, the connecting RNA strand is called a "hairpin loop." When the two strands are covalently connected by means other than a continuous chain of nucleotides between the 3' end of one strand and the 5' end of the other strand, the connecting structure is called a "linker." The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, the RNAi agent may contain one or more nucleotide overhangs. The term "siRNA" as used herein also refers to the RNAi agent described above.

[0028] In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, such as an HAO1 target mRNA sequence, to induce cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a RNase III-like enzyme, processes dsRNA into short interfering RNAs of 19-23 base pairs, each characterized by a two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). Then, siRNA is incorporated into RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). When it binds to the appropriate target mRNA, one or more endonucleases in RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev.15:188).

[0029] As used herein, "nucleotide overhang" refers to a non-base-paired nucleotide or a nucleotide protruding from the duplex structure of an RNAi agent when the 3'-end of one strand of the RNAi agent extends beyond the 5'-end of the other strand, or vice versa. "Blunt" or "blunt-ended" means that there are no non-base-paired nucleotides at the end of a double-stranded RNAi agent, i.e., there are no nucleotide overhangs. A "blunt-ended" RNAi agent is a dsRNA that is double-stranded throughout its entire length, i.e., there are no nucleotide overhangs at either end of the molecule. The RNAi agents of the present invention include RNAi agents that have a nucleotide overhang at one end (i.e., one end is an overhang and the other end is blunt), or have nucleotide overhangs at both ends.

[0030] The term "antisense strand" refers to the strand of a double-stranded RNAi agent that comprises a region that is substantially complementary to a target sequence (e.g., human HAO1 mRNA). As used herein, the term "region complementary to a portion of the mRNA encoding HAO1" refers to a region of the antisense strand that is substantially complementary to a portion of the HAO1 mRNA sequence. When the region of complementarity is not completely complementary to the target sequence, mismatches are most tolerated in the terminal regions, and if present, are generally in the terminal regions, or within, for example, 6, 5, 4, 3, or 2 nucleotides of the 5' and / or 3' end.

[0031] As used herein, the term "sense strand" refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.

[0032] The term "cleavage region" as used herein refers to the region located immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises either end of the cleavage site and three bases immediately adjacent to the cleavage site. In some embodiments, the cleavage region comprises either end of the cleavage site and two bases immediately adjacent to the cleavage site. In some embodiments, the cleavage site is specifically located at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13.

[0033] Unless otherwise specified, the term "complementary" as used herein, when used to describe a first nucleotide sequence relative to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize to an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions to form a duplex structure, as understood by those skilled in the art. Such conditions can be, for example, stringent conditions, such as: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 o C or 70 oC for 12-16 hours, followed by washing. Other conditions, such as physiologically relevant conditions that may be encountered in an organism, can be applied. For example, complementary sequences are sufficient to perform the relevant function of the nucleic acid, e.g., RNAi. Those skilled in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences according to the ultimate application of the hybridized nucleotides.

[0034] When there is base pairing between the nucleotides of the first nucleotide sequence and the nucleotides of the second nucleotide sequence throughout the entire length of the first and second nucleotide sequences, the sequences can be "fully complementary" to each other.However, as used herein, when a first sequence is described as "substantially complementary" to a second sequence, the two sequences can be completely complementary, or the two sequences can form one or more mismatched base pairs, not more than four, three, or two, during hybridization, while maintaining the ability to hybridize under the most suitable conditions for the final application.However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, these overhangs should not be considered as mismatches for determining complementarity.For example, a dsRNA comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, where the longer oligonucleotide comprises a 21-nucleotide sequence that is completely complementary to the shorter oligonucleotide, can still be said to be "fully complementary" for the purposes described herein.

[0035] As used herein, a "complementary" sequence may also contain or be formed entirely of non-Watson-Crick base pairs and / or base pairs formed from non-naturally occurring modified nucleotides, provided that the above requirements for the hybridization ability of complementary sequences are met, including, but not limited to, G:U wobble base pairing or Hoogstein base pairing.

[0036] As used herein, the terms "complementary," "fully complementary," and "substantially complementary" can refer to base matches between the sense and antisense strands of a dsRNA, or base matches between the antisense strand of a dsRNA and a target sequence, as understood from the context of their use.

[0037] As used herein, a polynucleotide "substantially complementary to at least a portion" of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding HAO1), including the 5' UTR, open reading frame (ORF), or 3' UTR. For example, a polynucleotide is complementary to at least a portion of an HAO1 mRNA if its sequence is substantially complementary to a contiguous portion of an mRNA encoding HAO1.

[0038] As used herein, the term "inhibit" is used interchangeably with "reduce," "silencing," "downregulate," "suppress," and other similar terms, and includes all levels of inhibition.

[0039] As used herein, the phrase "inhibiting expression of HAO1" includes inhibiting the expression of any HAO1 gene (e.g., mouse HAO1 gene, rat HAO1 gene, monkey HAO1 gene, or human HAO1 gene, etc.) and variants (e.g., naturally occurring variants) or mutants of the HAO1 gene. Thus, the HAO1 gene may be a wild-type HAO1 gene, a mutant HAO1 gene, or a recombinant HAO1 gene in the context of a genetically engineered cell, cell population, or organism.

[0040] "Inhibiting expression of the HAO1 gene" includes any level of inhibition of the HAO1 gene, for example, at least partial suppression of expression of the HAO1 gene, for example, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% inhibition.

[0041] The expression of the HAO1 gene can be evaluated based on the level of any variable associated with the expression of the HAO1 gene, such as the level of HAO1 mRNA or HAO1 protein in tissues, and / or the level of urinary oxalate. Inhibition can be evaluated by a decrease in the absolute or relative level of one or more of these variables compared to the control level. The control level can be any type of control level available in the art, such as a pre-drug baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (e.g., a buffer-only control or an inactive agent control).

[0042] As used herein, the phrase "contacting double-stranded RNAi agent with cell" includes contacting cell by any possible means.Contacting double-stranded RNAi agent with cell includes contacting RNAi agent with cell in vitro or contacting RNAi agent with cell in vivo.Such contact can be carried out directly or indirectly.Therefore, for example, RNAi agent can be physically contacted with cell by the person who carries out the method, or RNAi agent can be placed in a situation that allows or allows contact with cell later.

[0043] In vitro contact of cells can be achieved, for example, by incubating cells with an RNAi agent. In vivo contact of cells can be achieved, for example, by injecting an RNAi agent into the tissue where the cells are located or nearby, or by injecting an RNAi agent into another region, the bloodstream, or subcutaneous space, so that the RNAi agent can subsequently reach the tissue where the contacted cells are located. For example, the RNAi agent can contain and / or bind to a ligand, such as a GalNAc3 ligand, that directs the RNAi agent to the target site, for example, the liver. A combination of in vitro and in vivo contact methods is also possible. In connection with the method of the present invention, cells can be contacted with an RNAi agent in vitro and then transplanted into a subject.

[0044] As used herein, "subject" includes humans or non-human animals, preferably vertebrates, more preferably mammals.Subjects may include transgenic organisms.Most preferably, subjects are humans, for example, humans suffering from or susceptible to developing HAO1-related disorders.

[0045] As used herein, a "patient" or "subject" is intended to include a human or non-human animal, preferably a mammal, e.g., a human or a monkey. Most preferably, the subject or patient is a human.

[0046] As used herein, "HAO1-associated disorder" is intended to include any disorder that can be treated or prevented, or symptoms that can be alleviated, by inhibiting the expression of HAO1. Examples include, but are not limited to, primary oxaluria type 1 (PH1).

[0047] As used herein, a "therapeutically effective amount" is intended to include the amount of an RNAi agent that, when administered to a patient to treat an HAO1-associated disease, is sufficient to treat the disease (e.g., reduce, ameliorate, or maintain an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" may vary depending on the RNAi agent, the method by which the RNAi agent is administered, the disease and its severity and medical history, age, weight, family history, genetic makeup, the stage of the pathological process mediated by HAO1 expression, type of previous or concomitant therapy (if any), and other personal characteristics of the patient being treated.

[0048] As used herein, a "prophylactically effective amount" includes an amount of an RNAi agent sufficient to prevent or ameliorate an HAO1-associated disease or one or more of its symptoms when administered to a patient who does not have or exhibits symptoms of the disease but who may develop the disease. Ameliorating the disease includes delaying the progression of the disease or reducing the severity of subsequent disease development. A "prophylactically effective amount" may vary depending on the RNAi agent, the method by which the RNAi agent is administered, the degree of risk of the disease, and the patient's medical history, age, weight, family history, genetic makeup, type of previous or concurrent therapy (if any), and other personal characteristics of the patient being treated.

[0049] A "therapeutically effective amount" or "prophylactically effective amount" also includes that amount of an RNAi agent that provides some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agents utilized in the methods of the invention can be administered in amounts sufficient to provide a reasonable benefit / risk ratio applicable to such treatment.

[0050] As used herein, the term "sample" includes similar fluid, cell, or tissue collections isolated from a subject, as well as fluids, cells, or tissues present within a subject's body. Examples of biological fluids include blood, serum, and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, and saliva. Tissue samples can include samples from tissues, organs, or localized regions. For example, samples can be derived from specific organs, parts of organs, or fluids or cells within these organs. In certain embodiments, samples can be derived from the liver (e.g., the whole liver, a specific part of the liver, or a specific type of liver cell, e.g., hepatocytes). In some embodiments, a "sample derived from a subject" refers to blood or plasma taken from a subject. In further embodiments, a "sample derived from a subject" refers to liver tissue (or a subcomponent thereof) from a subject.

[0051] II. dsRNA iRNA Agents of the Invention Described herein are double-stranded RNAi agents that inhibit expression of the HAO1 gene in cells, e.g., cells of a subject, e.g., a mammal, e.g., a human having an HAO1-associated disorder, and uses of such double-stranded RNAi agents.

[0052] Thus, the present invention provides double-stranded RNAi agents having chemical modifications that are capable of inhibiting the expression of a target gene (ie, the HAO1 gene) in vivo.

[0053] As described in more detail below, in certain aspects of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA of the invention are modified. An iRNA of the invention in which "substantially all of the nucleotides are modified" may contain a majority, but not all, modified nucleotides, and may contain no more than five, no more than four, no more than three, no more than two, or no more than one unmodified nucleotide.

[0054] RNAi agents include a sense strand and an antisense strand. Each strand of an RNAi agent can be in the range of 12 to 30 nucleotides in length. For example, each strand can be 14 to 30 nucleotides in length, 17 to 30 nucleotides in length, 19 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length.

[0055] Each strand can be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. Each strand of an RNAi agent can be the same length or different lengths.

[0056] The sense and antisense strands typically form a duplex, double-stranded RNA (dsRNA), also referred to herein as an RNAi agent. The duplex region of an RNAi agent can be 12 to 30 nucleotide pairs in length. For example, the duplex region can be 14 to 30 nucleotide pairs in length, 17 to 30 nucleotide pairs in length, 27 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 21 nucleotide pairs in length, 17 to 19 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, 21 to 25 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length. In another example, the duplex region is selected from 15 nucleotide pairs in length, 16 nucleotide pairs in length, 17 nucleotide pairs in length, 18 nucleotide pairs in length, 19 nucleotide pairs in length, 20 nucleotide pairs in length, 21 nucleotide pairs in length, 22 nucleotide pairs in length, 23 nucleotide pairs in length, 24 nucleotide pairs in length, 25 nucleotide pairs in length, 26 nucleotide pairs in length, and 27 nucleotide pairs in length.

[0057] In one embodiment, an RNAi agent can include one or more overhang regions and / or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs can be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides in length. The overhangs can occur when one strand is longer than the other, or when two strands of the same length are offset. The overhangs can form mismatches with the target mRNA, or the overhangs can be complementary to the targeted gene sequence, or they can be a different sequence. The first and second strands can be linked, for example, by additional bases to form a hairpin, or they can be linked by other non-basic linkers.

[0058] In one embodiment, each nucleotide in the overhang region of an RNAi agent can be independently modified or unmodified, including, but not limited to, 2'-sugar modifications such as 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT can be the overhang sequence at either end of either strand. The overhang can form a mismatch with the target mRNA, or the overhang can be complementary to the targeted gene sequence, or it can be a different sequence.

[0059] The 5' or 3' overhang on the sense strand, the antisense strand, or both strands of an RNAi agent may be phosphorylated. In some embodiments, the overhang region comprises two nucleotides with a phosphorothioate between them, which may be the same or different nucleotides. In one embodiment, the overhang is present at the 3' end of the sense strand, the antisense strand, or both strands. In one embodiment, the 3' overhang is present on the antisense strand. In one embodiment, the 3' overhang is present on the sense strand.

[0060] An RNAi agent can have only one overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, the single-stranded overhang can be located at the 3'-end of the sense strand or the 3'-end of the antisense strand.RNAi can also have a blunt end located at the 5'-end of the antisense strand (or the 3'-end of the sense strand) (or vice versa).Generally, the antisense strand of RNAi has a nucleotide overhang at the 3'-end and is blunt at the 5'-end.Without wishing to be bound by theory, the asymmetric blunt end at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand favor the guide strand added in the RISC process.

[0061] Synthesis and Modification Any nucleic acid featured in the present invention, e.g., RNAi, 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, SLet et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, e.g., 5'-end modifications (phosphorylation, conjugation, reverse linkage) or 3'-end modifications (conjugation, DNA nucleotide, reverse linkage, etc.); base modifications, e.g., substitution with stabilizing bases, destabilizing bases, or bases that base pair with a wider range of partners, base removal (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, including modifications or substitutions of phosphodiester bonds; and / or backbone modifications. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or non-natural internucleoside linkages. The RNA having a modified backbone particularly includes the RNA that does not have a phosphorus atom in the backbone.In this specification, as sometimes referred to in the art, the modified RNA that does not have a phosphorus atom in its internucleoside backbone can also be considered as an oligonucleoside.In some embodiments, the modified iRNA has a phosphorus atom in its internucleoside backbone.

[0062] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with linear 3'-5' linkages, their 2'-5' linked analogs, and those with opposite polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0063] Representative United States patents that teach the formation of the above phosphorus-containing bonds include, but are not limited to, U.S. Pat. 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 ... Specification No. 78,302; Specification No. 5,286,717; Specification No. 5,321,131; Specification No. 5,399,676; Specification No. 5,405,939; Specification No. 5,453,496; Specification No. 5,455,233 Specification; Specification No. 5,466,677; Specification No. 5,476,925; Specification No. 5,519,126; Specification No. 5,536,821; Specification No. 5,541,316; Specification No. 5,550,111; Specification No. 5,5 Specification No. 63,253; Specification No. 5,571,799; Specification No. 5,587,361; Specification No. 5,625,050; Specification No. 6,028,188; Specification No. 6,124,445; Specification No. 6,160,109 Specification; Specification No. 6,169,170; Specification No. 6,172,209; Specification No. 6,239,265; Specification No. 6,277,603; Specification No. 6,326,199; Specification No. 6,346,614; Specification No. 6,4 Nos. 44,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Re. Pat. No. 39464.

[0064] Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These modified RNA backbones include backbones with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones with mixed N, O, S, and CH2 moieties.

[0065] Representative United States patents that teach the formation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,46 Nos. 6,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

[0066] In other embodiments, suitable RNA mimics are intended for use in iRNA. When used in this manner, both the sugar and internucleoside linkages, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are maintained and are linked directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the formation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire disclosures of each of which are incorporated herein by reference. Further PNA compounds suitable for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0067] Some embodiments featured herein include RNAs with phosphorothioate backbones, as well as oligonucleosides with heteroatom backbones, particularly -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- (known as methylene(methylimino) or MMI backbones), -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and N(CH3)-CH2-CH2- (natural phosphodiester backbones are represented as -OPO-CH2-) of the above-referenced U.S. Patent No. 5,489,677, and amide backbones of the above-referenced U.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Patent No. 5,034,506.

[0068] Modified RNAs can also contain one or more substituted sugar moieties. iRNAs, e.g., dsRNAs, featured in the present invention can contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl and alkynyl. Exemplary suitable modifications include O[(CH) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA includes one of the following at the 2' position: C1 to C 10The modifications include lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group for improving the pharmacological properties of iRNA, or group for improving the pharmacological properties of iRNA, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., O(CH2)2ON(CH3)2 group, 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, as described in the Examples herein below.

[0069] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in the RNA of an iRNA, particularly the 3' position of the sugar of the 3'-terminal nucleotide or 2'-5'-linked dsRNA, and the 5' position of the 5'-terminal nucleotide. An iRNA can also have, for example, a sugar mimic, such as a cyclobutyl moiety, in place of the pentofuranosyl sugar. Representative United States patents that teach the formation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; Nos. 7,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, some of which are owned by the present applicant, the entire disclosures of each of which are incorporated herein by reference.

[0070] iRNAs may also contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as deoxythymine (dT), 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, 6-azouracil, and 6-azouracil. Cytosine, and 6-azothymine, 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-deazaadenine, and 3-deazaguanine and 3-deazaadenine.Additional nucleobases include the nucleobases disclosed in US Patent No. 3,687,808, the nucleobases disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P.ed. Wiley-VCH, 2008; the nucleobases disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990; the nucleobases disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and the nucleobases disclosed in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993.These specific nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T., and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are further exemplary base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0071] Representative United States patents that teach the formation of the above-mentioned specific and other modified nucleobases include, but are not limited to, the above-mentioned U.S. Patent Nos. 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; and 5,552,540, the disclosures of each of which are incorporated herein by reference in their entirety. ; Specification No. 5,587,469; Specification No. 5,594,121; Specification No. 5,596,091; Specification No. 5,614,617; Specification No. 5,681,9 Specification No. 41; Specification No. 5,750,692; Specification No. 6,015,886; Specification No. 6,147,200; Specification No. 6,166,197; Specification No. 6, Nos. 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.

[0072] The RNA of iRNA can also be modified to contain one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with modified ribose moieties, which contain an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose into a 3' internal conformation. The addition of locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum 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).

[0073] Representative United States patents that teach the formation of locked nucleic acid nucleotides include, but are not limited to, U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the entire disclosures of each of which are incorporated herein by reference.

[0074] Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, and inverted base dT (idT), among others. Disclosure of this modification can be found in PCT Publication WO 2011 / 005861.

[0075] Modified iRNAs containing the motifs of the present invention In certain embodiments of the invention, double-stranded RNAi agents of the invention include agents having chemical modifications as disclosed, for example, in U.S. Provisional Patent Application No. 61 / 561,710, filed November 18, 2011, or International Application No. PCT / US2012 / 065691, filed November 16, 2012, published as WO 2013075035 A1, the entire disclosures of each of which are incorporated herein by reference.

[0076] As shown herein and in US Provisional Patent Application No. 61 / 561,710, excellent results can be obtained by introducing one or more motifs of three identical modifications of three consecutive nucleotides into the sense strand and / or antisense strand of RNAi agent, particularly at or near the cleavage site.In some embodiments, the sense strand and antisense strand of RNAi agent can be completely modified by other methods.By introducing these motifs, if present, the modification pattern of the sense strand and / or antisense strand can be interrupted.Optionally, RNAi agent can be conjugated with a GalNAc derivative ligand, for example, in the sense strand.The obtained RNAi agent shows excellent gene silencing activity.

[0077] More particularly, it has surprisingly been found that the gene silencing activity of an RNAi agent is maximally enhanced when the sense and antisense strands of the double-stranded RNAi agent are modified to have one or more motifs of three identical modifications of three consecutive nucleotides at or near the cleavage site of at least one strand of the RNAi agent.

[0078] In one embodiment, the RNAi agent is a 19-nucleotide double-ended bluntmer, wherein the sense strand contains at least one motif consisting of three 2'-F modifications at three consecutive nucleotides at the 5'-terminus: positions 7, 8, and 9. The antisense strand contains at least one motif consisting of three 2'-O-methyl modifications at three consecutive nucleotides at the 5'-terminus: positions 11, 12, and 13.

[0079] In another embodiment, the RNAi agent is a 20 nucleotide long blunt-ended duplex, wherein the sense strand contains at least one motif consisting of three 2'-F modifications at three consecutive nucleotides at the 5' end, positions 8, 9, and 10. The antisense strand contains at least one motif consisting of three 2'-O-methyl modifications at three consecutive nucleotides at the 5' end, positions 11, 12, and 13.

