HAO1 (hydroxyacid oxidase 1 (glycolate oxidase)) method for inhibiting gene expression

Abstract

A method of treating a human subject having primary hyperoxaluria type 1 (PH1) is provided.SOLUTION: Provided are methods of inhibiting the expression of hydroxyacid oxidase (HAO1) using RNAi agents and methods of treating a subject having, for example, primary hyperoxaluria type 1 (PH1).SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application Nos. 62 / 532,176, filed July 13, 2017; 62 / 581,565, filed November 3, 2017; 62 / 646,285, filed March 21, 2018; and 62 / 682,020, filed May 7, 2018, the entire disclosures of each of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing containing 15 sequences submitted electronically herewith in ASCII format, which is incorporated by reference in its entirety. The ASCII copy, created on July 12, 2018, is entitled 40491WO_sequencelisting.txt and is 8045 bytes in size. [Background technology]

[0003] 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 expressed primarily in the liver and is the 2-hydroxyacid oxidase most active on 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 to 4). The compositions and methods are described in (Patent Document 1), filed October 9, 2015. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Application No. PCT / US2015 / 054881 [Non-patent literature]

[0009] [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]

[0010] The present invention provides methods of using RNAi agents, e.g., double-stranded iRNA agents, that target HAO1 to inhibit HAO1 expression, to treat HAO1-associated disorders, e.g., PH1, and to increase plasma glycolate and decrease urinary oxalate in a human subject. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the nucleotide sequence of human (Homo sapiens) HAO1 mRNA (SEQ ID NO: 1). [Figure 2]FIG. 2 shows the reverse complement of the nucleotide sequence of human (Homo sapiens) HAO1 mRNA (SEQ ID NO:2). [Figure 3] FIG. 3 is a graph showing dose-dependent inhibition of HAO1 mRNA by ALN-65585 in primary cynomolgus monkey hepatocytes. [Figure 4] FIG. 4 is two graphs showing HAO1 mRNA and serum glycolate levels after a single dose treatment with ALN-GO1 in mice. [Figure 5] FIG. 5 is a graph showing the duration of HAO1 mRNA silencing after a single dose treatment with ALN-GO1 in mice. [Figure 6] FIG. 6 is a graph showing HAO1 mRNA and serum glycolate levels after a single dose treatment with ALN-GO1 in rats. [Figure 7-1] FIG. 7 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 7-2] FIG. 7 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 8A] FIG. 8A is a graph showing HAO1 mRNA levels in a rat model of primary oxaluria type I after a single administration of ALN-GO1. [Figure 8B] FIG. 8B is a graph showing urinary oxalate levels in a rat model of primary oxaluria type I after a single administration of ALN-GO1. [Figure 9] FIG. 9 shows 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 10] FIG. 10 is two graphs showing HAO1 mRNA and urinary glycolate levels after repeated administration in non-human primates. [Figure 11]FIG. 11 is a graph showing plasma glycolate levels in healthy human subjects treated with HAO1 siRNA ALN-GO1. [Figure 12] FIG. 12 is a graph showing the recovery period of plasma glycolate in healthy human subjects treated with HAO1 siRNA ALN-GO1. [Figure 13] FIG. 13 is a graph showing 24-hour urinary oxalate levels in Cohort 1 after administration of ALN-GO1. [Figure 14] FIG. 14 is a graph showing 24-hour urinary oxalate levels in Cohort 2 after 29 days of administration of ALN-GO1. [Figure 15] FIG. 15 is a graph showing the reduction in urinary oxalate levels in patients after administration of ALN-GO1. [Figure 16] FIG. 16 is a graph showing years since diagnosis and ESRD. [Figure 17] FIG. 17 is a graph using the PK-PD model showing the dose-dependent increase in plasma glycolate levels. [Figure 18] Figure 18 is a graph showing oxalate response in PH1 patients using the PK-PD model. Solid line = median; shaded area = 5th to 95th percentiles; arrow = dose administration; symbols = observed values. Negative time values ​​represent time before the start of the active ALN-GO1 dosing regimen. Simulations were performed assuming a baseline urinary oxalate median of 1.7 (mmol / 24 h / 1.73 m). [Figure 19] Figure 19 shows a graph predicting the relationship between dose and steady-state GO enzyme inhibition in PH1 patients using a PK-PD model. Solid line = median; shaded area = 5th to 95th percentiles. The model-estimated inhibition of the rate of glycolate oxidation and the rate of oxalate degradation is considered to be the same as GO enzyme inhibition. [Figure 20]Figure 20 is a graph predicting the relationship between dose and steady-state reduction in urinary oxalate in PH1 patients using a PK-PD model. Light dotted line = 1.5 × ULN for oxalate: 0.7 mmol / 24 h / 1.73 m²; dark dotted line = ULN for urinary oxalate: 0.46 mmol / 24 h / 1.73 m²; solid line = median; shaded area = 5th to 95th percentile. DETAILED DESCRIPTION OF THE INVENTION

[0012] 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 expression of HAO1 and to treat HAO1-associated disorders.

[0013] 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.

[0014] 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.

[0015] The word "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0016] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless expressly stated to the contrary.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] "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 by other moieties without substantially altering the base pairing properties of oligonucleotides containing nucleotides having 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.

[0022] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent," used interchangeably herein, refer to an agent that contains RNA as defined herein and mediates the cleavage of a target RNA transcript through the RNA-induced silencing complex (RISC) pathway. iRNA induces sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates, e.g., inhibits, the expression of HAO1 in cells, e.g., cells of a subject, e.g., cells of a mammalian subject.

[0023] 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.

[0024] 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 is 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.

[0025] 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 a sequence that is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more contiguous nucleotides of any one of the antisense nucleotide sequences described herein, e.g., 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.

[0026] 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," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "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 a "sense" and "antisense" direction 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.

[0027] 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 include 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.

[0028] 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.

[0029] 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).

[0030] 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.

[0031] The term "antisense strand" refers to the strand of a double-stranded RNAi agent that includes 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 perfectly 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' termini.

[0032] 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.

[0033] As used herein, the term "cleavage region" refers to a 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 includes either end of the cleavage site and three bases immediately adjacent to the cleavage site. In some embodiments, the cleavage region includes 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 includes nucleotides 11, 12, and 13.

[0034] 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.

[0035] When there is base pairing between the nucleotides of the first and second nucleotide sequences 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 being "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 conditions most suitable for the final application. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs should not be considered as mismatches for determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides long and another oligonucleotide 23 nucleotides long, 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] As used herein, the term "inhibit" is used interchangeably with "reduce," "silencing," "downregulate," "suppress," and other similar terms, and includes all levels of inhibition.

[0040] As used herein, the phrase "inhibiting the 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.

[0041] "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.

[0042] Expression of the HAO1 gene can be assessed based on the level of any variable associated with HAO1 gene expression, such as the level of HAO1 mRNA or protein in a tissue, and / or the level of urinary oxalate. Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared 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 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).

[0043] As used herein, the phrase "contacting a cell with a double-stranded RNAi agent" includes contacting a cell by any possible means. Contacting a cell with a double-stranded RNAi agent includes contacting a cell with an RNAi agent in