[0080] In another embodiment, the RNAi agent is a blunt-ended duplex 21 nucleotides in length, wherein the sense strand contains at least one motif consisting of three 2'-F modifications at three consecutive nucleotides at the 5'-terminus: positions 9, 10, and 11. The antisense strand contains at least one motif consisting of three 2'-O-methyl modifications at three consecutive nucleotides at the 5'-terminus: positions 11, 12, and 13.

[0081] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif consisting of three 2'-F modifications of three consecutive nucleotides at positions 9, 10, and 11 of the 5'-end; the antisense strand comprises at least one motif consisting of three 2'-O-methyl modifications of three consecutive nucleotides at positions 11, 12, and 13 of the 5'-end, and one end of the RNAi agent is blunt, while the other end comprises a 2-nucleotide overhang.Preferably, the 2-nucleotide overhang is at the 3'-end of the antisense strand.When the 2-nucleotide overhang is at the 3'-end of the antisense strand, there may be two phosphorothioate internucleotide bonds between the three nucleotides at the end, and two of the three nucleotides are overhanging nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further comprises two phosphorothioate internucleotide bonds between the three nucleotides at the end of both the 5'-end of the sense strand and the 5'-end of the antisense strand.In one embodiment, each nucleotide of the sense strand and the antisense strand of the RNAi agent is a modified nucleotide, including the nucleotide that is part of a motif.In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in an alternating motif.Optionally, the RNAi agent further comprises a ligand (preferably GalNAc3).

[0082] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the RNAi agent comprising a first strand at least 25 and at most 29 nucleotides in length, and a second strand at most 30 nucleotides in length having at least one motif consisting of three 2'-O-methyl modifications at three consecutive nucleotides located at positions 11, 12, and 13 from the 5' end; the 3' end of the first strand and the 5' end of the second strand form a blunt end, the second strand is 1-4 nucleotides longer at the 3' end than the first strand, the duplex region is at least 25 nucleotides in length, the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotides in length of the second strand such that the RNAi agent reduces expression of the target gene when introduced into a mammalian cell, and Dicer cleavage of the RNAi agent results in an siRNA that preferentially includes the 3' end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a ligand.

[0083] In one embodiment, the sense strand of the RNAi agent contains at least one motif consisting of three identical modifications in three consecutive nucleotides, one of the motifs being at the cleavage site of the sense strand.

[0084] In one embodiment, the antisense strand of the RNAi agent may also contain at least one motif consisting of three identical modifications in three consecutive nucleotides, one of the motifs being at or near the cleavage site on the antisense strand.

[0085] In RNAi agents having a duplex region of 17 to 23 nucleotides in length, the cleavage site of the antisense strand is typically near positions 10, 11, and 12 from the 5' end. Thus, motifs consisting of three identical modifications may be present at positions 9, 10, and 11; 10, 11, and 12; 11, 12, and 13; 12, 13, and 14; and 13, 14, and 15 of the antisense strand, counting from the first nucleotide from the 5' end of the antisense strand or from the first paired nucleotide in the duplex region at the 5' end of the antisense strand. The cleavage site of the antisense strand may also vary depending on the length of the duplex region of the 5' end of the RNAi agent.

[0086] The sense strand of RNAi agent can comprise at least one motif consisting of three identical modifications in three consecutive nucleotides at the cleavage site of its strand; and the antisense strand can have at least one motif consisting of three identical modifications in three consecutive nucleotides at or near the cleavage site of its strand.When sense strand and antisense strand form a dsRNA duplex, sense strand and antisense strand can be aligned so that one motif consisting of three nucleotides in sense strand and one motif consisting of three nucleotides in antisense strand overlap by at least one nucleotide, that is, at least one of the three nucleotides in the motif of sense strand and at least one of the three nucleotides in the motif of antisense strand form base pairs.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0087] In one embodiment, the sense strand of an RNAi agent can contain two or more motifs consisting of three identical modifications of three consecutive nucleotides. The first motif can be located at or near the cleavage site of the sense strand, and the other motif can be a wing modification. As used herein, the term "wing modification" refers to a motif located in another portion of the same strand, away from the motif at or near the cleavage site of the strand. The wing modifications can be located near the first motif or separated by at least one or more nucleotides. If the motifs are closer to each other than their chemical nature, the motifs are different from each other; if the motifs are separated by one or more nucleotides than their chemical nature, the motifs can be the same or different. Two or more wing modifications can be present. For example, if two wing modifications are present, each wing modification can be located at one end of the first motif at or near the cleavage site, or on either side of the lead motif.

[0088] Similar to the sense strand, the antisense strand of the RNAi agent can comprise two or more motifs consisting of three identical modifications of three consecutive nucleotides, at least one of which is present at or near the cleavage site of the antisense strand.The antisense strand can also comprise one or more wing modifications with the same sequence as the wing modifications that can be present in the sense strand.

[0089] In one embodiment, wing modifications of the sense or antisense strand of an RNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0090] In another embodiment, wing modifications of the sense or antisense strand of an RNAi agent typically do not include the first one or two base-pairing nucleotides in the duplex region at the 3' end, 5' end, or both ends of the strand.

[0091] When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modifications may be at the same end of the duplex region and have an overlap of 1, 2, or 3 nucleotides.

[0092] When the sense and antisense strands of an RNAi agent each comprise at least two wing modifications, the sense and antisense strands can be aligned such that: two modifications on one strand are located at one end of the duplex region and overlap by one, two, or three nucleotides; two modifications on one strand are located at the other end of the duplex region and overlap by one, two, or three nucleotides; and two modifications on one strand are located on either side of the lead motif and overlap the duplex region by one, two, or three nucleotides.

[0093] In one embodiment, each nucleotide in the sense and antisense strands of an RNAi agent, including nucleotides that are part of a motif, can be modified. Each nucleotide can be modified with the same or different modifications, which can include one or more changes to one or both of the unlinked phosphate oxygen and / or one or more linking phosphate oxygens; changes to components of the ribose sugar, such as the 2' hydroxyl of the ribose sugar; bulk replacement of the phosphate moiety with a "dephospho" linker; modifications or substitutions of natural bases; and substitutions or modifications of the ribose-phosphate backbone.

[0094] Because nucleic acids are polymers of subunits, many modifications, such as modifications of bases, phosphate moieties, or non-linked Os in phosphate moieties, occur at repeated positions within nucleic acids. In some cases, modifications occur at all desired positions within a nucleic acid, but often not. For example, modifications may occur only at the 3' or 5' terminal positions, or only in terminal regions, such as terminal nucleotide positions or the last two, three, four, five, or ten nucleotides of a chain. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in double-stranded regions of an RNA or only in single-stranded regions of an RNA. For example, phosphorothioate modifications at non-linked oxygen positions may occur only at one or both ends, or only in terminal regions, such as terminal nucleotide positions or the last two, three, four, five, or ten nucleotides of a chain, or in both double-stranded and single-stranded regions, especially at the ends. The 5' or both ends may be phosphorylated.

[0095] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, e.g., the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, e.g., with the modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar using modifications known in the art, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F), or 2'-O-methyl modifications rather than ribosugars in the nucleobase, and modifications of the phosphate group, e.g., phosphothioate modifications. The overhang need not be homologous to the target sequence.

[0096] In one embodiment, each residue of sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl or 2'-fluoro.Sense strand and antisense strand can contain two or more modifications.In one embodiment, each residue of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0097] At least two different modifications are typically present in the sense and antisense strands, and these two modifications may be 2'-O-methyl or 2'-fluoro, or others.

[0098] In one embodiment, N a and / or N b includes an alternating pattern of modifications. As used herein, the term "alternating motif" refers to a motif having one or more modifications, with each modification occurring at alternating nucleotides in a strand. The alternating nucleotides may refer to one at every other nucleotide, one at every third nucleotide, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif could be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0099] The types of modifications included in the alternating motifs can be the same or different. For example, if A, B, C, and D each represent one type of modification of a nucleotide, the alternation pattern, i.e., the modifications of every other nucleotide, can be the same, but the sense or antisense strand can each choose from several possibilities for modifications within the alternating motif, e.g., "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0100] In one embodiment, an RNAi agent of the present invention comprises an alternating motif modification pattern in the sense strand that is shifted relative to the alternating motif modification pattern in the antisense strand. This shift can be such that a modification group on a nucleotide in the sense strand corresponds to a different modification group on a nucleotide in the antisense strand, or vice versa. For example, when the sense strand base-pairs with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can begin with "ABABAB" from 5' to 3' of the sense strand, and the alternating motif in the antisense strand can begin with "BABABA" from 5' to 3' of the antisense strand within the duplex region. As another example, the alternating motif in the sense strand can begin with "AABBAABB" from 5' to 3' of the sense strand, and the alternating motif in the antisense strand can begin with "BBAABBAA" from 5' to 3' of the antisense strand within the duplex region, resulting in a complete or partial shift in the modification pattern between the sense and antisense strands.

[0101] In one embodiment, the RNAi agent initially has a pattern of alternating 2'-O-methyl and 2'-F modifications in the sense strand, and this pattern initially has a shift with respect to the pattern of alternating 2'-O-methyl and 2'-F modifications in the antisense strand, i.e., the 2'-O-methyl modified nucleotides in the sense strand form base pairs with the 2'-F modified nucleotides in the antisense strand, and vice versa.Position 1 of the sense strand can start with 2'-F modification, and position 1 of the antisense strand can start with 2'-O-methyl modification.

[0102] The introduction of one or more motifs consisting of three identical modifications at three consecutive nucleotides into the sense strand and / or antisense strand disrupts the original modification pattern present in the sense strand and / or antisense strand. This disruption of the original modification pattern present in the sense strand and / or antisense strand by the introduction of one or more motifs consisting of three identical modifications at three consecutive nucleotides into the sense strand and / or antisense strand surprisingly enhances gene silencing activity against the target gene.

[0103] In one embodiment, when a motif consisting of three identical modifications at three consecutive nucleotides is introduced in either strand, the modification of the nucleotide next to the motif is a different modification than the modification of the motif. For example, a portion of a sequence containing a motif may be represented by "...N a YYYN b ...', where 'Y' represents a motif modification consisting of three identical modifications in three consecutive nucleotides, and 'N a " and "N b " represents a modification of the next nucleotide of the motif "YYY" that is different from the modification of Y, and N a and N b may be the same or different modifications, or N a and / or N b may or may not be present if wing modifications are present.

[0104] RNAi agent can further comprise at least one phosphorothioate or methylphosphonate internucleotide bond.The phosphorothioate or methylphosphonate internucleotide bond modification can be present at any nucleotide in sense strand and / or antisense strand, or at any position of both strands.For example, internucleotide bond modification can be present at any nucleotide in sense strand and antisense strand; each internucleotide bond modification can be present in an alternating pattern in sense strand and / or antisense strand; or sense strand or antisense strand can comprise both internucleotide bond modifications in an alternating pattern.The alternating pattern of internucleotide bond modification in sense strand can be the same or different from that of antisense strand, and the alternating pattern of internucleotide bond modification in sense strand can have a shift relative to the alternating pattern of internucleotide bond modification in antisense strand.

[0105] In one embodiment, the RNAi agent comprises a phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region. For example, the overhang region can comprise two nucleotides with a phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides. The internucleotide bond modification can also be formed to allow the overhang nucleotide to be bound to the terminal base-pairing nucleotide in the duplex region. For example, at least two, three, four, or all of the overhang nucleotides can be bound by a phosphorothioate or methylphosphonate internucleotide bond, and optionally, there can be an additional phosphorothioate or methylphosphonate internucleotide bond that connects the overhang nucleotide to the next base-pairing nucleotide. For example, there can be at least two phosphothioate internucleotide bonds between the terminal three nucleotides, two of which are overhanging nucleotides, and the third nucleotide is the next base-pairing nucleotide of the overhanging nucleotide. These terminal three nucleotides can be the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and / or the 5' end of the antisense strand.

[0106] In one embodiment, the 2-nucleotide overhang is at the 3'-end of the antisense strand, and there are two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of the three nucleotides are overhanging nucleotides, and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide.Optionally, the RNAi agent can further have two phosphorothioate internucleotide bonds between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.

[0107] In one embodiment, the RNAi agent comprises mismatches with the target, mismatches within the duplex, or a combination thereof. Mismatches can occur in overhang regions or duplex regions. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., based on the free energy of binding or dissociation of a particular pairing; the simplest approach is to evaluate base pairs on an individual base pair basis, but affinity or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical pairings or non-canonical pairings (described elsewhere herein), are preferred over canonical pairings (A:T, A:U, G:C); and base pairs containing universal bases are preferred over canonical pairings.

[0108] In one embodiment, the RNAi agent comprises at least one of the first one, two, three, four, or five base pairs in the duplex region from the 5' end of the antisense strand selected from the group of A:U, G:U, I:C, and a mismatch pairing, e.g., a non-canonical or non-canonical pairing, or a pairing including a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0109] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of antisense strand is selected from the group consisting of A, dA, dU, U and dT.Alternatively, at least one of the first one, two or three base pairs in the double-stranded region from the 5' end of antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of antisense strand is an AU base pair.

[0110] In one embodiment, the sense strand sequence can be represented by formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -Na -n q 3' (I) During the ceremony, i and j are each independently 0 or 1; p and q are each independently 0 to 6; each N a independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b independently represent an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p and n q independently represent overhanging nucleotides; Nb and Y do not have the same modification; and XXX, YYY, and ZZZ each independently represent a motif consisting of three identical modifications in three consecutive nucleotides. Preferably, YYY is a 2'-F modified nucleotide.

[0111] In one embodiment, N a and / or N b includes alternating pattern modifications.

[0112] In one embodiment, the YYY motif is located at or near the cleavage site of the sense strand. For example, if the RNAi agent has a duplex region of 17 to 23 nucleotides in length, the YYY motif can be located at or near the cleavage site of the sense strand (e.g., at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13), counting from the first nucleotide from the 5' end; or optionally, counting from the first base-pairing nucleotide in the duplex region from the 5' end.

[0113] In one embodiment, i is 1 and j is 0, i is 0 and j is 1, or both i and j are 1. Thus, the sense strand can be represented by the formula: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' (Ib) 5'n p -N a -XXX-N b -YYY-N a -n q 3' (Ic); or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' (Id).

[0114] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0115] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-5, 0-4, 0-2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0116] When the sense strand is represented by formula (Id), each N b independently represent an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5, or 6. Each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0117] Each of X, Y, and Z may be the same as or different from one another.

[0118] In other embodiments, i is 0, j is 0, and the sense strand can be represented by the formula: 5'n p -N a -YYY-N a -n q 3' (Ia).

[0119] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0120] In one embodiment, the antisense strand sequence of an RNAi agent can be 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) During the ceremony, k and l are each independently 0 or 1; p' and q' are each independently 0 to 6; each N a ' independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p ' and n q ' independently represents an overhanging nucleotide; N b ' and Y' do not have the same modification; and X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a motif consisting of three identical modifications in three consecutive nucleotides.

[0121] In one embodiment, N a ' and / or N b ' includes alternating pattern modifications.

[0122] The Y'Y'Y' motif is located at or near the cleavage site of the antisense strand. For example, the RNAi agent has a double-stranded region of 17-23 nucleotides in length, and the Y'Y'Y' motif can be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide from the 5' end; or optionally, counting from the first base-pairing nucleotide in the double-stranded region from the 5' end.Preferably, the Y'Y'Y' motif is located at positions 11, 12, 13.

[0123] In one embodiment, the Y'Y'Y' motif is a 2'-OMe modified nucleotide.

[0124] In one embodiment, k is 1 and l is 0, k is 0 and l is l, or both k and l are 1.

[0125] Thus, the antisense strand can be represented by the 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).

[0126] When the antisense strand is represented by formula (IIb), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0127] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0128] When the antisense strand is represented by formula (IId), each N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5, or 6.

[0129] In other embodiments, k is 0, 1 is 0, and the antisense strand can be represented by the formula: 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3' (Ia).

[0130] When the antisense strand is represented by formula (IIa), each N a' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0131] Each of X', Y', and Z' may be the same as or different from one another.

[0132] Each nucleotide of sense strand and antisense strand can be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl or 2'-fluoro.For example, each nucleotide of sense strand and antisense strand can be independently modified with 2'-O-methyl or 2'-fluoro.Particularly, X, Y, Z, X', Y' and Z' can respectively represent 2'-O-methyl modification or 2'-fluoro modification.

[0133] In one embodiment, the sense strand of the RNAi agent can include a YYY motif at positions 9, 10, and 11 of the sense strand when the duplex region is 21 nucleotides, counting from the first nucleotide from the 5' end, or optionally counting from the first base-pairing nucleotide in the duplex region from the 5' end; and Y represents a 2'-F modification. The sense strand can additionally include a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region; and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0134] In one embodiment, the antisense strand can include a Y'Y'Y' motif at positions 11, 12, and 13 of the sense strand, counting from the first nucleotide from the 5' end, or optionally counting from the first base-pairing nucleotide in the duplex region from the 5' end; and Y' represents a 2'-O-methyl modification. The antisense strand can additionally include an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the duplex region; and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0135] Each of the sense strands represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with an antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId).

[0136] Thus, the RNAi agent used in the methods of the invention can include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi duplex can be represented by 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) During the ceremony: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each N a and N a ' independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p ',n p , n q ', and n q each may be present or absent and independently represent an overhanging nucleotide; and XXX, YYY, ZZZ, X'X'X', Y'Y'Y', Z'Z'Z' each independently represent a motif consisting of three identical modifications of three consecutive nucleotides.

[0137] 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 both i and j are 0; or both i and j are 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 both k and l are 0; or both k and l are 1.

[0138] Exemplary combinations of sense and antisense strands that form RNAi duplexes include the following formulas: 5'n p -N a -YYY-N a -n q 3' 3'n p '-N a '-Y'Y'Y'-N a 'n q '5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'np '-N a '-Y'Y'Y'-N b '-Z'Z'Z'-N a 'n q '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'-N a '-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'-N a -n q '5' (IIId)

[0139] When the RNAi agent is represented by formula (IIIa), each N a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0140] When the RNAi agent is represented by formula (IIIb), each N b independently represent an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0141] When the RNAi agent is represented by formula (IIIc), each N b , Nb ' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0142] When the RNAi agent is represented by formula (IIId), each N b , N b ' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b , N b ' independently includes alternating pattern modifications.

[0143] In formulae (III), (IIIa), (IIIb), (IIIc), and (IIId), X, Y, and Z may be the same as or different from each other.

[0144] When the RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides can be base-paired with one of the Y' nucleotides, alternatively, at least two of the Y nucleotides are base-paired with the corresponding Y' nucleotide; or all three of the Y nucleotides are base-paired with the corresponding Y' nucleotide.

[0145] When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can be base-paired with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides are base-paired with the corresponding Z' nucleotide; or all three of the Z nucleotides are base-paired with the corresponding Z' nucleotide.

[0146] When the RNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides can be base-paired with one of the X' nucleotides, or at least two of the X nucleotides can be base-paired with the corresponding X' nucleotide; or all three of the X nucleotides can be base-paired with the corresponding X' nucleotide.

[0147] In one embodiment, the modification of a Y nucleotide is different from the modification of a Y' nucleotide, the modification of a Z nucleotide is different from the modification of a Z' nucleotide, and / or the modification of an X nucleotide is different from the modification of an X' nucleotide.

[0148] In one embodiment, when the RNAi agent has formula (IIId), N a The modification is a 2'-O-methyl modification or a 2'-fluoro modification. In another embodiment, when the RNAi agent has formula (IIId), N a The modification is a 2'-O-methyl modification or a 2'-fluoro modification, p '>0 and at least one n p In yet another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to the adjacent nucleotide by a phosphorothioate bond. a The modification is a 2'-O-methyl modification or a 2'-fluoro modification, p '>0 and at least one n p In another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to adjacent nucleotides by phosphorothioate bonds, and the sense strand is conjugated to one or more GalNAc derivatives attached by a bivalent or trivalent branched linker. aThe modification is a 2'-O-methyl modification or a 2'-fluoro modification, p '>0 and at least one n p ' is linked to adjacent nucleotides by phosphorothioate bonds, the sense strand comprises at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives attached by a bivalent or trivalent branched linker.

[0149] In one embodiment, when the RNAi agent has formula (IIIa), N a The modification is a 2'-O-methyl modification or a 2'-fluoro modification, p '>0 and at least one n p ' is linked to adjacent nucleotides by phosphorothioate bonds, the sense strand comprises at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives attached by a bivalent or trivalent branched linker.

[0150] In one embodiment, the RNAi agent is a multimer comprising at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each duplex may target the same gene or two different genes; or each duplex may target the same gene at two different target sites.

[0151] In some embodiments, the RNAi agent is a multimer comprising three, four, five, six, or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each duplex may target the same gene or two different genes; or each duplex may target the same gene at two different target sites.

[0152] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at their 5' ends, and one or both of their 3' ends are optionally conjugated with a ligand. The RNAi agents may each target the same gene or two different genes; alternatively, the RNAi agents may each target the same gene at two different target sites.

[0153] A variety of publications describe multimeric RNAi agent, and can be used in the method of the present invention.Such publications include WO2007 / 091269, United States Patent (USP) 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, each of which is incorporated herein by reference in its entirety.

[0154] RNAi agents containing one or more sugar moieties conjugated to the RNAi agent can optimize one or more properties of the RNAi agent. Often, the sugar moiety is attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, such as a non-carbohydrate carrier (preferably cyclic), to which a sugar ligand is attached. Ribonucleotide subunits in which the ribose sugar of the subunit has been replaced in this manner are referred to herein as ribose-replacement modified subunits (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., a ring system in which all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., a ring system in which one or more ring atoms can be a heteroatom, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, e.g., fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0155] The ligand can be attached to the polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tether attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally to a bond available and suitable for incorporation of the carrier into the backbone of a ribonucleic acid, e.g., a phosphate backbone, or, e.g., a sulfur-containing modified phosphate backbone. In some embodiments, a "tether attachment point" (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. The selected moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is connected to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain functional groups, such as amino groups, or generally allow for attachment suitable for incorporation or tethering of another chemical entity, such as a ligand, to the constituent ring.

[0156] The RNAi agent can be conjugated to the ligand by a carrier, which is a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0157] In certain embodiments, the RNAi agent used in the methods of the invention is an agent selected from the group of agents listed in either Table 1 or Table 2. In one embodiment, when the agent is an agent listed in Table 1, the agent may lack a terminal dT.

[0158] The present invention further includes double-stranded RNAi agents comprising any one of the sequences listed in either Table 1 or Table 2, which comprise a 5' phosphate or phosphate mimetic in the antisense strand (see, e.g., PCT Publication WO 2011005860). Additionally, the present invention includes double-stranded RNAi agents comprising any one of the sequences listed in either Table 1 or Table 2, which comprise a 2' fluoro group in place of a 2'-OMe group at the 5' end of the sense strand.

[0159] Further motifs In certain embodiments, the double-stranded RNAi agents described herein comprise a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise fewer than 11, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, or fewer than 5 2'-deoxyfluoro.

[0160] In certain embodiments, the double-stranded RNAi agents described herein comprise a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, or fewer than 4 phosphorothioate internucleotide linkages.

[0161] In certain embodiments, the double-stranded RNAi agents described herein comprise a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise fewer than 10 2'-deoxyfluoro and fewer than 6 phosphorothioate internucleotide linkages.

[0162] In certain embodiments, the double-stranded RNAi agents described herein comprise a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise fewer than 8 2'-deoxyfluoro and fewer than 6 phosphorothioate internucleotide linkages.

[0163] In certain embodiments, the double-stranded RNAi agents described herein comprise a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise fewer than 9 2'-deoxyfluoro and fewer than 6 phosphorothioate internucleotide linkages.

[0164] Ligand The double-stranded RNAi agent of the present invention can optionally be conjugated to one or more ligands.Ligand can be attached to sense strand, antisense strand, or both strands at 3'-end, 5'-end, or both ends.For example, ligand can be conjugated to sense strand.In some embodiments, ligand is conjugated to 3'-end of sense strand.In one embodiment, ligand is GalNAc ligand.In some particular embodiments, ligand is GalNAc3.This ligand is directly or indirectly bound through intervening tether, preferably covalently bound.

[0165] In some embodiments, a ligand alters the distribution, targeting, or lifetime of a molecule into which it is incorporated. In some embodiments, a ligand increases affinity for a selected target, e.g., a molecule, a cell or cell type, a compartment, a receptor, e.g., a cell or organ compartment, a tissue, an organ, or a region of the body, e.g., compared to a species in which such ligand is absent. Ligands that increase affinity for a selected target are also referred to as targeting ligands.

[0166] Some ligands may have endosomolytic properties. Endosomolytic ligands promote the lysis of endosomes and / or the transport of the compositions of the present invention or their components from endosomes to the cytoplasm of cells. Endosomolytic ligands may be polyanionic peptides or peptidomimetics that exhibit pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand is presumed to adopt its active conformation at endosomal pH. An "active" conformation is a conformation in which the endosomolytic ligand promotes the lysis of endosomes and / or the transport of the compositions of the present invention or their components from endosomes to the cytoplasm of cells. Exemplary endosomolytic ligands include GALA peptide (Subbarao et al., Biochemistry, 1987, 26:2964-2972), EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118:1581-1586), and derivatives thereof (Turk et al., Biochem. Biophys. Acta, 2002, 1559:56-68). In one embodiment, the endosomolytic component may contain a chemical group (e.g., an amino acid) that undergoes a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

[0167] The ligands can improve the transport, hybridization, and specificity properties, and can also improve the nuclease resistance of the resulting natural or modified oligoribonucleotides, or polymer molecules comprising any combination of the monomers and / or natural or modified ribonucleotides described herein.

[0168] Ligands generally may include therapeutic modifiers, e.g., to enhance uptake; diagnostic compounds or reporter groups, e.g., to monitor distribution; cross-linking agents; and moieties that confer nuclease resistance. Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics.

[0169] Ligands can include naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids, or oligonucleotides (e.g., aptamers). Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolized) copolymer, divinyl ether maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0170] The ligand can also include a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid, or protein, e.g., an antibody, that binds to a specific cell type, such as a kidney cell. The targeting group can be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine, multivalent mannose, multivalent fucose, glycosylated polyamino acid, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, biotin, RGD peptide, RGD peptidomimetic, or aptamer.

[0171] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelating agents (e.g., EDTA), lipophilic molecules, such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-( oleoyl), cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0172] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules with specific affinity for a co-ligand, or antibodies, e.g., antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. Ligands can also include non-peptide species, e.g., lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, or aptamers. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-κB.

[0173] The ligand can be a substance, e.g., a drug, that can increase cellular uptake of an iRNA agent, e.g., by disrupting the cytoskeleton of a cell, e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0174] Ligands can increase cellular uptake of oligonucleotides, for example, by activating an inflammatory response. Exemplary ligands that can have such an effect include tumor necrosis factor alpha (TNFα), interleukin-1β, or gamma interferon.

[0175] In one embodiment, the ligand is a lipid or lipid-based molecule.Such lipid or lipid-based molecule preferably binds to serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows conjugate to be distributed to target tissue, for example, non-renal target tissue of the body.For example, target tissue can be the liver, including liver parenchymal cells.Other molecules that can bind to HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can (a) increase the resistance of conjugate to degradation, (b) increase targeting or transport to target cell or cell membrane, and / or (c) be used to regulate the binding to serum protein, for example, HSA.

[0176] Lipid-based ligand can be used to regulate (for example, control) the binding of conjugate to target tissue.For example, the lipid or lipid-based ligand that binds more strongly to HSA is less likely to target kidney, and therefore is less likely to be removed from body.The lipid or lipid-based ligand that binds less strongly to HSA can be used to make conjugate target kidney.

[0177] In one embodiment, the lipid-based ligand binds to HSA. Preferably, the lipid-based ligand binds to HSA with sufficient affinity so that the conjugate is preferably distributed in non-renal tissues. In one embodiment, the affinity is such that the HSA-ligand bond can be reversed. In another embodiment, the lipid-based ligand binds weakly or not at all to HSA, so that the conjugate is preferably distributed in the kidney. Other moieties that target kidney cells can also be used instead of or in addition to the lipid-based ligand.

[0178] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, e.g., proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HAS, low-density lipoprotein (LDL), and high-density lipoprotein (HDL).

[0179] In another embodiment, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide, such as tat or antennopedia. When the agent is a peptide, modifications can be made, including peptidyl mimetics, invertomers, non-peptide or pseudo-peptide bonds, and the use of D-amino acids. Preferably, the helical cell-penetrating agent is an alpha-helical agent, preferably having a lipophilic phase and a lipophobic phase.

[0180] The ligand may be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule that can fold into a defined three-dimensional structure similar to a natural peptide. The peptide or peptidomimetic portion may be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. The peptide or peptidomimetic may be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide portion may be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide portion may contain a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). A hydrophobic MTS-containing RFGF analog (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) can also be a targeting moiety. The peptide moiety can also be a "delivery" peptide, which can transport large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, the sequence (GRKKRRQRRRPPQ) (SEQ ID NO: 11) from the HIV Tat protein and the sequence (RQIKIWFQNRRMKWKK) (SEQ ID NO: 12) from the Drosophila Antennapedia protein have been shown to function as delivery peptides. Peptides or peptidomimetics, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries, can also be encoded by random sequences of DNA (Lam et al., Nature, 354:82-84, 1991). Preferably, the peptide or peptidomimetic linked to the iRNA agent via the incorporated monomer unit is a cell-targeting peptide, e.g., an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimetic. The peptide portion can range from about 5 amino acids in length to about 40 amino acids in length.The peptide moiety can have a structural change, for example, to enhance stability or direct conformational properties. Any of the following structural changes can be utilized: RGD peptide moieties can be used to target tumor cells, such as endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can promote targeting of iRNA agents to tumors in various other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, RGD peptides promote targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic and can be modified, e.g., glycosylated or methylated, to promote targeting to specific tissues. For example, glycosylated RGD peptides can be α-. v iRNA agents can be delivered to tumor cells expressing β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001). Peptides targeting markers abundant in proliferating cells can be used. For example, RGD-containing peptides and RGD-containing peptidomimetics can target cancer cells, particularly cells that display integrins. Thus, RGD peptides, cyclic peptides containing RGD, RGD peptides containing D-amino acids, and synthetic RGD mimetics can be used. In addition to RGD, other moieties that target integrin ligands can also be used. Generally, such ligands can be used to control proliferating cells and angiogenesis. Some conjugates of this type of ligand target PECAM-1, VEGF, or other oncogenes, such as those described herein.

[0181] A "cell-penetrating peptide" can penetrate cells, such as microbial cells, e.g., bacterial or fungal cells, or mammalian cells, e.g., human cells. Peptides that penetrate microbial cells can be, for example, α-helical linear peptides (e.g., LL-37 or seropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two predominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bipartite amphipathic peptide, e.g., MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0182] In one embodiment, the targeting peptide may be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, esculentinis-1, and caerin. Preferably, multiple factors are considered to maintain the integrity of helix stability. For example, utilize a maximum number of helix-stabilizing residues (e.g., leu, ala, or lys) and a minimum number of helix-destabilizing residues (e.g., proline, or cyclic monomer units). Capping residues are also considered (e.g., Gly is an exemplary N-capping residue, and / or C-terminal amidation can be used to provide additional hydrogen bonds to stabilize the helix). Stabilization can be achieved by the formation of salt bridges between oppositely charged residues spaced at positions i±3, or i±4. Cationic residues, such as lysine, arginine, homo-arginine, ornithine, or histidine, can form salt bridges with the anionic residues glutamic acid or aspartic acid.

[0183] Peptide and peptidomimetic ligands include ligands having natural or modified peptides, such as D- or L-peptides; alpha, beta, or gamma peptides; N-methyl peptides; azapeptides; peptides having one or more amide bonds, i.e., peptidic bonds, replaced by one or more urea, thiourea, carbamate, or sulfonylurea bonds; or cyclic peptides.

[0184] The targeting ligand may be any ligand capable of targeting a specific receptor. Examples include folate, GalNAc, galactose, mannose, mannose-6P, glycoclusters such as GalNAc clusters, mannose clusters, galactose clusters, or aptamers. A cluster is a combination of two or more glycoconjugates. Targeting ligands also include integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL ligands, and HDL ligands. The ligand may be based on a nucleic acid, for example, an aptamer. The aptamer may be unmodified or may have any combination of modifications disclosed herein.

[0185] Endosomal release agents include imidazoles, poly- or oligoimidazoles, PEI, peptides, fusogenic peptides, polycaboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketyals, orthoesters, polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges.

[0186] PK modulators refer to pharmacokinetic modulators. PK modulators include lipophilics, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides containing multiple phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides containing multiple phosphorothioate linkages in the backbone, for example, oligonucleotides of about 5, 10, 15, or 20 bases, are also applicable as ligands (e.g., as PK-modulating ligands) in the present invention.

[0187] In addition, aptamers that bind to serum components (eg, serum proteins) are also applicable to the present invention as PK-regulating ligands.

[0188] Other ligand conjugates applicable to the present invention are described in U.S. patent application Ser. Nos. 10 / 916,185, filed Aug. 10, 2004; 10 / 946,873, filed Sep. 21, 2004; 10 / 833,934, filed Aug. 3, 2007; 11 / 115,989, filed Apr. 27, 2005; and 11 / 944,227, filed Nov. 21, 2007, the entire disclosures of which are incorporated herein by reference.

[0189] When two or more ligands are present, the ligands may all have the same properties, all may have different properties, or some ligands may have the same properties while others have different properties. For example, the ligands may have targeting properties, endosomal activity, or PK modulating properties. In one embodiment, all of the ligands have different properties.

[0190] The ligand can be attached to the oligonucleotide at various positions, e.g., the 3'-terminus, the 5'-terminus, and / or an internal position. In some embodiments, the ligand is attached to the oligonucleotide via an intervening tether, e.g., a carrier described herein. The ligand or tethered ligand may be present on the monomer when the monomer is incorporated into a growing chain. In some embodiments, the ligand can be incorporated by attachment to a "precursor" monomer after the "precursor" monomer has been incorporated into a growing chain. For example, an amino-terminated tether (i.e., no ligand attached), e.g., TAP-(CH2) n Monomers bearing an NH can be incorporated into a growing oligonucleotide chain. Subsequent to this, i.e., after the precursor monomer is incorporated into the chain, a ligand bearing an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can then be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the tether of the precursor monomer.

[0191] In another example, monomers bearing chemical groups suitable for participating in click chemistry reactions can be incorporated into tethers / linkers, for example, azide- or alkyne-terminated tethers / linkers. Subsequent to this, i.e., after the precursor monomers are incorporated into the chain, a ligand bearing a complementary chemical group, e.g., an alkyne or azide, can be attached to the precursor monomer by linking the alkyne and azide together.

[0192] In some embodiments, the ligand can be conjugated to the nucleobase, sugar moiety, or internucleoside linkage of a nucleic acid molecule. Conjugation to a purine nucleobase or its derivative can occur at any position, including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of a purine nucleobase is bound to a conjugate moiety. Conjugation to a pyrimidine nucleobase or its derivative can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a conjugate moiety. Conjugation to a sugar moiety of a nucleoside can occur at any carbon atom. Examples of carbon atoms of the sugar moiety that can be bound to a conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be bound to a conjugate moiety, for example, an abasic residue. The internucleoside linkage can also carry a conjugate moiety. In the case of phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, and phosphoramidate), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. In the case of internucleoside linkages containing amines or amides (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0193] GalNAc Ligands and Linkers In some embodiments, siRNA targeting HAO1 gene is conjugated to carbohydrate, for example, monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, polysaccharide. In some embodiments, siRNA is conjugated to N-acetylgalactosamine (GalNAc) ligand. This promotes efficient delivery to hepatocytes after subcutaneous administration. Methods for conjugating carbohydrate, for example, N-acetylgalactosamine, to siRNA, for example, are well known to those skilled in the art. Examples can be found in U.S. Patent No. 8,106,022 and WO 2014 / 025805.

[0194] In some embodiments, the siRNA targeting the HAO1 gene is conjugated to a ligand, such as GalNAc, via a linker. For example, the ligand can be one or more GalNAc (N-acetylglucosamine) derivatives attached by a bivalent or trivalent branched linker.

[0195] In one embodiment, the dsRNA of the invention is conjugated to bivalent and trivalent branched linkers comprising the structure shown in any of the following formulas (V) to (VII): [ka] During the ceremony, q 2A , q 2B , q 3A , q 3B , q4 A , q 4B , q 5A , q 5B , and q 5C represents independently for each occurrence 0 to 20, and the repeat units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C represents, independently for each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q5A , Q 5B , Q 5C represents independently at each occurrence absent, alkylene, or substituted alkylene, and one or more methylenes are O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C, or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C are independently present in each occurrence: NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B , and L 5C represents a ligand; i.e., each occurrence independently represents a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R a is H or an amino acid side chain.

[0196] Trivalent conjugated GalNAc derivatives, such as those of formula (VII), are particularly useful in conjunction with RNAi agents to inhibit expression of target genes: [ka] In the formula, L 5A , L 5B , and L 5Crepresents a monosaccharide, for example, a GalNAc derivative. Suitable bivalent and trivalent branched linker group conjugated GalNAc derivatives include, but are not limited to, the following compounds:

[0197] Suitable bivalent and trivalent branched linker group conjugated GalNAc derivatives include, but are not limited to, the following compounds: [ka] [ka] [ka]

[0198] Further Ligands In some embodiments, the ligand is selected from one of the following: [ka] [ka]

[0199] III. Delivery of iRNA of the Invention Delivery of an iRNA agent of the invention to a cell, e.g., a cell of a subject, e.g., a human subject (e.g., a subject in need of delivery of an iRNA agent, e.g., a subject with an HAO1-associated disorder), can be achieved in a variety of ways. For example, delivery can be achieved by contacting an iRNA of the invention with a cell in vitro or in vivo. In vivo delivery can also be achieved directly by administering a composition containing an iRNA, e.g., a dsRNA, to the subject. Alternatively, in vivo delivery can be achieved indirectly by administering one or more vectors that encode the iRNA and induce expression of the iRNA. These alternatives are further described below.

[0200] Generally, any method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNAs of the present invention (see, e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and WO 94 / 02595, the entire disclosures of each of which are incorporated herein by reference). For in vivo delivery, factors to consider for delivering iRNA molecules include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, e.g., direct injection or implantation into tissue, or by local administration of a formulation. Local administration at the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may otherwise be damaged or degrade the agent, and can reduce the total dose of iRNA molecules to be administered. Several studies have demonstrated successful knockdown of gene products when iRNAs are administered locally. For example, intraocular delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ., et al (2004) Retina 24:132-138) and subretinal injection in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) has been shown to prevent neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice can reduce tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and prolong 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 be delivered locally to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P.H., et al. (2005) Gene Ther. 12:59-66; Makimura, H., et al. (2002) BMC Neurosci. 3:18; Shishkina, G.T., et al. (2004) Neuroscience 129:521-528; Thakker, E.R., et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602), and to the lungs by intranasal administration (Howard, K.A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55) have also shown success. When administering iRNA systemically for disease treatment, the RNA can be modified or delivered using a drug delivery system; both of these methods act to prevent rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of the RNA or pharmaceutical carrier can enable targeting of iRNA compositions to target tissues while also avoiding undesired off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups, such as cholesterol, to promote cellular uptake and prevent degradation. For example, systemic injection of ApoB-directed iRNA 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). Binding of iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J.O., et al. (2006) Nat. Biotechnol. 24:1005-1015).In an alternative embodiment, iRNA can be delivered using a drug delivery system, such as a nanoparticle, dendrimer, polymer, liposome, or cationic delivery system. Positively charged cation delivery systems promote binding of iRNA molecules (which are negatively charged) and also promote interaction with the negatively charged cell membrane, allowing for efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can bind to iRNA or be induced to form vesicles or micelles that surround iRNA (see, for example, Kim SH., et al. (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents degradation of iRNA when administered systemically. Methods for forming and administering cation-iRNA complexes are well within the capabilities of those skilled in the art (see, e.g., Sorensen, D.R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U.N., et al. (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A.S. et al. (2007) J. Hypertens. 25:197-205, the entire disclosures of each of which are incorporated herein by reference).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, D.R., et al (2003), supra; Verma, U.N., et al (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T.S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P.Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M.E., et al (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D. A., et al. (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al. (1999) Pharm. Res. 16:1799-1804). In some embodiments, the iRNA is complexed with cyclodextrin for systemic administration. Methods for administering iRNA and cyclodextrin and pharmaceutical compositions can be found in U.S. Pat. No. 7,427,605, the entire disclosure of which is incorporated herein by reference.

[0201] Vectors encoding the iRNAs of the present invention iRNAs targeting the HAO1 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, for example, Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., PCT International Publication No. 00 / 22113; Conrad, PCT International Publication No. 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (ranging from a few hours to a few weeks) or persistent (weeks to months or longer) depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrative or non-integrative. Transgenes can also be constructed to allow for inheritance as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0202] Individual strands or multiple strands of iRNA can be transcribed from the promoter of expression vector.For example, when two separate strands are expressed to form dsRNA, two separate expression vectors can be simultaneously introduced into target cells (for example, by transfection or injection).Alternatively, each individual strand of dsRNA can be transcribed by the promoters located in the same expression plasmid.In one embodiment, dsRNA is expressed as an inverted repeat polynucleotide linked by a linker polynucleotide sequence, so that this dsRNA has a stem and loop structure.

[0203] iRNA expression vectors are generally DNA plasmids or viral vectors.Recombinant constructs for expressing the iRNAs described herein can be prepared using expression vectors compatible with eukaryotic cells, preferably expression vectors compatible with vertebrate cells.Eukaryotic cell expression vectors are well known in the art and are available from numerous commercial sources.Typically, such vectors are provided with convenient restriction sites for the insertion of desired nucleic acid segments.The iRNA expression vector can be delivered systemically, for example, by intravenous or intramuscular administration, by administration to target cells removed from a patient and then reintroduced into the patient, or by other means that allow introduction into desired target cells.

[0204] iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdown targeting different regions of a target RNA over a period of one week or more are also contemplated by the present invention. Successful introduction of the vector into host cells can be monitored using various known methods. For example, transient transfection can be signaled by a reporter, such as a fluorescent marker, e.g., green fluorescent protein (GFP). Stable transfection of cells ex vivo can be confirmed using markers that provide transfected cells with resistance to certain environmental factors (e.g., antibiotics and drugs), e.g., hygromycin B resistance.

[0205] Viral vector systems that can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including, but not limited to, lentiviral vectors, Moloney murine leukemia virus, and the like; (c) adeno-associated viral vectors; (d) herpes simplex viral vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopoxviruses, e.g., vaccinia virus vectors, or avipox, e.g., canarypox or fowlpox; and (j) helper-dependent or gutless adenoviruses. Replication-deficient viruses may also be advantageous. Various vectors may or may not integrate into the cellular genome. The constructs may contain viral sequences for transfection, as desired. Alternatively, the constructs may be incorporated into vectors that allow episomal replication, e.g., EPV and EBV vectors. Constructs for recombinant expression of iRNA generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure expression of the iRNA in target cells. Other aspects of vectors and constructs to consider are further described below.

[0206] Vectors useful for delivery of iRNA contain sufficient regulatory elements (promoters, enhancers, etc.) for expression of the iRNA in the desired target cells or tissues. Regulatory elements can be selected to provide constitutive or regulated / inducible expression.

[0207] The expression of iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to specific physiological regulators, such as circulating blood glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24).Such inducible expression systems suitable for regulating dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, dimerization of chemical inducers, and regulation by isopropyl-β-D1-thiogalactopyranoside (IPTG).Those skilled in the art will be able to select an appropriate regulatory / promoter sequence based on the intended use of the iRNA transgene.

[0208] Viral vectors containing nucleic acid sequences encoding iRNAs can be used. For example, retroviral vectors can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for accurate packaging of the viral genome and integration into host cell DNA. The nucleic acid sequences encoding iRNAs are cloned into one or more vectors, which facilitates delivery of the nucleic acid to patients. Further details about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the delivery of the mdr1 gene to hematopoietic stem cells using retroviral vectors to enhance stem cell resistance to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy include: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV-based vectors described in U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, which are incorporated herein by reference.

[0209] Adenoviruses are also contemplated for use in delivering iRNAs of the present invention. Adenoviruses are particularly attractive vehicles for delivering genes to respiratory epithelia, for example. Adenoviruses naturally infect respiratory epithelia, causing mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), provide an overview of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994), demonstrated gene transfer to the respiratory epithelia of rhesus monkeys using adenovirus vectors. Other examples of the use of adenovirus in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT International Publication No. WO 94 / 12649; and Wang et al., Gene Therapy 2:775-783 (1995). Suitable AV vectors for expressing iRNAs featured in the present invention, 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.

[0210] Adeno-associated virus (AAV) vectors can also be used to deliver iRNAs of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No. 5,436,146). In one embodiment, the iRNAs can 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 dsRNA featured in the present invention, 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. WO 94 / 13788; and International Patent Application Publication No. WO 93 / 24641, the entire disclosures of each of which are incorporated herein by reference.

[0211] Another viral vector suitable for delivery of the iRNA of the invention is a poxvirus, e.g., a vaccinia virus, e.g., an attenuated vaccinia, e.g., Modified Virus Ankara (MVA) or NYVAC, an avipox, e.g., canarypox or fowlpox.

[0212] The tropism of viral vectors can be changed by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by replacing various viral capsid proteins.For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, and Mokola, etc.AAV vectors can be engineered to target various cells by engineering vectors that express various capsid protein serotypes: see, for example, Rabinowitz JE et al.(2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0213] The vector pharmaceutical preparation can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which form the gene delivery system.

[0214] IV. Pharmaceutical Compositions of the Present Invention The present invention also includes pharmaceutical compositions and pharmaceutical preparations comprising the iRNA of the present invention. In one embodiment, a pharmaceutical composition comprising the iRNA described herein and a pharmaceutically acceptable carrier is provided herein. Pharmaceutical compositions comprising iRNA are useful for treating HAO1-related diseases or disorders. Such pharmaceutical compositions can be formulated based on the mode of delivery.

[0215] The pharmaceutical composition comprising the RNAi agent of the present invention can be, for example, a solution with or without buffer, or a composition comprising pharmaceutically acceptable carrier.Such compositions include, for example, water-soluble or crystalline compositions, liposome preparations, micelle preparations, emulsions, and gene therapy vectors.

[0216] In the methods of the present invention, the RNAi agent can be administered dissolved in a solution. The free RNAi agent can be administered dissolved in a non-buffered solution, such as saline or water. Alternatively, the free siRNA can be administered dissolved in a suitable buffer. The buffer can include acetate, citrate, prolamin, carbonate, phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS). The pH and osmolality of the buffer containing the RNAi agent can be adjusted to be suitable for administration to a subject.

[0217] In some embodiments, the buffer solution further comprises an agent for controlling the osmolality of the solution so that the osmolality is maintained at a desired value, for example, the physiological value of human plasma. Solutes that can be added to the buffer solution to control the osmolality include, but are not limited to, proteins, peptides, amino acids, non-metabolizable polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the agent for controlling the osmolality of the solution is a salt. In certain embodiments, the agent for controlling the osmolality of the solution is sodium chloride or potassium chloride.

[0218] The pharmaceutical compositions of the present invention can be administered in a dose sufficient to inhibit the expression of the HAO1 gene.

[0219] dose In general, suitable doses of iRNAs of the present invention range from about 0.001 to about 200.0 mg per kg of recipient body weight per day, and generally range from about 0.1 to 10 mg or 1 to 50 mg per kg of body weight per day. For example, dsRNAs can be administered at about 0.01 mg / kg, about 0.05 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg per administration.

[0220] In another embodiment, the RNAi agent, e.g., dsRNA, is administered at a concentration of about 0.1 to about 50 mg / kg, about 0.25 to about 50 mg / kg, about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / mg, about 1.5 to about 50 mg / kb, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 36 to about 50 mg / kg, about 37 to about 50 mg / kg, about 38 to about 50 mg / kg, about 39 to about 50 mg / kg, about 40 to about 50 mg / kg, about 41 to about 50 mg / kg, about 42 to about 50 mg / kg, about 43 to about 50 mg / kg, about 44 to about 50 mg / kg, about 45 to about 50 mg / kg, about 46 to about 50 mg / kg, about 47 to about 50 mg / kg, about 48 to about 50 mg / kg, about 49 to about 50 mg / kg, about 50 to about 50 mg / kg, about 51 to about 51 mg / kg, about 52 to about 52 mg / kg, about 53 to about 53 mg / 0mg / kg, about 20 to about 50mg / kg, about 25 to about 50mg / kg, about 25 to about 50mg / kg, about 30 to about 50mg / kg, about 35 to about 50mg / kg, about 40 to about 50mg / kg, about 45 to about 50mg / kg, about 0.1 to about 45mg / kg, about 0.25 to about 45mg / kg, about 0.5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / mg, about 1.5 to about 45 mg / kb, about 2 to about 45 mg / kg, about 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 4 5mg / kg, about 35 to about 45mg / kg, about 40 to about 45mg / kg, about 0.1 to about 40mg / kg, about 0.25 to about 40mg / kg, about 0.5 to about 40mg / kg, about 0.75 to about 40mg / kg, about 1 to about 40mg / mg, about 1.5 to about 40mg / kb, about 2 to about 40m g / kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, About 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg, about 0.1 to about 30 mg / kg, about 0.25 to about 30 mg / kg, about 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30mg / kg, about 5 to about 30mg / kg, about 7.5 to about 30mg / kg, about 10 to about 30mg / kg, about 15 to about 30mg / kg, about 20 to about 30mg / kg, about 20 to about 30mg / kg, about 25 to about 30mg / kg, about 0.1 to about 20m g / kg, about 0.25 to about 20 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / kg, about 1.5 to about 20 mg / kg, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Intermediate values ​​and ranges to the recited values ​​are also intended to be part of the present invention.

[0221] Whereas RNAi concentration, dsRNA is 0.01mg / kg, 0.02mg / kg, 0.03mg / kg, 0.04mg / kg .05mg / kg、0.06mg / kg、0.07mg / kg、0.08mg / kg、0.09mg / kg、0.1mg / kg、0.2m g / kg、0.3mg / kg、0.4mg / kg、0.5mg / kg、0.6mg / kg、0.7mg / kg、0.8mg / kg、0.9 mg / kg、1.0mg / kg、1.1mg / kg、1.2mg / kg、1.3mg / kg、1.4mg / kg、1.5mg / kg、1. 6mg / kg、1.7mg / kg、1.8mg / kg、1.9mg / kg、2.0mg / kg、2.1mg / kg、2.2mg / kg、2 .3mg / kg、2.4mg / kg、2.5mg / kg、2.6mg / kg、2.7mg / kg、2.8mg / kg、2.9mg / kg、 3.0mg / kg、3.1mg / kg、3.2mg / kg、3.3mg / kg、3.4mg / kg、3.5mg / kg、3.6mg / kg 、3.7mg / kg、3.8mg / kg、3.9mg / kg、4.0mg / kg、4.1mg / kg、4.2mg / kg、4.3mg / kg 、4.4mg / kg、4.5mg / kg、4.6mg / kg、4.7mg / kg、4.8mg / kg、4.9mg / kg、5.0mg / kg g、5.1mg / kg、5.2mg / kg、5.3mg / kg、5.4mg / kg、5.5mg / kg、5.6mg / kg、5.7mg / kg、5.8mg / kg、5.9mg / kg、6.0mg / kg、6.1mg / kg、6.2mg / kg、6.3mg / kg、6.4mg / kg、6.5mg / kg、6.6mg / kg、6.7mg / kg、6.8mg / kg、6.9mg / kg、7.0mg / kg、7.1mg / kg、7.2mg / kg、7.3mg / kg、7.4mg / kg、7.5mg / kg、7.6mg / kg、7.7mg / kg、7.8m g / kg、7.9mg / kg、8.0mg / kg、8.1mg / kg、8.2mg / kg、8.3mg / kg、8.4mg / kg、8.5 mg / kg、8.6mg / kg、8.7mg / kg、8.8mg / kg、8.9mg / kg、9.0mg / kg、9.1mg / kg、9. 2mg / kg、9.3mg / kg、9.4mg / kg、9.5mg / kg、9.6mg / kg、9.7mg / kg、9.8mg / kg、9.9mg / kg, 10mg / kg, 10.5mg / kg, 11mg / kg, 11.5mg / kg, 12mg / kg, 12.5mg / kg, 13mg / kg, 13.5mg / kg, 14mg / kg, 14.5mg / kg, 15mg / kg, 15.5mg / kg, 16mg / kg, 16.5mg / kg, 17mg / kg , 17.5mg / kg, 18mg / kg, 18.5mg / kg, 19mg / kg, 19.5mg / kg, 20mg / kg, 20.5mg / kg, 21mg / kg, 21.5mg / kg, 22mg / kg, 22.5mg / kg, 23mg / kg, 23.5mg / kg, 24mg / kg, 24.5mg / kg, 25m The compound may be administered at a dose of about 25.5 mg / kg, 26 mg / kg, 26.5 mg / kg, 27 mg / kg, 27.5 mg / kg, 28 mg / kg, 28.5 mg / kg, 29 mg / kg, 29.5 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, or about 50 mg / kg. Intermediate values ​​and ranges to the recited values ​​are also intended to be part of the invention.

[0222] In certain embodiments of the invention, for example, when a double-stranded RNAi agent comprises a modification (e.g., one or more motifs consisting of three identical modifications of three consecutive nucleotides, with one such motif at or near the cleavage site of the agent), six phosphorothioate linkages, and a ligand, such an agent may be administered at a concentration of 0.01-0.5 mg / kg, about 0.01-0.4 mg / kg, about 0.01-0.3 mg / kg, about 0.01-0.2 mg / kg, about 0.01-0.1 mg / kg, about 0.01 mg / kg to 0.09 mg / kg, or about 0.01 mg / kg. g / kg to 0.08mg / kg, approximately 0.01mg / kg to 0.07mg / kg, approximately 0.01mg / kg to 0.06mg / kg, approximately 0.01mg / kg to 0.05mg / kg, approximately 0.02 to 0.5mg / kg, approximately 0.02 to 0.4mg / kg, approximately 0.02 to 0.3mg / kg, approximately 0.02 to 0.2mg / kg, approximately 0.02 to 0.1mg / kg, approximately 0.02mg / kg to 0.09mg / kg, approximately 0.02mg / kg to 0.08mg / kg, approximately 0.02mg / kg to 0.07mg / kg, approximately 0.02mg / kg to 0.06mg / kg, approximately 0.02mg / kg to 0.06mg / kg 0.05mg / kg, approximately 0.03 to 0.5mg / kg, approximately 0.03 to 0.4mg / kg, approximately 0.03 to 0.3mg / kg, approximately 0.03 to 0.2mg / kg, approximately 0.03 to 0.1mg / kg, approximately 0.03mg / kg to 0.09mg / kg, approximately 0.03mg / kg to 0.08mg / kg, approximately 0.03mg / kg to 0.07mg / kg, approximately 0.03mg / kg to 0.06mg / kg, approximately 0.03mg / kg to 0.05mg / kg, approximately 0.04 to 0.5mg / kg, approximately 0.04 to 0.4mg / kg, approximately 0.04 to 0.3mg / kg, approximately 0.04 to 0.2mg / kg, It is administered at a dose of about 0.04-0.1 mg / kg, about 0.04 mg / kg-0.09 mg / kg, about 0.04 mg / kg-0.08 mg / kg, about 0.04 mg / kg-0.07 mg / kg, about 0.04 mg / kg-0.06 mg / kg, about 0.05-0.5 mg / kg, about 0.05-0.4 mg / kg, about 0.05-0.3 mg / kg, about 0.05-0.2 mg / kg, about 0.05-0.1 mg / kg, about 0.05 mg / kg-0.09 mg / kg, about 0.05 mg / kg-0.08 mg / kg, or about 0.05 mg / kg-0.07 mg / kg.Intermediate values ​​and ranges to the above-listed values ​​are also intended to be part of the invention, for example, an RNAi agent can be administered to a subject at a dose of from about 0.015 mg / kg to about 0.45 mg / mg.

[0223] For example, the RNAi agent, e.g., the RNAi agent in the pharmaceutical composition, may be about 0.01 mg / kg, 0.0125 mg / kg, 0.015 mg / kg, 0.0175 mg / kg, 0.02 mg / kg, 0.0225 mg / kg, 0.025 mg / kg, 0.0275 mg / kg, 0.03 mg / kg, 0.0325 mg / kg, 0.035 mg / kg, 0.03 75mg / kg, 0.04mg / kg, 0.0425mg / kg, 0.045mg / kg, 0.0475mg / kg, 0.05mg / kg, 0.0525mg / kg, 0.0 55mg / kg, 0.0575mg / kg, 0.06mg / kg, 0.0625mg / kg, 0.065mg / kg, 0.0675mg / kg, 0.07mg / kg, 0.0 725mg / kg, 0.075mg / kg, 0.0775mg / kg, 0.08mg / kg, 0.0825mg / kg, 0.085mg / kg, 0.0875mg / kg, 0 .09mg / kg, 0.0925mg / kg, 0.095mg / kg, 0.0975mg / kg, 0.1mg / kg, 0.125mg / kg, 0.15mg / kg, 0.17 It can be administered at a dose of 5 mg / kg, 0.2 mg / kg, 0.225 mg / kg, 0.25 mg / kg, 0.275 mg / kg, 0.3 mg / kg, 0.325 mg / kg, 0.35 mg / kg, 0.375 mg / kg, 0.4 mg / kg, 0.425 mg / kg, 0.45 mg / kg, 0.475 mg / kg, or about 0.5 mg / kg. Values ​​intermediate to the foregoing recited values ​​are also intended to be part of this invention.

[0224] Treatment Plan The pharmaceutical composition may be administered once daily, or the iRNA may be administered in two or more subdoses at appropriate intervals throughout the day, or continuous infusion or continuous delivery may be used via a controlled-release formulation. In this case, the amount of iRNA contained in each subdose must be correspondingly less to achieve the total daily dose. The dosage unit may also be formulated for delivery over several days, for example, using a conventional sustained-release formulation that provides sustained release of the iRNA over a period of several days. Sustained-release formulations are well known in the art and are particularly useful for delivering agents at specific sites and can be used, for example, with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0225] In other embodiments, a single administration of the pharmaceutical composition can be sustained such that subsequent administrations occur at intervals of 3 days or less, 4 days or less, or 5 days or less, or at intervals of 1 week or less, 2 weeks or less, 3 weeks or less, or 4 weeks or less. In some embodiments of the present invention, a single administration of the pharmaceutical composition of the present invention occurs once a week. In other embodiments of the present invention, a single administration of the pharmaceutical composition of the present invention occurs every other month.

[0226] Those skilled in the art will understand that certain factors, including but not limited to, the severity of the disease or disorder, previous treatment, the overall health and / or age of the subject, and other existing diseases, can affect the dosage and timing required to effectively treat a subject. Furthermore, treating a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. The effective amount and in vivo half-life of each iRNA encompassed by the present invention can be estimated using conventional methodology.

[0227] The effective dose and in vivo half-life of each iRNA included in the present invention can also be estimated based on in vivo testing using suitable animal models.For example, the advancement of mouse genetics has led to the creation of a large number of mouse models for the study of various human diseases, for example, disorders related to the expression of HAO1.Such models can be used for the in vivo testing of iRNA and the determination of therapeutically effective dose.Suitable mouse models are known in the art, and include, for example, the animal models described herein.

[0228] Administration method The pharmaceutical composition of the present invention can be administered in various ways depending on whether local administration or systemic treatment is desired and the area to be treated.Administration can be topical (for example, transdermal patch), pulmonary, including by inhalation or injection, including by powder or aerosol sprayer; intratracheal, intranasal, epidermal and transdermal, oral, or parenteral.Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous, for example, by implantation device; or intracranial, for example, by intraparenchymal, intrathecal, or intraventricular administration.

[0229] iRNA can be delivered in a manner that targets a specific tissue, for example, the liver.

[0230] formulation Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome containing formulations. These compositions may be generated from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids.

[0231] The pharmaceutical preparation of the present invention, which can be conveniently provided in unit dosage form, can be prepared by conventional techniques well known in the pharmaceutical industry.Such techniques include combining active ingredients with pharmaceutical carriers or excipients.Generally, the preparation is prepared by uniformly and intimately combining active ingredients with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0232] The compositions of the present invention can be formulated into any of a variety of possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous, or mixed media.Aqueous suspensions can further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran.Suspensions can also contain stabilizers.

[0233] The compositions of the present invention can be formulated for oral administration; parenteral, intraparenchymal (intracerebral), intrathecal, intracerebroventricular or intrahepatic administration, and / or topical administration.

[0234] Oral administration compositions and formulations include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets, or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders may be desirable. In some embodiments, the oral formulation is an oral formulation in which the dsRNA of the present invention is administered with one or more penetration-enhancing surfactants and chelating agents. Suitable surfactants include fatty acids and / or esters or their 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, 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-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or monoglycerides, diglycerides, or their pharmaceutically acceptable salts (e.g., sodium). In some embodiments, a combination of penetration enhancers is used, for example, a fatty acid / salt combined with bile acid / salt. An exemplary combination is lauric acid, capric acid, and the sodium salt of UDCA. Additional penetration enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The dsRNA featured in the present invention can be delivered orally in the form of granules, including spray-dried particles, or complexed to form microparticles or nanoparticles.dsRNA complexing agents include polyamino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG), and starch; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pullulan, cellulose, and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methyl cyanoacrylate), poly(ethyl cyanoacrylate), poly(butyl cyanoacrylate), poly(isobutyl cyanoacrylate), poly(isohexyl cyanoacrylate), DEAE-methacrylate, DEAE-hexyl acrylate, ... Examples of suitable dsRNA formulations include DEAE-acrylamide, DEAE-albumin, and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid) (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. Patent Application Publication No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

[0235] Compositions and formulations for parenteral, intraparenchymal (intracerebral), intrathecal, intraventricular, or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives, such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0236] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms and gloves may also be useful. Suitable topical formulations include those in which the iRNA featured in the present invention is mixed with a topical delivery agent, 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, distearolyphosphatidylcholine (distearolyphosphatidyl choline), and liposomes containing iRNA. choline), anionic (e.g., dimyristoyl phosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNA featured in the present invention may be encapsulated within or complexed with liposomes, particularly cationic liposomes. Alternatively, the iRNA may be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1-20 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.

[0237] iRNA formulations containing membrane-like molecular assemblies The iRNA used in the compositions and methods of the present invention can be formulated for delivery in membranous molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids, composed of at least one bilayer, e.g., one or more bilayers. Liposomes include unilamellar and multilamellar vesicles, whose membranes are formed from a lipophilic material and an aqueous interior. The aqueous portion contains the RNAi composition. The lipophilic material separates the aqueous interior from the aqueous exterior, which typically does not contain the RNAi composition, although in some cases it may. Liposomes are useful for transporting and delivering active ingredients to the site of action. Because the liposome membrane is structurally similar to biological membranes, upon contact with tissue, the liposome bilayer fuses with the cell membrane bilayer. Upon integration of the liposome with the cell, the internal aqueous component containing the RNAi is delivered into the cell, where it can specifically bind to the target RNA and mediate RNAi. In some cases, the liposomes are specifically targeted, for example, to direct RNAi to a particular cell type.

[0238] Liposomes containing RNAi agents can be prepared in various ways. In one example, the lipid components of the liposome are dissolved in a detergent, thereby forming micelles with the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. Then, the RNAi agent preparation is added to the micelles containing the lipid components. The cationic groups on the lipids interact with the RNAi agent and condense around the RNAi agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposome preparation of the RNAi agent.

[0239] If necessary, carrier compounds that aid condensation can be added during the condensation reaction, for example, by controlled addition.For example, the carrier compounds can be polymers other than nucleic acids (for example, spermine or spermidine).The pH can also be adjusted to be suitable for condensation.

[0240] Methods for forming stable polynucleotide delivery vehicles that include polynucleotide / cationic lipid complexes as structural components of the delivery vehicle are further described in WO 96 / 37194, the entire disclosure of which is incorporated herein by reference. Formation of liposomes is described by Felgner, PLet al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Patent No. 4,897,355; U.S. Pat. al.Biochim.Biophys.Acta 557:9,1979;Szoka,et al.Proc.Natl.Acad.Sci.75:4194,1978;Mayhew,et al.Biochim.Biophys.Acta 775:169,1984;Kim,et al.Biochim.Biophys.Acta 728:339,1983; and Fukunaga, et al. The present invention may also include one or more embodiments of the exemplary method described in [Endocrinol. 115:757, 1984]. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50-200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Such methods are easily adapted for packaging RNAi agent preparations into liposomes.

[0241] Liposomes are broadly classified into two types: cationic liposomes, which are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes; the positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are internalized within endosomes; the acidic pH within the endosomes causes the liposomes to rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0242] pH-sensitive or negatively charged liposomes entrap nucleic acids rather than complexing them. Because both the nucleic acid and the lipid are similarly charged, repulsion occurs rather than complex formation. However, some nucleic acid is entrapped in the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the foreign gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0243] One important type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholine. Neutral liposome compositions can be formed, for example, from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, and anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0244] Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0245] Nonionic liposomal systems, especially those containing nonionic surfactants and cholesterol, have also been tested to determine their usefulness in delivering drugs to the skin.Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-steryl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-steryl ether) nonionic liposomal formulations were used to deliver cyclosporine A to the dermis of mouse skin.The results showed that such nonionic liposomal systems were effective in promoting the deposition of cyclosporine A in various layers of the skin (Hu et al. STPPharma.Sci., 1994, 4(6)466).

[0246] Liposomes also include "sterically stabilized" liposomes, a term used herein to refer to liposomes containing one or more specialized lipids that, when incorporated within the liposome, have an increased circulation lifetime compared to liposomes lacking the specialized lipids. Examples of sterically stabilized liposomes include those in which (A) a portion of the vesicle-forming lipid portion of the liposome is one or more glycolipids, e.g., monosialoganglioside G M1or (B) liposomes derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without wishing to be bound by any particular theory, it is believed in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the improved circulation half-life of these sterically stabilized liposomes is due to reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0247] 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 reported the ability of (1) sphingomyelin and (2) ganglioside G to improve the blood half-life of liposomes. These findings were detailed by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). WO 88 / 04924 and U.S. Pat. No. 4,837,028, both to Allen et al., reported the ability of (1) sphingomyelin and (2) ganglioside G to improve the blood half-life of liposomes. M1 or galactocerebroside sulfate esters. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. WO 97 / 13499 (Lim et al.) discloses liposomes containing 1,2-sn-dimyristoylphosphatidylcholine.

[0248] In one embodiment, cationic liposome is used.Cationic liposome has the advantage that it can fuse with cell membrane.Non-cationic liposome cannot fuse with plasma membrane efficiently, but it can be taken up by macrophage in vivo, so it can be used to deliver RNAi agent to macrophage.

[0249] Additional advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a variety of water-soluble and lipid-soluble drugs; and liposomes can protect incorporated RNAi agents within the internal compartment from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger, and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size, and aqueous volume of the liposomes.

[0250] A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that naturally interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with negatively charged lipids in the plasma membrane of tissue culture cells, resulting in delivery of RNAi agents (see, e.g., Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987, and U.S. Pat. No. 4,897,355, which describes DOTMA and its use with DNA).

[0251] The DOTMA analog, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP), can be used in combination with phospholipids to form vesicles that complex with DNA. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to living tissue culture cells, which contain positively charged DOTMA liposomes that naturally interact with negatively charged polynucleotides to form complexes. When sufficiently positively charged liposomes are used, the net charge of the resulting complex is also positive. The positively charged complexes thus prepared naturally adhere to negatively charged cell surfaces, fuse with the plasma membrane, and effectively deliver functional nucleic acids to, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moiety is attached by an ester rather than an ether bond.

[0252] Other reported cationic lipid compounds include those conjugated to one of two types of lipids and conjugated to various moieties, including compounds such as carboxyspermine containing 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

[0253] Another cationic lipid conjugate comprises lipid derivatives with cholesterol ("DC-Chol") formulated in liposomes in combination with DOPE (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine formed by conjugating polylysine to DOPE has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). In certain cell lines, such liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and achieve more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland).Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0254] Liposome preparations are particularly suitable for topical administration, and liposomes have several advantages over other preparations.These advantages include the reduction of the side effects associated with the high systemic absorption of administered drugs, the increased accumulation of administered drugs in desired targets, and the ability to administer RNAi agents to skin.In some implementations, liposomes are also used to deliver RNAi agents to epithelial cells and promote the penetration of RNAi agents into skin tissues, such as skin.For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been demonstrated (e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, RJ and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276, 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R M and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C Y and See Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

[0255] Nonionic liposome systems have also been tested to determine their utility in delivering drugs to the skin, particularly in systems containing nonionic surfactants and cholesterol. Nonionic liposome formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver drugs to the dermis of mouse skin. Such formulations containing RNAi agents are useful for treating skin disorders.

[0256] Liposomes containing RNAi can be formed to be highly deformable. Such deformability can allow the liposomes to enter pores smaller than the average radius of the liposomes. For example, transfersomes are a type of deformable liposome. Transfersomes can be prepared by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Transfersomes containing RNAi agents can be delivered, for example, by subcutaneous injection to deliver the RNAi agent to keratinocytes in the skin. To pass through intact mammalian skin, lipid vesicles must pass through a series of micropores, each with a diameter of less than 50 nm, under the influence of an appropriate transdermal gradient. In addition, due to the properties of lipids, such transfersomes can be self-optimizing (e.g., adapting to the shape of skin pores), self-repairing, and often reach their target without fragmentation, and are often self-loading.

[0257] Other formulations applicable to the present invention are described in U.S. Provisional Patent Applications Nos. 61 / 018,616, filed January 2, 2008; 61 / 018,611, filed January 2, 2008; 61 / 039,748, filed March 26, 2008; 61 / 047,087, filed April 22, 2008; and 61 / 051,528, filed May 8, 2008. International Application No. PCT / US2007 / 080331, filed October 3, 2007, also describes formulations applicable to the present invention.

[0258] Transferosomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transferosomes can be described as liquid droplets that are so highly deformable that they can easily penetrate pores smaller than lipid droplets. Transferosomes are adaptable to the environment in which they are used, for example, they self-optimize (conform to the shape of skin pores), self-repair, frequently reach their target without fragmentation, and often self-add. To form transferosomes, surface edge-activating agents, usually surfactants, can be added to standard liposome compositions. Transferosomes have been used to deliver serum albumin to the skin. Transferosome-mediated delivery of serum albumin has been shown to be effective as a subcutaneous injection of a solution containing serum albumin.

[0259] Surfactants find a wide range of applications in formulations, such as emulsions (including microemulsions) and liposomes.The most common method of classifying and ranking the properties of many different types of surfactants, both natural and synthetic, is by using hydrophilic / lipophilic balance (HLB).The nature of hydrophilic group (also known as "head") provides the most useful means for classifying different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p.285).

[0260] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. Their HLB values ​​generally range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters, such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this class. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0261] When surfactant molecule has negative charge when dissolved or dispersed in water, this surfactant molecule is classified as anionic.Anionic surfactants include carboxylates such as soap, acyl lactate, acyl amide of amino acid, esters of sulfuric acid such as alkyl sulfate and ethoxylated alkyl sulfate, sulfonates such as alkyl benzene sulfonate, acyl isethionate, acyl taurate and sulfosuccinate, and phosphate.The most important members of anionic surfactant class are alkyl sulfate and soap.

[0262] If a surfactant molecule has a positive charge when dissolved or dispersed in water, the surfactant molecule is classified as cationic.Cationic surfactants include quaternary ammonium salts and ethoxylated amines.Quaternary ammonium salts are the most commonly used members of this class.

[0263] If a surfactant molecule has the ability to carry either a positive or negative charge, it is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0264] The use of surfactants in drug products, formulations, and emulsions has been reviewed (Rieger, in "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0265] RNAi used herein can also be provided as a micelle formulation.In this specification, " micelle " is defined as a specific type of molecular assembly, in which amphiphilic molecules are configured in a spherical structure, so that all hydrophobic parts of amphiphilic molecules face inward, and hydrophilic parts contact with the surrounding aqueous phase.When the environment is hydrophobic, they exist in the opposite configuration.

[0266] Mixed micelle formulations suitable for transdermal delivery are prepared by combining an aqueous solution of the siRNA composition with alkali metal ions (C8-C6). 22 It can be prepared by mixing alkyl sulfate with a micelle-forming compound.Exemplary micelle-forming compounds include lecithin, hyaluronic acid, hyaluronic acid pharmaceutically acceptable salts, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocholanylglycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ether and its analogs, polidocanol alkyl ether and its analogs, chenodeoxycholate, deoxycholate, and mixtures thereof.The micelle-forming compound can be added simultaneously with or after the addition of alkali metal alkyl sulfate.Mixed micelles can be formed by mixing virtually any type of components, but vigorously mixing is required to provide smaller micelles.

[0267] In one method, a first micelle composition is prepared, which contains siRNA composition and at least alkali metal alkyl sulfate.Then, this first micelle composition is mixed with at least three kinds of micelle-forming compounds to form a mixed micelle composition.In another method, the micelle composition is prepared by mixing siRNA composition, alkali metal alkyl sulfate, and at least one kind of micelle-forming compound, and then adding the remaining micelle-forming compounds and vigorously mixing.

[0268] Phenol and / or m-cresol can be added to the mixed micelle composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol can be added together with the micelle-forming components. An isotonicity agent, such as glycerin, can be added after the mixed micelle composition is formed.

[0269] To deliver a micelle formulation as a spray, the formulation can be placed in an aerosol dispenser, which is filled with a propellant. The propellant under pressure is in liquid form within the dispenser. The ratio of components is adjusted so that the aqueous phase and the propellant phase are combined, i.e., only one phase is present. If two phases are present, the dispenser must be shaken before dispensing a portion of the contents, for example, via a metering valve. The dispensed amount of pharmaceutical agent is sprayed as a fine mist from the metering valve.

[0270] Propellants can include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

[0271] The specific concentrations of the essential components can be determined by relatively simple experimentation. For absorption via the oral cavity, it is often desirable to increase the dosage administered by injection or via the gastrointestinal tract, e.g., by at least two or three times.

[0272] lipid particles The iRNA, e.g., dsRNA, of the invention can be fully encapsulated within a lipid formulation, e.g., an LNP or other nucleic acid-lipid particle.

[0273] As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle. LNPs contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). LNPs exhibit long circulatory life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the administration site), making them highly useful for systemic applications. LNPs include "pSPLPs" containing encapsulated condensing agent-nucleic acid complexes, as described in PCT International Publication No. WO 00 / 03683. The particles of the present invention 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. Additionally, nucleic acids present in the nucleic acid-lipid particles of the present invention are resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Patent Application Publication No. 2010 / 0324120; and PCT Publication WO 96 / 40964.

[0274] In one embodiment, the lipid to drug ratio (mass / mass ratio (e.g., lipid to dsRNA)) ranges from about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above-listed ranges are also considered part of the invention.

[0275] Examples of cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleyl kaolin, and the like. 1,2-Dilinoleyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or similar. the compound, (3aR,5s,6aS)-N,N-dimethyl 1-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3) 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid may comprise about 20 mol% to about 50 mol%, or about 40 mol% of the total lipid present in the particle.

[0276] In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles.The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in International Application PCT / US2009 / 061897, published as WO2010 / 048536, which is incorporated herein by reference.

[0277] In one embodiment, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane:10% DSPC:40% cholesterol:10% PEG-C-DOMG (mol percent), have a particle size of 63.0±20 nm, and 0.027 siRNA / lipid.

[0278] Ionized / non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidyl The lipids may be anionic or neutral lipids, including diethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. When cholesterol is included, the non-cationic lipid may be about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the particle.

[0279] The conjugated lipid that inhibits particle aggregation can be, for example, a polyethylene glycol (PEG)-lipid, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C]8). The conjugated lipid that prevents particle aggregation can be 0 mol% to about 20 mol%, or about 2 mol%, of the total lipid present in the particle.

[0280] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, for example, from about 10 mol % to about 60 mol %, or about 48 mol % of the total lipid present in the particle.

[0281] In one embodiment, lipid-dsRNA nanoparticles (i.e., LNP01 particles) can be prepared using lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids). Stock solutions can be prepared in ethanol 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 mixed in a molar ratio of, for example, 42:48:10. The mixed lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate at pH 5) to a final ethanol concentration of about 35-45% and a final sodium acetate concentration of about 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 thermobarrel extruder, such as the 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, for example, with phosphate-buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4. LNP01 formulations are described, for example, in WO 2008 / 042973, which is incorporated herein by reference.

[0282] Further exemplary lipid-dsRNA formulations are shown in Table A.

[0283] [Table 1]

[0284] [Table 2]

[0285] Abbreviations in Table A include the following: DSPC: distearoylphosphatidylcholine; DPPC: dipalmitoylphosphatidylcholine; PEG-DMG: PEG-dimyristoylglycerol (C14-PEG, or PEG-C14) (PEG with an average molecular weight of 2000); PEG-DSG: PEG-distyrylglycerol (C18-PEG, or PEG-C18) (PEG with an average molecular weight of 2000); PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with an average molecular weight of 2000).

[0286] DLinDMA (1,2-dilinolenyloxy-N,N-dimethylaminopropane) containing formulations are described in WO 2009 / 127060, filed April 15, 2009, which is incorporated herein by reference.

[0287] XTC containing formulations are described, for example, in U.S. Provisional Patent Application Nos. 61 / 148,366, filed January 29, 2009; 61 / 156,851, filed March 2, 2009; U.S. Provisional Patent Application Nos. 61 / 228,373, filed July 24, 2009; and 61 / 239,686, filed September 3, 2009; and International Application No. PCT / US2010 / 022614, filed January 29, 2010, which are incorporated herein by reference.

[0288] MC3 containing formulations are described, for example, in US Patent Application Publication No. 2010 / 0324120, filed June 10, 2010, the entire disclosure of which is incorporated herein by reference.

[0289] ALNY-100 containing formulations are described, for example, in International Application No. PCT / US09 / 63933, filed November 10, 2009, which is incorporated herein by reference.

[0290] C12-200 containing formulations are described in U.S. Provisional Patent Application No. 61 / 175,770, filed May 5, 2009, and International Application No. PCT / US10 / 33777, filed May 5, 2010, which are incorporated herein by reference.

[0291] Further formulations i. Emulsion The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets, usually greater than 0.1 μm in diameter (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 199; 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 (See Banker (Eds.), 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 intimately mixed and dispersed within one another. Generally, emulsions can be water-in-oil (w / o) or oil-in-water (o / w). When an aqueous phase is finely divided and dispersed as minute droplets within a bulk oil phase, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when an oil phase is finely divided and dispersed as minute droplets within a bulk aqueous phase, the resulting composition is called an oil-in-water (o / w) emulsion.Emulsions may contain additional components in addition to the dispersed phase, including active agents, which may be present in the aqueous phase, the oil phase, or as a separate phase. Pharmaceutical excipients, such as emulsifiers, stabilizers, dyes, and antioxidants, may also be present in the emulsion as needed. Pharmaceutical emulsions may also be multiple emulsions, consisting of three or more phases, such as oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer specific advantages over simple two-phase emulsions. A multiple emulsion in which individual oil droplets of an o / w emulsion surround small water droplets constitutes a w / o / w emulsion. Similarly, a system of oil droplets surrounded by small globules of water stabilized by an oily continuous phase constitutes an o / w / o emulsion.

[0292] Emulsions are characterized by little or no thermodynamic stability.In many cases, the dispersed or discontinuous phase of an emulsion is well dispersed within the external or continuous phase, and is maintained in this form by emulsifiers or the viscosity of the formulation.Either phase of an emulsion can be semi-solid or solid, as in the case of emulsion-type ointment bases and creams.Another means of stabilizing emulsions involves the use of emulsifiers, which can be included in either phase of the emulsion. Emulsifiers can be broadly classified into four types: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0293] Synthetic surfactants, also known as surface active agents, find widespread application in the formulation of emulsions and have been discussed in 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 include hydrophilic and hydrophobic proteins. The ratio of the hydrophilicity of surfactant to the hydrophobicity is called hydrophilic / lipophilic balance (HLB), and is a useful tool in classifying and selecting surfactants in the preparation of formulations.Surfactants can be classified into different classes based on the nature of hydrophilic group: nonionic, anionic, cationic and amphoteric (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams&Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.285).

[0294] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and acacia. Absorbent bases, such as anhydrous lanolin and hydrophilic petrolatum, are hydrophilic and can absorb water to form water-in-oil emulsions while maintaining a semi-solid consistency. Finely divided solids, especially in combination with surfactants, are also used as good emulsifiers in viscous formulations. Examples of these include polar inorganic solids, such as heavy metal hydroxides, non-swelling clays, such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and non-polar solids, such as carbon or glyceryl tristearate.

[0295] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty 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).

[0296] Hydrophilic colloids, or hydrocolloids, include naturally occurring gums and synthetic polymers, such as polysaccharides (e.g., acacia, agar, alginate, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomer, cellulose ethers, and carboxyvinyl polymers), which disperse or swell in water to form colloidal solutions that form strong interfacial films around droplets of the dispersed phase, stabilizing the emulsion by increasing the viscosity of the external phase.

[0297] Emulsion often contains many components, such as carbohydrates, proteins, sterols and phosphatides, which can easily support the growth of microorganisms, so these preparations often contain preservatives.The commonly used preservatives that are included in emulsion preparations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid and boric acid.Antioxidants are also commonly added to emulsion preparations to prevent the deterioration of preparations.The antioxidants used can be free radical scavengers, such as tocopherol, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents, such as ascorbic acid and sodium metabisulfite, and antioxidant synergists, such as citric acid, tartaric acid and lecithin.

[0298] The application of emulsion formulations via dermal, oral, and parenteral routes, as well as methods for their preparation, have been discussed 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 very widely used due to their ease of formulation and effectiveness in terms of absorption and bioavailability (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutrients are among the materials commonly administered orally as o / w emulsions.

[0299] ii. Microemulsions In one embodiment of the present invention, iRNA and nucleic acid compositions are formulated as microemulsions.Microemulsions can be defined as a system of water, oil, and amphiphilic substances that is a single optically isotropic and thermodynamically stable solution (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.245).Typically, microemulsions are prepared by first dispersing oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, generally a medium-chain alcohol, to form a transparent system. Therefore, microemulsions are also described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions are generally prepared by combining 3 to 5 components, including oil, water, surfactant, cosurfactant, and electrolyte. 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 and the structure and geometric packing 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).

[0300] The phenomenological approach using phase diagrams has been extensively studied and has provided those skilled in the art with comprehensive knowledge of how to formulate microemulsions (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 dissolving water-insoluble drugs in a formulation of spontaneously forming, thermodynamically stable droplets.

[0301] Surfactants used in preparing microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ether, fatty acid polyglycerol esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sesquioleate (SO750), and decaglycerol decaoleate (DAO750), either alone or in combination with cosurfactants. Cosurfactants, usually short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, act to increase interfacial fluidity by penetrating the surfactant film through interstitial spaces formed between surfactant molecules, resulting in the formation of irregular films. However, microemulsions can be prepared without the use of cosurfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG 300, PEG 400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) monoglycerides, diglycerides, and triglycerides, polyoxyethylenated fatty acid glyceryl esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0302] Microemulsions are particularly important in terms of drug solubilization and enhancing drug absorption.Lipid-based microemulsions (both o / w and w / o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see, for example, U.S. Patent Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth.Find.Exp.Clin.Pharmacol., 1993, 13, 205). Microemulsions offer the following advantages: improved drug solubilization, drug protection from enzymatic hydrolysis, potential for enhanced drug absorption due to surfactant-mediated changes in membrane fluidity and permeability, ease of preparation, easier oral administration than solid dosage forms, improved clinical efficacy, and reduced toxicity (see, e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). In many cases, microemulsions can spontaneously form when their components are mixed at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides, or iRNAs. Microemulsions are also useful for transdermal delivery of active ingredients in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to promote increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract, as well as improve local cellular uptake of iRNA and nucleic acids.

[0303] The microemulsions of the present invention may also contain additional ingredients and additives, such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers, to improve formulation properties and increase absorption of the iRNA and nucleic acids of the present invention. The penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes is described above.

[0304] iii. Particulates The RNAi agent of the present invention can be contained in particles, for example, microparticles.Microparticles can be formed by spray drying, but can also be formed by other methods, including freeze-drying, evaporation, fluidized bed drying, vacuum drying, or a combination of these techniques.

[0305] iv. Penetration enhancers In one embodiment, the present invention utilizes various penetration enhancers to efficiently deliver nucleic acids, particularly iRNA, to the skin of animals. Most drugs exist in solution in both ionized and non-ionized forms. However, typically, only lipid-soluble or lipophilic drugs can easily pass through cell membranes. It has been found that non-lipophilic drugs can also pass through cell membranes when the cell membrane to be passed through is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs through cell membranes, penetration enhancers also promote the permeability of lipophilic drugs.

[0306] Penetration enhancers can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, e.g., Malmsten, M., Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above classes of penetration enhancers is described in detail below.

[0307] Surfactants (or "surface-active agents") are chemicals that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the solution and another liquid, thereby facilitating 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 (see, e.g., Malmsten, M., Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0308] 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, tricapric acid, monoolein (1-monooleyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, and their C 1-20 These include alkyl esters (e.g., methyl, isopropyl, and t-butyl), and their mono- and diglycerides (e.g., 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).

[0309] The physiological role of bile includes promoting the dispersion and absorption of lipids and fat-soluble vitamins (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts and their synthetic derivatives act as penetration enhancers. Thus, the term "bile salt" includes any naturally occurring component of bile and any synthetic derivative thereof.Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glycolic acid (sodium glycolate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE) (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Delivery). 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; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0310] Chelating agents used in connection with the present invention can be defined as compounds that remove metal ions from solution by forming complexes with the metal ions, thereby facilitating absorption of iRNA through mucous membranes. Regarding the use of chelating agents as penetration enhancers in the present invention, chelating agents have the added advantage of also acting as DNase inhibitors, since most characterized DNA nucleases require divalent metal ions for catalysis and are therefore inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetic acid (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylic acid, and homovanillate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of β-diketones (enamines) (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).

[0311] As used herein, non-chelating non-surfactant penetration enhancers can be defined as compounds that exhibit only slight activity as chelating agents or surfactants, but still promote the absorption of iRNA through the gastrointestinal mucosa (see, for example, Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl-alkanone derivatives, and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory drugs, such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0312] The agent that promotes the uptake of iRNA at the cellular level can also be added to the pharmaceutical composition and other compositions of the present invention.For example, cationic lipids such as lipofectin (Junichi et al., U.S. Patent No. 5,705,188), cationic glycerol derivatives, and polycationic molecules such as polylysine (Lollo et al., PCT International Patent WO97 / 30731) are also known to promote the cellular uptake of dsRNA.Examples of commercially available transfection reagents include, for example, Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000, among others. CD (Invitrogen; Carlsbad, CA), Lipofectamine(TM) (Invitrogen; Carlsbad, CA), RNAiMAX(Invitrogen; Carlsbad, CA), Oligofectamine(TM) (Invitrogen; Carlsbad, CA), Optifect(TM) (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™ (Invitrogen; San Diego, CA, USA), PerFectin transfection reagent (Genlantis; San Diego, CA, USA), NeuroPORTER transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin transfection reagent (Genlantis; San Diego, CA, USA), BaculoPORTER transfection reagent (Genlantis; San Diego, CA, USA), TroganPORTER™ transfection reagent (Genlantis; San Examples of suitable fusion proteins include RiboFect (Bioline; Taunton, MA, USA), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), or HiFect™ (B-Bridge International, Mountain View, CA, USA).

[0313] Other agents can be utilized to enhance penetration of the administered nucleic acid, including glycols such as ethylene glycol and propylene glycol, pyrroles such as 2-pyrrole, azone, and terpenes such as limonene and menthone.

[0314] v. Carrier Certain compositions of the present invention also include a carrier compound in the formulation. As used herein, "carrier compound" or "carrier" can refer to nucleic acids or their analogs, which are inert (i.e., have no biological activity themselves) but are recognized as nucleic acids by in vivo processes that reduce the bioavailability of biologically active nucleic acids, for example, by degrading the biologically active nucleic acid or promoting its removal from the circulation. Co-administration of nucleic acids and carrier compounds, typically in excess of the latter substance, can substantially reduce the amount of nucleic acid recovered in the liver, kidney, or other extracorporeal circulation reservoir, possibly due to competition between the carrier compound and nucleic acid for a common receptor. For example, the recovery of partially phosphorothioate dsRNA in liver tissue can be reduced when coadministered with polyinosinic acid, dextran sulfate, polycytidylic acid, or 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0315] vi. excipients In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. Excipients can be liquid or solid and are selected for the intended mode of administration, taking into account the desired bulk, consistency, etc., when combined with the nucleic acids and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (such as pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, and the like); disintegrants (such as starch, sodium starch glycolate, and the like); and wetting agents (such as sodium lauryl sulfate, and the like).

[0316] Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not adversely react with nucleic acid can also be used to formulate the composition of the present invention.Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

[0317] The preparation for local administration of nucleic acid can include but is not limited to sterile and non-sterile aqueous solution, common solvent, non-aqueous solution in alcohol, or nucleic acid solution in liquid or solid oil base.These solutions can also contain buffer, diluent and other suitable additives.Can use pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not adversely react with nucleic acid.

[0318] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0319] vii. Other ingredients The composition of the present invention can further comprise other adjuvant components that are conventionally found in pharmaceutical compositions, at the use level established in the art.Therefore, for example, this composition can comprise additional compatible pharmaceutically active substances, such as antipruritic agents, astringents, local anesthetics or anti-inflammatory agents, or can comprise additional substances that are useful for the physical formulation of various dosage forms of the composition of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners and stabilizers.However, when added, such substances should not excessively inhibit the biological activity of the components of the composition of the present invention.These preparations can be stabilized, and if desired, can be mixed with auxiliary substances that do not adversely interact with the nucleic acid of the preparation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, coloring agents, flavoring agents and / or aromatic substances.

[0320] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0321] In some embodiments, pharmaceutical compositions featured in the invention are provided that include (a) one or more iRNA compounds and (b) one or more agents that function by a non-RNAi mechanism and are useful in the treatment of, e.g., PH1.

[0322] Composition Testing The toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred.

[0323] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of the compositions featured herein in the present invention is generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending on the dosage form and route of administration utilized. For any compound used in the methods featured in the present invention, the therapeutically effective dose can be estimated initially from cell culture assays. The dose can be determined based on the IC 50 (i.e., the concentration of test compound that achieves half-maximal inhibition of symptoms), or this IC 50 The polypeptide product of the target sequence can be formulated in animal models to achieve a circulating plasma concentration range (e.g., to achieve a reduced concentration of the polypeptide). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0324] In addition to these administrations, as described above, the iRNAs featured in the present invention can be administered in combination with other known agents effective in treating pathological processes mediated by iron overload and treatable by inhibiting the expression of HAO1. In either case, the administering physician can adjust the amount and timing of iRNA administration based on the results observed using standard measures of efficacy known in the art or described herein.

[0325] V. Methods of Inhibiting Expression of HAO1 The present invention provides a method for inhibiting expression of HAO1 (hydroxyacid oxidase 1) in a cell, the method comprising contacting the cell with an RNAi agent, e.g., a double-stranded RNAi agent, in an amount effective to inhibit expression of HAO1 in the cell, thereby inhibiting expression of HAO1 in the cell.

[0326] The contacting of the double-stranded RNAi agent with the cell can be carried out in vitro or in vivo. The step of contacting the RNAi agent with the cell in vivo includes contacting the RNAi agent with a cell or group of cells in the body of a subject, for example, a human subject. A combination of in vitro and in vivo contacting methods is also possible. The contacting can be carried out directly or indirectly as described above. Furthermore, the step of contacting the cell can be achieved by a targeting ligand, including any ligand described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, for example, a GalNAc3 ligand, or other ligand that directs the RNAi agent to a desired site, for example, the liver of a subject.

[0327] As used herein, the term "inhibit" is used interchangeably with "reduce," "silencing," "downregulate," and other similar terms, and includes any level of inhibition.

[0328] The phrase "inhibiting the expression of HAO1" refers to inhibiting the expression of any HAO1 gene (e.g., mouse HAO1 gene, rat HAO1 gene, monkey HAO1 gene, or human HAO1 gene) and variants or mutants of the HAO1 gene. Thus, the HAO1 gene can be a wild-type HAO1 gene, a mutant HAO1 gene, or a transgenic HAO1 gene in the context of a genetically engineered cell, cell population, or organism.

[0329] "Inhibiting the expression of the HAO1 gene" includes any level of inhibition of the HAO1 gene, for example, at least partial suppression of the expression of the HAO1 gene. The expression of the HAO1 gene can be evaluated based on the level of any variable associated with the expression of the HAO1 gene, for example, the level of HAO1 mRNA, the level of HAO1 protein, or a change in this level. This level can be evaluated in individual cells or a group of cells, including, for example, a sample derived from a subject.

[0330] Inhibition can be assessed by a reduction in the absolute or relative level of one or more variables associated with HAO1 expression relative to a control level. The control level can be any type of control level available in the art, such as a pre-dose baseline level or a level determined from similar subjects, cells, or samples that are untreated or treated with a control (e.g., a buffer-only control or an inactive agent control).

[0331] In some embodiments of the methods of the present invention, expression of the HAO1 gene is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0332] Inhibition of the expression of the HAO1 gene can be indicated by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells can be present, for example, in a sample derived from a subject). This first cell or group of cells has been treated (for example, by contacting one or more cells with an RNAi agent of the present invention, or by administering an RNAi agent of the present invention to a subject in which this cell is or was present) to inhibit the expression of the HAO1 gene compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but is not treated. In some embodiments, this inhibition is assessed by expressing the level of mRNA in the treated cells as a percentage of the level of mRNA in the control cells using the following formula:

number

[0333] Alternatively, the inhibition of HAO1 gene expression can be evaluated in terms of the parameter functionally linked to the expression of HAO1 gene, for example, the reduction of the expression of HAO1 protein.HAO1 gene silencing can be determined by any assay known in the art in any cell that constitutively or by genome manipulation expresses HAO1.The liver is the main site of HAO1 expression.Other important sites of expression include kidney and uterus.

[0334] Inhibition of HAO1 protein expression can be indicated by a decrease in the level of HAO1 protein expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As described above for assessing mRNA suppression, inhibition of protein expression levels in treated cells or groups of cells can similarly be expressed as a percentage of the protein levels in control cells or groups of cells.

[0335] The control cell or control cell group that can be used to evaluate the inhibition of the expression of HAO1 gene includes the cell or cell group that is not contacted with the RNAi agent of the present invention.For example, the control cell or control cell group can be derived from an individual subject (e.g., human or animal subject) before the subject is treated with the RNAi agent.

[0336] The level of HAO1 mRNA expressed by a cell or group of cells can be determined using any method known in the art for assessing mRNA expression. In one embodiment, the expression level of HAO1 in a sample is determined by detecting a transcribed polynucleotide, such as the mRNA of the HAO1 gene, or a portion thereof. RNA can be extracted from cells using RNA extraction techniques, such as acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kit (Qiagen), or PAXgene (PreAnalytix, Switzerland). Typical assay formats that utilize ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray analysis.

[0337] In one embodiment, the expression level of HAO1 is determined using a nucleic acid probe. As used herein, the term "probe" refers to any molecule that can selectively bind to a specific HAO1. Probes can be synthesized by those skilled in the art or derived from appropriate biological specimens. Probes can also be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0338] The isolated mRNA can be used in hybridization or amplification assays, including but not limited to Southern analysis, Northern analysis, polymerase chain reaction (PCR) analysis, and probe assays. In one method for determining mRNA levels, the isolated mRNA is contacted with a nucleic acid molecule (probe) that can hybridize to HAO1 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe is immobilized on a solid surface and the mRNA is contacted with the probe, for example, on an Affymetrix gene chip array. Those skilled in the art can easily adapt known mRNA detection methods for use in determining HAO1 mRNA levels.

[0339] In alternative methods for determining the expression level of HAO1 in a sample, nucleic acid amplification of, for example, mRNA and / or reverse transcriptase (to prepare cDNA) processes in the sample can be performed using, for example, RT-PCR (Mullis, 1987, an experimental embodiment described in U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033), or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when such molecules are present in very low numbers. In certain embodiments of the present invention, the expression level of HAO1 is determined by quantitative fluorescent RT-PCR (i.e., TaqMan™ system).

[0340] The expression level of HAO1 mRNA can be monitored using membrane blots (such as those used in hybridization analysis, e.g., Northern, Southern, and dot), or microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acids). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. Determining the expression level of HAO1 can also include using a nucleic acid probe in solution.

[0341] In some embodiments, mRNA expression levels are assessed using branched DNA (bDNA) assays or real-time PCR (qPCR). The use of such methods is described and exemplified in the Examples described herein.

[0342] The expression level of HAO1 protein can be determined using any method known in the art for measuring protein levels, including, for example, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), high-diffusion chromatography, liquid or gel precipitation, spectrophotometry, colorimetry, spectrophotometric assay, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence analysis, and electrochemiluminescence analysis.

[0343] The term "sample," as used herein, refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject's body. Examples of biological fluids include blood, serum and serous fluid, plasma, lymph, urine, cerebrospinal fluid, saliva, and ocular fluid. A tissue sample may include a sample from a tissue, organ, or localized area. For example, a sample may be derived from a specific organ, part of an organ, or fluid or cells within these organs. In certain embodiments, a sample may be derived from the liver (e.g., the whole liver or a specific part of the liver, or a specific type of liver cell, such as a hepatocyte). In some embodiments, a "sample derived from a subject" refers to blood or plasma obtained from a subject. In further embodiments, a "sample derived from a subject" refers to liver tissue derived from a subject.

[0344] In some embodiments of the method of the present invention, the RNAi agent is administered to the subject so that the RNAi agent is delivered to a specific site in the subject's body.The inhibition of HAO1 expression can be evaluated by measuring the level or change of the level of HAO1 mRNA or HAO1 protein in the sample derived from fluid or tissue from a specific site in the subject's body.In some embodiments, this site is the liver.This site can also be a small section or a small group of cells from any one of the aforementioned sites.This site can also include cells that express a specific type of receptor.

[0345] VI. METHODS FOR TREATING OR PREVENTING HAO1-ASSOCIATED DISORDERS The present invention also provides methods for treating or preventing diseases and conditions that can be regulated by the expression of the HAO1 gene. For example, the compositions described herein can be used to treat any disorder associated with PH1.

[0346] The effectiveness of disease treatment or prevention can be assessed, for example, by measuring disease progression, disease remission, symptom severity, pain relief, quality of life, the dosage of a drug required to maintain therapeutic efficacy, the level of a disease marker, or other measurable parameters appropriate for the given disease being treated or targeted for prevention. It is well within the capabilities of one of ordinary skill in the art to monitor the effectiveness of treatment or prevention by measuring any one of such parameters or any combination of parameters.

[0347] The effectiveness of treatment or prevention is revealed when there is a statistically significant improvement in one or more parameters of disease state, or when there is no worsening or onset of symptoms that would normally be expected.As an example, at least 10%, preferably at least 20%, 30%, 40%, or 50% or more favorable change in measurable parameters of disease can indicate effective treatment.The effectiveness of a given iRNA drug or the formulation of this drug can also be evaluated using experimental animal models of a given disease known in the art.When using experimental animal models, the effectiveness of treatment is revealed when a statistically significant reduction in markers or symptoms is observed.

[0348] Alternatively, efficacy can be measured by a reduction in disease severity, as determined by one of ordinary skill in the art, the diagnosis being based on a clinically accepted disease severity rating scale.

[0349] In some embodiments of the methods of the invention, HAO1 expression is reduced over an extended period of time, e.g., at least 1 week, 2 weeks, 3 weeks, or 4 weeks or more. For example, in certain cases, expression of the HAO1 gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% by administration of an iRNA agent described herein. In some embodiments, the HAO1 gene is suppressed by at least about 60%, 70%, or 80% by administration of an iRNA agent. In some embodiments, the HAO1 gene is suppressed by at least about 85%, 90%, or 95% by administration of a double-stranded oligonucleotide. In another embodiment, the HAO1 gene remains suppressed for 7 days, 10 days, 20 days, or 30 days or more following administration.

[0350] Administration The RNAi agent of the present invention can be administered by any administration method known in the art, including, but not limited to, subcutaneous administration, intravenous administration, intramuscular administration, intraocular administration, intrabronchial administration, intrapleural administration, intraperitoneal administration, intraarterial administration, lymphatic administration, cerebrospinal administration, and any combination thereof.In some embodiments, the RNAi agent is administered subcutaneously.

[0351] In some embodiments, administration is performed by depot injection. Depot injection can consistently release RNAi agent over a long period of time. Therefore, depot injection can reduce the frequency of administration required to achieve a desired effect, for example, the desired inhibition of HAO1, or a therapeutic or preventive effect. Depot injection can also achieve a more constant serum concentration. Depot injection can include subcutaneous injection or intramuscular injection. In some embodiments, the depot injection is a subcutaneous injection.

[0352] In some embodiments, the administration is performed by a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. The infusion pump can be used for intravenous, subcutaneous, arterial, or epidural injection. In some embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the RNAi agent to the liver.

[0353] Other administration methods include epidural administration, intracerebral administration, intraventricular administration, nasal administration, intraarterial administration, intracardiac administration, intraosseous injection, subarachnoid administration, intravitreal administration and intrapulmonary administration.Administration method can be selected based on whether local treatment or systemic treatment is desired and the area to be treated.Administration route and site can be selected to improve targeting.

[0354] The method includes administering an iRNA agent, e.g., at a dose sufficient to suppress HAO1 mRNA levels for at least 5 days, preferably 7, 10, 14, 21, 25, 30, or 40 days; and, optionally, administering a second single dose of dsRNA, which second single dose is administered at least 5 days, preferably 7, 10, 14, 21, 25, 30, or 40 days after the first single dose, thereby inhibiting expression of the HAO1 gene in the subject.

[0355] In one embodiment, a dose of an iRNA agent of the invention is administered no more than once every four weeks, no more than once every three weeks, no more than once every two weeks, or no more than once a week. In another embodiment, administration can continue for one month, two months, three months, or six months, or for more than a year. In another embodiment, a dose of an iRNA agent of the invention is administered once a week for three weeks.

[0356] Generally, the iRNA agent does not activate the immune system, e.g., does not increase cytokine levels, e.g., TNF-α or IFN-α levels. For example, when measured by an assay, e.g., an in vitro PBMC assay as described herein, the increase in TNF-α or IFN-α levels is less than 30%, less than 20%, or less than 10% of that in control cells treated with a control dsRNA, e.g., a dsRNA that does not target HAO1.

[0357] For example, a subject can be administered a therapeutic amount of an iRNA agent, such as 0.3 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, or 3 mg / kg of dsRNA. The iRNA agent can be administered by intravenous injection over a period of time, for example, 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes. Administration can be repeated, for example, periodically, for example, every two weeks (i.e., every two weeks) for one, two, three, or four months or more. After the initial treatment regimen, treatment can be administered less frequently. For example, after three months of biweekly administration, administration can be repeated once a month for six months or one year or more. Administration of an iRNA agent can, for example, reduce the level of HAO1 in a patient's cells, tissues, blood, urine, or other compartment by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0358] Prior to administration of the full dose of the iRNA agent, the patient can administer a small amount, e.g., an amount that results in an infusion reaction of less than 5%, and monitor for adverse effects, e.g., allergic reactions, or elevated lipid levels or blood pressure. In another example, the patient can monitor for unwanted immune stimulatory effects, e.g., elevated levels of cytokines (e.g., TNF-α or INF-α).

[0359] Patients who need HAO1 RNAi agent can be identified by taking family history.Healthcare provider, for example, doctor, nurse or family member, can take family history before prescribing or administering HAO1 dsRNA.Before HAO1 RNAi agent is administered to patients, DNA test can also be carried out on patients to identify mutation in AGT1 gene.The diagnosis of PH1 can be confirmed by any test known to those skilled in the art.

[0360] Therapeutic or preventive effects are evident when there is a statistically significant improvement in one or more parameters of the disease state, or when the onset or worsening of symptoms that would normally be expected does not occur.As an example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, or 50% or more in measurable parameters of the disease may indicate effective treatment.The effectiveness of a given iRNA agent of the present invention or the formulation of this iRNA agent can also be evaluated using an experimental animal model of a given disease known in the art.When using an experimental animal model, the effectiveness of treatment is evident when a statistically significant decrease in markers or symptoms is observed.

[0361] The dose of an RNAi agent administered to a subject can be adjusted to balance the risks and benefits of a particular dose, for example, to achieve a desired level of HAO1 gene suppression (e.g., as assessed based on HAO1 mRNA suppression, HAO1 protein expression, or reduced oxalate levels) or a desired therapeutic or prophylactic effect while avoiding undesirable side effects.

[0362] In some embodiments, the RNAi agent is administered in two or more separate doses. To facilitate repeated or frequent infusions, a delivery device, such as a pump, implantation of a semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal, or intracapsular), or a reservoir may be desirable. In some embodiments, the number or amount of subsequent administrations depends on achieving the desired effect, e.g., suppression of the HAO1 gene, or achieving a therapeutic or prophylactic effect, e.g., reduction of iron overload. In some embodiments, the RNAi agent is administered according to a schedule. For example, the RNAi agent can be administered once a week, twice a week, three times a week, four times a week, or five times a week. In some embodiments, the schedule includes administration at regular intervals, e.g., every hour, every four hours, every six hours, every eight hours, every 12 hours, daily, every two days, every three days, every four days, every five days, weekly, every two weeks, or monthly. In other embodiments, the schedule involves administration at short intervals, followed by a longer period during which the agent is not administered. For example, the schedule may include an initial administration set administered at a relatively short period (e.g., about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, or about every 72 hours), followed by a longer period during which the RNAi agent is not administered (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks). In one embodiment, the RNAi agent is initially administered hourly, and then administered at longer intervals (e.g., once daily, once weekly, once every two weeks, or once monthly). In another embodiment, the RNAi agent is initially administered daily, and then administered at longer intervals (e.g., once weekly, once every two weeks, or once monthly). In certain embodiments, the longer period increases over time or is determined based on achieving the desired effect. In one particular embodiment, the RNAi agent is administered once daily for the first week, followed by once weekly administration beginning on day 8 of administration. In another particular embodiment, the RNAi agent is administered every other day for the first week, followed by once weekly administration beginning on day 8 of administration.

[0363] In some embodiments, the RNAi agent is administered according to a dosing schedule that includes a "loading phase" with closely spaced administrations, which may be followed by a "maintenance phase" in which the RNAi agent is administered at longer intervals. In one embodiment, the loading phase includes administering the RNAi agent five times per day for the first week. In another embodiment, the maintenance phase includes administering the RNAi agent once or twice per week. In a further embodiment, the maintenance phase lasts for five weeks.

[0364] Any of these schedules can optionally be repeated one or more times, the number of repetitions being determined by achieving a desired effect, e.g., suppression of the HAO1 gene, and / or achieving a therapeutic or prophylactic effect, e.g., reducing oxalate levels or alleviating symptoms of PH1.

[0365] In another aspect, the invention features a method for instructing an end user, e.g., a caregiver or subject, how to administer an iRNA agent described herein. The method optionally includes providing one or more doses of an iRNA agent to the end user and instructing the end user to administer the iRNA agent in a regimen described herein, thereby instructing the end user.

[0366] VII. Kit The present invention also provides kits for using any iRNA agent and / or for carrying out any method of the present invention. Such kits include one or more RNAi agents and instructions for use, for example, instructions for inhibiting the expression of HAO1 in cells by contacting the cells with an amount of RNAi agent that is effective to inhibit the expression of HAO1. Optionally, the kit can further include a means for contacting the RNAi agent with cells (e.g., an injection device) or a means for measuring the inhibition of HAO1 (e.g., a means for measuring the inhibition of HAO1 mRNA or protein). Such a means for measuring the inhibition of HAO1 can include a means for obtaining a sample from a subject, for example, a plasma sample. Optionally, the kit of the present invention can further include a means for administering the RNAi agent to a subject or a means for determining a therapeutically effective amount or a prophylactically effective amount.

[0367] VII. Diagnostic Markers for PH1 and Related Conditions Also described herein are markers and methods for the diagnosis of disease states caused by the overproduction of oxalate, particularly PH1 and related conditions, and agents for the treatment of said conditions.

[0368] In another aspect, the present invention relates to a method for treating a subject with PH1 condition (lithiasis, particularly PH1). The diagnostic method includes: (a) knocking down the expression of HAO1 in a subject; (b) obtaining biological serum from the subject; and (b) determining the level of glycolate in the serum. It should be understood that an increase in the level of glycolate in the serum compared to a negative control indicates the inhibition of glycolate oxidase enzyme, which prevents the production of oxalate caused by PH1 condition.

[0369] In one embodiment, a kit for diagnosing a PH1 condition is described herein, the kit comprising: (a) an agent for determining the presence of an analyte of interest in serum, wherein the analyte of interest is one of glycolate; and (b) a calibration means. For example, the analyte of interest is glycolate and the agent is an siRNA targeting HAO1.

[0370] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used to carry out or test the iRNA and methods featured in this invention, and suitable methods and materials are described below.All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety.In case of conflict, the present specification, including definitions, shall prevail.In addition, materials, methods, and examples are merely illustrative and are not intended to be limiting. [Example]

[0371] material and method The following materials and methods were used in the examples: As used herein, "HAO" and "GO" are used interchangeably.

[0372] Synthesis of siRNA Single-stranded RNA was produced by solid-phase synthesis at a 1 μmole scale using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as the solid support. RNA and RNA containing 2'-O-methyl nucleotides were produced by solid-phase synthesis using the corresponding phosphoramidites and 2'-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated into selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry as described in Current Protocols in Nucleic Acid Chemistry, Beaucage, S. Lett. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA. Phosphorothioate linkages were introduced by exchanging the iodine oxidizer solution with a solution of Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further auxiliary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).

[0373] Deprotection and purification of crude oligoribonucleotides by anion-exchange HPLC were performed according to established procedures. Yields and concentrations were determined by UV absorbance of the respective RNA solutions at a wavelength of 260 nm using a spectrophotometer (DU 640B, Beckman Coulter GmbH, Unterschleissheim, Germany).

[0374] Double-stranded RNA was prepared by mixing equimolar solutions of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heating in a water bath at 85–95°C for 3 min, and cooling to room temperature over 3–4 h. The annealed RNA solution was stored at −20°C until use.

[0375] In some cases, the duplex (dsRNA) was synthesized more than once. Different batches were labeled with different extensions. For example, AD-62933.1 and AD-62933.2 are different batches of the same duplex.

[0376] Cell culture and transfection Primary cynomolgus monkey hepatocytes (PCH) and primary mouse hepatocytes (PMH) were used. PCH (Celsis #M003055, lot CBT) or PMH (freshly isolated) were transfected by adding 14.8 μl of Opti-MEM and 0.2 μl of Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, cat #13778-150) per well to 5 μl of siRNA duplex per well of a 96-well plate and incubating for 15 minutes at room temperature. Approximately 2 × 10 4 80 μl of InVitroGRO CP Rat medium (InVitro Technologies) containing 100 μL of PCH or PMH cells was added to the siRNA mixture. Cells were incubated for 24 h before RNA purification. Single-dose experiments were performed at final duplex concentrations of 10 or 20 nM and 0.1 or 0.2 nM, and dose-response experiments were performed at a dose range of 10 nM to 36 fM final duplex concentrations, with 8- and 6-fold dilutions.

[0377] Total RNA isolation Total RNA was isolated using the DYNABEADS mRNA Isolation Kit (Invitrogen, part #:610-12). Cells were harvested and lysed with 150 μl of lysis / binding buffer, then mixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). A mixture of 10 μl of magnetic beads and 80 μl of lysis / binding buffer was added to a round-bottom plate and mixed for 1 minute. The magnetic beads were captured with a magnetic stand, and the supernatant was removed without disturbing the beads. After removing the supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing the supernatant, the magnetic beads were washed twice with 150 μl of wash buffer A and mixed for 1 minute. The beads were again captured, and the supernatant was removed. The beads were then washed with 150 μl of wash buffer B, captured, and the supernatant was removed. Next, the beads were washed with 150 μl of elution buffer, captured, and the supernatant was removed. The beads were allowed to dry for 2 minutes. After drying, 50 μl of elution buffer was added and mixed at 70° C. for 5 minutes. The beads were captured on a magnet for 5 minutes. 40 μl of the supernatant was removed and added to another 96-well plate.

[0378] cDNA synthesis Synthesis of cDNA was performed using the ABI High Performance cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat #4368813).

[0379] Per reaction, a master mix of 2 μl of 10× buffer, 0.8 μl of 25× dNTPs, 2 μl of random primers, 1 μl of reverse transcriptase, 1 μl of RNase inhibitor, and 3.2 μl of HO was added to 10 μl of total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) by the following steps: 25°C for 10 min, 37°C for 120 min, 85°C for 5 s, and a 4°C hold.

[0380] Real-time PCR Two microliters of cDNA was added to each well of a 384-well 50-well plate (Roche cat # 04887301001) containing master mix containing 0.5 μl of mouse GAPDH (cat # 4352339E Life Technologies) or custom-designed cynomolgus GAPDH TaqMan probe: (F-GCATCCTGGGCTACACTGA (SEQ ID NO: 13), R-TGGGTGTCGCTGTTGAAGTC (SEQ ID NO: 14), probe-CCAGGTGGTCTCCTCC (SEQ ID NO: 15)), 0.5 μl of human or mouse HAO1 (HS00213909_M1, cross-reactive with cynomolgus HOA1; Mm 00439249_ml for mouse assays, Life Technologies), and 5 μl of Lightcycler 480 probe master mix (Roche cat # 04887301001). Real-time PCR was performed on a LightCycler480 Real-Time PCR System (Roche) using the ΔΔCt (RQ) assay. Unless otherwise noted in the summary table, each duplex was tested in two independent transfections, and each transfection was assayed in duplicate.

[0381] To calculate relative fold changes, real-time data were analyzed using the ΔΔCt method and normalized to assays performed with 10 nM AD-1955-transfected or mock-transfected cells. IC50s were calculated with a four-parameter fit model using XL Fit and normalized to AD-1955-transfected or naive cells.

[0382] The sense and antisense sequences of AD-1955 are: SENSE: 5'-cuuAcGcuGAGuAcuucGAdTsdT-3' (SEQ ID NO: 16); and ANTISENSE: 5'-UCGAAGuACUcAGCGuAAGdTsdT-3' (SEQ ID NO: 17).

[0383] [Table 3]

[0384] [Table 4]

[0385] [Table 5]

[0386] Example 1. siRNA design, specificity, and efficacy prediction siRNA design was performed to identify siRNAs targeting human, cynomolgus monkey, mouse, and rat HAO1 transcripts annotated in the NCBI Gene database ( http: / / www.ncbi.nlm.nih.gov / gene / ).

[0387] The following transcripts from the NCBI RefSeq collection were used in the design: human (Homo sapiens) HAO1 mRNA is NM_017545.2; cynomolgus monkey (Macaca fascicularis) HAO1 mRNA is XM_005568381.1; house mouse (Mus musculus) HAO1 mRNA is NM_010403.2; and brown rat (Rattus norvegicus) HAO1 mRNA is XM_006235096.1.

[0388] Due to the diversity of higher primate / rodent sequences, siRNA duplexes were designed in several separate batches, including but not limited to: human and cynomolgus monkey transcripts only; human, cynomolgus monkey, mouse, and rat transcripts only; and batches containing duplexes matching only mouse and rat transcripts. All siRNA duplexes that share 100% identity with the listed human transcripts and transcripts of other species considered in each design batch (above) were designed.

[0389] The specificity of all possible 19-mers was predicted from each sequence. Candidate 19-mers lacking repeats longer than 7 nucleotides were then selected. These 1,069 candidate human / cynomolgus monkey, 184 human / cynomolgus monkey / mouse / rat, and 579 mouse / rat siRNAs were subjected to a comprehensive search against the appropriate transcriptome (defined as the set of NM_ and XM_ records within the NCBI Refseq set for human, cynomolgus monkey, mouse, or rat) using an exhaustive "brute-force" algorithm implemented in the Python script "BruteForce.py." The script then analyzed the transcript-oligo alignment to generate a score based on the position and number of mismatches between the siRNA and any potential "off-target" transcripts. The off-target score is weighted to emphasize differences in the siRNA "seed" region at positions 2-9 of the 5' end of the molecule. Each oligo-transcript pair from the brute-force search was assigned a mismatch score by adding their respective mismatch scores; a mismatch at positions 2 to 9 was counted as 2.8, a mismatch at cleavage sites 10 to 11 was counted as 1.2, and a mismatch at region 12 to 19 was counted as 1.0. Further off-target predictions were made by comparing the frequencies of heptamers and octamers derived from three different seed-derived hexamers for each oligo. Two heptamers and one octamers were generated using the hexamers at positions 2 to 7 relative to the 5' start site. Heptamers 1 was generated by adding a 3' A to the hexamer; heptamers 2 was generated by adding a 5' A to the hexamer; and the octamers were generated by adding A's to the 5' and 3' ends of the hexamer. We previously calculated the frequencies of octamers and heptamers in the 3'UTROME (defined as the end of the coding sequence, or "CDS," as a subsequence of the transcriptome from NCBI's Refseq database) of human, cynomolgus monkey, mouse, or rat. Octamer frequencies were normalized to heptamer frequencies using the mean value of the octamer frequency range.The "mirSeedScore" was then determined by calculating the sum of ((3 x normalized octamer counts) + (2 x heptamer2 counts) + (1 x heptamer1 counts)).

[0390] Both siRNA strands were assigned to specificity categories according to their calculated scores: scores greater than 3 were considered highly specific, scores equal to 3 were considered highly specific, and scores between 2.2 and 2.8 were considered moderately specific. siRNAs were classified according to the specificity of the antisense strand. Duplexes from the human / cynomolgus monkey set and mouse / rat set were selected in which the antisense oligo lacked GC at position 1, G at both positions 13 and 14, and three or more Us or As in the seed region (duplex characteristics predicted for high efficacy). Similarly, duplexes from the human / cynomolgus monkey / mouse set and human / cynomolgus monkey / mouse / rat set with three or more Us or As in the seed region were selected.

[0391] Candidate GalNAc-conjugated duplexes of 21 and 23 nucleotides in length for the sense and antisense strands, respectively, were designed by extending the antisense 19-mer with four additional nucleotides in the 3' direction (maintaining perfect complementarity with the target transcript). The sense strand was designated as the reverse complement of the first 21 nucleotides of the antisense 23-mer. Duplexes that maintained perfect matches to all transcripts of the selected species across all 23 nucleotides were selected.

[0392] The antisense strand containing C or G at the first 5' position was modified to have U at the first 5' position if the modification did not result in the introduction of a series of four or more consecutive Us (5'→3'), and was modified to have A at the first 5' position if the modification resulted in the introduction of a series of four or more consecutive Us. The sense strand that pairs with these "UA exchange" antisense strands to form a duplex was modified accordingly to maintain complementarity. Examples described below include AD-62989 and AD-62993.

[0393] A total of 31 sense and 31 antisense oligos derived from human / cynomolgus monkey 21 / 23-mer oligos, 19 sense and 19 antisense oligos derived from human / cynomolgus monkey / mouse / rat 21 / 23-mer oligos, and 48 sense and 48 antisense oligos derived from mouse / rat 21 / 23-mer oligos were synthesized to form GalNAc-conjugated duplexes.

[0394] The sequences of the sense and antisense strands of the modified duplex are shown in Table 1, and the sequences of the sense and antisense strands of the unmodified duplex are shown in Table 2.

[0395] [Table 6]

[0396] [Table 7]

[0397] [Table 8]

[0398] [Table 9]

[0399] [Table 10]

[0400] [Table 11]

[0401] [Table 12]

[0402] [Table 13]

[0403] [Table 14]

[0404] [Table 15]

[0405] [Table 16]

[0406] [Table 17]

[0407] [Table 18]

[0408] [Table 19]

[0409] Example 2. In vitro single dose screening in primary monkey hepatocytes The modified, conjugated HAO1 siRNA duplexes were evaluated for efficacy by transfection assays in primary monkey hepatocytes. HAO1 siRNA was transfected at two doses: 10 nM and 0.1 nM. The results of these assays are shown in Table 3, where the data are expressed as the fraction of message remaining in cells transfected with siRNA targeting HAO1 relative to cells transfected with the negative control siRNA, AD-1955 ± standard deviation (SD).

[0410] The results are also shown in Figure 3A. Figure 3B illustrates the dose response using one of the most active conjugates (#31) (AD-62933) from the first two-dose screen: IC50 was approximately 19 pM.

[0411] [Table 20]

[0412] [Table 21]

[0413] [Table 22]

[0414] [Table 23]

[0415] [Table 24]

[0416] Example 3. In vitro single dose screening in primary mouse hepatocytes The modified, conjugated HAO1 siRNA duplexes were evaluated for efficacy by transfection assays in primary mouse hepatocytes. HAO1 siRNA was transfected at two doses: 20 nM and 0.2 nM. The results of these assays are shown in Table 4, with data expressed as the fraction of message remaining in cells transfected with siRNA targeting HAO1 relative to cells transfected with the negative control siRNA, AD-1955 ± standard deviation (SD).

[0417] [Table 25]

[0418] [Table 26]

[0419] [Table 27]

[0420] [Table 28]

[0421] Example 4. Dose-response screening in primary monkey hepatocytes The IC50 of the modified and conjugated HAO1 siRNA duplexes was determined in primary monkey hepatocytes. HAO1 siRNA was transfected at 8- and 6-fold dilutions, with a dose range of 10 nM to 36 fM final duplex concentrations. The results of these assays are shown in Table 5.

[0422] [Table 29]

[0423] [Table 30]

[0424] Example 5. Dose-response screening in primary monkey hepatocytes The IC50 of the modified and conjugated HAO1 siRNA duplexes was determined in primary mouse hepatocytes. HAO1 siRNA was transfected at 8- and 6-fold dilutions, with a dose range of 10 nM to 36 fM final duplex concentrations. The results of these assays are shown in Table 6.

[0425] [Table 31]

[0426] [Table 32]

[0427] [Table 33]

[0428] [Table 34]

[0429] [Table 35]

[0430] [Table 36]

[0431] Example 6. In vivo evaluation of GO-GalNAc conjugates in C57B6 mice GO-GalNAc conjugates were administered subcutaneously to C57B6 mice at 10 mg / kg, 5 mg / kg, 2.5 mg / kg, or 1.25 mg / kg, and liver mRNA knockdown was assessed by qPCR 72 hours after administration. The single-dose ED50 was approximately 1.25 mg / kg for compound A (AD-62994) and approximately 2.5 mg / kg for compound B (AD-62933). In a repeated-dose study, the conjugates were administered subcutaneously weekly (QW) for four weeks, and liver GO mRNA levels were assessed 72 hours after the fourth dose. The repeated-dose ED50 was approximately 0.3 mg / kg for both compounds. The results are shown in Figure 4.

[0432] Example 7. In vivo evaluation of GO knockdown in AGXT KO mice and the effect on oxalate levels GO siRNA (AD-40257) in lipid nanoparticles (LNPs) was administered intravenously at 1 mg / kg to AGXT KO mice (Salido et al. (2006) PNAS 103:18249). Urinary oxalate or glycolate levels were measured on day 15 using ion chromatography / mass spectrometry. The results are shown in Figure 5. Data are expressed relative to pre-dose values ​​and normalized to creatinine (Cr) to adjust for urine dilution. N = 4 mice per group, and error bars represent standard deviation.

[0433] Example 8. In vivo evaluation of GO-GalNAc conjugates in a rat AGXT knockdown model To generate the rat PH1 model, AGXT siRNA (AD-63102) in LNP (AF-011-63102) was administered intravenously to female Sprague-Dawley rats at 1 mg / kg on days 1 and 7 to maintain AGXT knockdown in the rat liver. 1% ethylene glycol was added to the drinking water to further simulate oxalate production. On days 0 and 7, some rats also received a GO GalNAc-siRNA (AD-62994) conjugate or a PBS control. The results are shown in Figure 6. Figure 6A shows quantification of liver AGXT mRNA levels 72 hours after a single administration of 1 mg / kg of AGXT siRNA in LNP. In Figure 6B, urinary oxalate levels were quantified from urine collected over 24 hours from the previous day to day 0, day 3-4, day 5-6, and day 7-8. Data were normalized to creatinine to adjust for urine dilution. N = 3 for the AGXT group and N = 2 for the PBS control group. In Figure 6C, these same rats (shown in Figure 6B) were treated with weekly doses of both AF-011-63102 and AD-62994 on days 14 and 21, as indicated, and with 24-hour urine collections through day 49. Ethylene glycol remained in the drinking water until day 28. In Figure 6D, the duration of HAO1 knockdown in rats is shown by measuring mRNA levels 1 week or 4 weeks after the last of four doses (corresponding to days 28 and 49 in Figure 6C), expressed relative to levels seen in PBS-treated rats. Error bars indicate the overall standard deviation.

[0434] [Table 37]

[0435] Example 9. In vivo evaluation of GO-GalNAc conjugates Six- to eight-week-old female C57BL / 6 mice were given a single subcutaneous injection of the GO siRNA-GalNac conjugates listed in Table 7. 72 hours later, mice were sacrificed and livers were assayed for HAO mRNA by bDNA analysis. The results are shown in Figure 13.

[0436] [Table 38]

[0437] Example 10. In vivo evaluation of GO-GalNAc conjugates in mice Female C57BL / 6 mice received a single subcutaneous dose of 3 mg / kg of multiple GO siRNA-GalNac conjugates described herein or a PBS control. 72 hours later, mice were sacrificed and liver HAO1 mRNA knockdown was assessed using qPCR. Results are shown in Figure 14 and are expressed relative to the PBS control.

[0438] Example 11. Dose-response evaluation of GO-GalNAc conjugates in mice Female C57BL / 6 mice were administered a single subcutaneous dose of 1 mg / kg or 3 mg / kg of one of the GO siRNA-GalNAc conjugates Compound A (AD-62994), Compound B (AD-62933), Compound C (AD-65644), Compound D (AD-65626), Compound E (AD-65590), Compound F (AD-65585), or a PBS control. Ten days later, mice were sacrificed, and liver HAO1 mRNA knockdown was assessed using qPCR. In a repeat-dose study, Compounds C, D, F, or a PBS control were administered subcutaneously weekly (QW) for four weeks, and liver HAO1 mRNA levels were assessed 10 days after the last dose. Results from a single dose are shown in Figure 15, and results from a repeat-dose study are shown in Figure 16, expressed relative to the PBS control. These data demonstrated improved potency of compounds AD-65644 and AD-65626 over AD-62933, and improved potency of compounds AD-65590 and AD-65585 over AD-62994.

[0439] Example 12. Dose-response evaluation of Compound D in mice Female C57BL / 6 mice were administered a single subcutaneous dose of 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg of AD-65626 or a PBS control. Ten days later, mice were sacrificed and liver HAO1 mRNA knockdown was assessed using qPCR; the results, expressed relative to the PBS control, are shown in Figure 17. These results demonstrate a greater than three-fold improvement in efficacy compared to compound AD-62933.

[0440] Example 13. Relationship between mRNA knockdown and serum glycolate levels in mice Female C57BL / 6 mice received a single subcutaneous dose of 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg AD-65585 or a PBS control. Ten days later, mice were sacrificed, and liver HAO1 mRNA knockdown was assessed using qPCR; results are expressed relative to the PBS control. Glycolate levels in serum samples from these same mice were quantified using ion chromatography with mass spectrometry as previously described (Knight et al., Anal. Biochem. 2012 February 1;421(1):121-124). The results of these experiments are shown in Figure 18.

[0441] These results demonstrate that AD-65585 is as potent as AD-65626, both with a single-dose ED50 of approximately 0.3 mg / kg in WT mice. In addition, silencing of HAO1 mRNA resulted in a dose-response serum glycolate increase of up to 4-fold (approximately 200 μM) at the two highest doses.

[0442] Example 14. Relationship between mRNA knockdown and serum glycolate levels in rats Male Sprague Dawley rats were administered a single subcutaneous dose of 1 mg / kg, 3 mg / kg, or 10 mg / kg of AD-65626 or a PBS control. After 14 days, the rats were sacrificed, and liver HAO1 mRNA knockdown was assessed using qPCR. Results were expressed relative to the PBS control. Glycolate levels in serum samples from these same rats collected before administration and on day 14 were quantified using ion chromatography with mass spectrometry, similar to that previously described (Knight et al., Anal. Biochem. 2012 February 1;421(1):121-124). The results of these experiments are shown in Figure 19.

[0443] As observed in wild-type mice, these results demonstrate that HAO1 mRNA silencing in Sprague Dawley rats results in a dose-response serum glycolate increase of up to 12-fold (approximately 140 μM) at the highest dose.

[0444] Example 15. Pharmacological testing with ALN-65585 Primary cynomolgus monkey hepatocytes were transfected at 10 nM with serial dilutions of AD-65585 (ALN-65585, "ALN-GO1") or a non-targeting mRNA luciferase control (AD1955) using RNAimax (Invitrogen). Relative levels of HAO1 mRNA were determined by normalizing to GAPDH mRNA levels quantified by real-time RT-PCR. Data were plotted to calculate an IC50 value of 10 pM. The results are shown in Figure 20.

[0445] In vitro transfection of AD-65585 demonstrates an ED50 of approximately 10 pM in primary cynomolgus monkey hepatocytes.

[0446] Single-dose pharmacology in mice The pharmacology of ALN-GO1 was evaluated in mice by quantifying liver HAO1 mRNA and serum glycolate levels (Figure 21). A single SC administration of ALN-GO1 resulted in dose-dependent suppression of HAO1 mRNA at a dose of 10 mg / kg, resulting in ED90 silencing. The ED50 dose for GO1 silencing in mice was estimated to be 0.3 mg / kg. Serum glycolate levels increased in a dose-response manner, with a maximum level approximately fourfold above baseline levels. The results are shown in Figure 21, which illustrates liver HAO1 mRNA and serum glycolate levels 10 days after a single subcutaneous administration of ALN-65585 in C57BL / 6 mice. Bars represent the average of three or four animals, and error bars indicate standard deviation.

[0447] Duration of single administration in mice GO1 silencing was sustained and reversible after a single SC administration (Figure 22). A single SC administration of 3 mg / kg of ALN-GO1 in mice resulted in over 70% mRNA silencing for approximately 6 weeks, followed by a return of mRNA levels to baseline levels 12 weeks after administration. The results are shown in Figure 22: Liver HAO1 mRNA levels at multiple time points after a single subcutaneous administration of ALN-65585 to C57BL / 6 mice. Each data point represents the average of three animals, and error bars indicate the standard deviation.

[0448] Single-dose pharmacology in rats The pharmacology of ALN-GO1 was also evaluated in rats by quantifying hepatic HAO1 mRNA levels (Figure 23). A single SC administration of ALN-GO1 to male Sprague Dawley rats resulted in dose-dependent suppression of HAO1 mRNA at 3 mg / kg or higher, resulting in ED90 silencing. The results are shown in Figure 23: HAO1 mRNA levels in liver 10 days after a single subcutaneous administration of ALN-65585 to Sprague Dawley rats. Bars represent the mean of three animals, and error bars indicate standard deviation. The ED50 dose for GO1 silencing in rats was estimated to be 0.3 mg / kg.

[0449] Single-dose pharmacology in AGXT KO mice The effect of ALN-GO1 on oxalate levels was evaluated in the AGXT KO mouse model of PH1. The results are shown in Figure 24: 24-hour urinary oxalate (top) and glycolate (bottom) excretion in Agxt KO mice after a single subcutaneous dose of ALN-65585. Different letters indicate significant differences among the three treatment groups (n=3 per treatment) in each particular week. Urinary excretion did not change significantly over time in PBS control animals (n=1).

[0450] Urinary oxalate levels showed a dose-dependent decrease after a single dose of ALN-GO1, with a maximum approximately 50% decrease in oxalate at a dose of 3 mg / kg, which took more than 3 weeks to return to pre-dose levels.Urinary glycolate levels showed a dose-dependent increase after a single dose of ALN-GO1, with a maximum approximately 5-fold increase at a dose of 3 mg / kg, which continued for more than 4 weeks.

[0451] Single-dose pharmacology in the PH1-induced rat model ALN-GO1 was evaluated in a second PH1 rodent model by inhibiting liver AGXT in rats using siRNA and stimulating oxalate levels with ethylene glycol (Figures 25A and 25B). Liver HAO1 mRNA and 24-hour urinary oxalate were quantified to determine the degree of HAO1 reduction required for maximal oxalate reduction. The results are shown in Figures 25A and 25B: Liver HAO1 mRNA levels in a rat induced model of PH1 after 14 days of a single subcutaneous dose of ALN-65585 and weekly administration of AF-011 AGXT siRNA (two doses of 1 mg / kg). 24-hour urinary oxalate was normalized to urinary creatinine. Bars represent the mean of three animals, and error bars indicate standard deviation. Correlation plots of mRNA and oxalate reduction represent individual animals from multiple experiments.

[0452] A single dose of ALN-GO1 in this model demonstrated a dose-responsive reduction in mRNA and urinary oxalate, with a maximum reduction in mRNA of approximately 85% and a maximum reduction in urinary oxalate of approximately 90% observed at the highest dose of ALN-GO1 (Figures 25A and 25B). In this rat model of PH1 induction, the reduction in mRNA and urinary oxalate showed a 1:1 correlation.

[0453] Multiple-dose pharmacology in the PH1-induced rat model The efficacy of ALN-GO1 was evaluated in normal rats (an induced model of PH1) in which AGXT activity and ethylene glycol were inhibited by quantifying hepatic HAO1 mRNA and 24-hour urinary oxalate. The results are shown in Figure 26: Hepatic HAO1 mRNA levels in a rat induced model of PH1 28 days after repeated subcutaneous administration of ALN-65585 and repeated IV administration of AF-011-AGXT siRNA (four doses of 1 mg / kg). 24-hour urinary oxalate was normalized to urinary creatinine. Bars represent the average of two or three animals, and error bars indicate standard deviation.

[0454] Treatment with ALN-GO1 sustained a reduction in urinary oxalate in all treatment groups for approximately 3 weeks. After 28 days of repeated ALN-GO1 administration (and four doses of AF-011-AGXT), all groups showed a >95% reduction in mRNA and a >85% reduction in urinary oxalate.

[0455] Multiple-dose pharmacology in NHPs The pharmacology of ALN-GO1 was evaluated in cynomolgus monkeys (non-human primates (NHPs)) by quantification of HAO1 mRNA in liver biopsies and serum glycolate levels. The table below provides a summary of the NHP pharmacology studies detailing the dose levels and dosing schedules.

[0456] [Table 39]

[0457] The results are shown in Figure 27. Serum glycolate levels in NHPs in all groups were obtained up to day 85; data represent group means of three animals per group, with lines indicating standard deviations. HAO1 mRNA in liver biopsies on day 29; lines represent group means, with symbols representing individual animal mRNA levels relative to PBS controls on day 29.

[0458] After the first month of treatment (day 29), dose-responsive mRNA silencing was observed in all groups, with a maximum of 99% mRNA silencing in groups 6 and 7 treated with 4 mg / kg monthly or 2 mg / kg weekly. Maximum elevated serum glycolate levels of approximately 70 μM persisted for at least 3 weeks in group 6 treated with 4 mg / kg monthly. Intermediate serum glycolate

[0459] Example 16: Additional siRNA sequences Further siRNA design was carried out to identify siRNAs targeting HAO1 NM_017545.2.

[0460] [Table 40]

[0461] Table 41

[0462] Table 42

[0463] Table 43

[0464] Table 44

[0465] Table 45

[0466] Table 46

[0467] Table 47

[0468] Table 48

[0469] Table 49

[0470] Table 50

[0471] Table 51

[0472] Table 52

[0473] Table 53

[0474] Table 54

[0475] Table 55

[0476] Table 56

[0477] Table 57

[0478] Table 58

[0479] Table 59

[0480] Table 60

[0481] Table 61

[0482] Table 62

[0483] Table 63

[0484] Table 64

[0485] Table 65

[0486] Table 66

[0487] Table 67

[0488] Table 68

[0489] Table 69

[0490] Table 70

[0491] Table 71

[0492] Table 72

[0493] Table 73

[0494] Table 74

[0495] Table 75

[0496] Table 76

[0497] Table 77

[0498] Table 78

[0499] Table 79

[0500] Table 80

[0501] Table 81

[0502] Table 82

[0503] Table 83

[0504] Table 84

[0505] Table 85

[0506] Table 86

[0507] Table 87

Claims

1. A composition for inhibiting the expression of hydroxy acid oxidase (HAO1) ​​in cells, comprising an oligonucleotide or a salt thereof containing a nucleotide sequence in which the nucleotide sequence 5'-usAfsuauUfuCfCfaggaUfgAfaagucscsa-3' of SEQ ID NO: 330 differs from three or fewer modified nucleotides, a, g, c, and u are 2'-O-methyl (2'-OMe) modified A, G, C, and U nucleotides, respectively; Af, Cf, and Uf are 2'-fluoro A, C, and U modified nucleotides, respectively; and s is a phosphorothioate bond. composition.

2. The composition according to claim 1, wherein the nucleotide sequence differs from the nucleotide sequence 5'-usAfsuauUfuCfCfaggaUfgAfaagucscsa-3' of SEQ ID NO: 330 by two or fewer modified nucleotides.

3. The composition according to claim 1, wherein the nucleotide sequence differs from the nucleotide sequence 5'-usAfsuauUfuCfCfaggaUfgAfaagucscsa-3' of SEQ ID NO: 330 by one or fewer modified nucleotides.

4. The composition according to claim 1, wherein the nucleotide sequence comprises the nucleotide sequence 5'-usAfsuauUfuCfCfaggaUfgAfaagucscsa-3' of SEQ ID NO:

330.

5. The composition according to claim 1, wherein the nucleotide sequence consists of the nucleotide sequence 5'-usAfsuauUfuCfCfaggaUfgAfaagucscsa-3' of SEQ ID NO:

330.

6. A composition for inhibiting the expression of hydroxy acid oxidase (HAO1) ​​in cells, comprising an oligonucleotide or a salt thereof containing at least 19 consecutive nucleotides from the nucleotide sequence 5'-usAfsuauUfuCfCfaggaUfgAfaagucscsa-3' of SEQ ID NO: 330, a, g, c, and u are 2'-O-methyl (2'-OMe) modified A, G, C, and U nucleotides, respectively; Af, Cf, and Uf are 2'-fluoro A, C, and U modified nucleotides, respectively; and s is a phosphorothioate bond. composition.

7. The composition according to claim 6, wherein the oligonucleotide or a salt thereof comprises at least 20 consecutive nucleotides from the nucleotide sequence 5'-usAfsuauUfuCfCfaggaUfgAfaagucscsa-3' of SEQ ID NO:

330.

8. The composition according to claim 6, wherein the oligonucleotide or a salt thereof comprises at least 21 consecutive nucleotides from the nucleotide sequence 5'-usAfsuauUfuCfCfaggaUfgAfaagucscsa-3' of SEQ ID NO:

330.

9. The composition according to claim 6, wherein the oligonucleotide or a salt thereof comprises at least 22 consecutive nucleotides from the nucleotide sequence 5'-usAfsuauUfuCfCfaggaUfgAfaagucscsa-3' of SEQ ID NO:

330.

10. A composition for inhibiting the expression of hydroxy acid oxidase (HAO1) ​​in cells, comprising an oligonucleotide or a salt thereof containing the nucleotide sequence 5'-usAfsuauUfuCfCfaggaUfgAfaagucscsa-3' of SEQ ID NO: 330, a, g, c, and u are 2'-O-methyl (2'-OMe) modified A, G, C, and U nucleotides, respectively; Af, Cf, and Uf are 2'-fluoro A, C, and U modified nucleotides, respectively; and s is a phosphorothioate bond. composition.

11. A composition for inhibiting the expression of hydroxy acid oxidase (HAO1) ​​in cells, comprising an oligonucleotide or a salt thereof consisting of the nucleotide sequence 5'-usAfsuauUfuCfCfaggaUfgAfaagucscsa-3' of SEQ ID NO: 330, a, g, c, and u are 2'-O-methyl (2'-OMe) modified A, G, C, and U nucleotides, respectively; Af, Cf, and Uf are 2'-fluoro A, C, and U modified nucleotides, respectively; and s is a phosphorothioate bond. composition.

12. A cell comprising the composition according to any one of claims 1 to 11.

13. A pharmaceutical composition for inhibiting the expression of hydroxy acid oxidase (HAO1) ​​in cells, comprising the composition described in any one of claims 1 to 11.

14. The pharmaceutical composition according to claim 13, wherein an oligonucleotide or a salt thereof is present in a non-buffer, and optionally the non-buffer is physiological saline or water.

15. An oligonucleotide or its salt is present in the buffer solution. The buffer may optionally contain acetate, citrate, prolamin, carbonate, phosphate, or any combination thereof, and / or If desired, the buffer solution may be phosphate-buffered saline (PBS). The pharmaceutical composition according to claim 13.

16. A pharmaceutical composition for use in treating subjects having HAO1-related disorders, the composition according to any one of claims 1 to 11 or the pharmaceutical composition according to any one of claims 13 to 15.

17. The pharmaceutical composition according to claim 16, wherein the subject is a human.

18. The pharmaceutical composition according to claim 17, wherein the human has primary oxaluria type 1 (PH1).

19. The pharmaceutical composition according to claim 16, wherein the composition or pharmaceutical composition is administered in two or more doses.

20. The pharmaceutical composition according to claim 16, wherein the composition or pharmaceutical composition is administered subcutaneously.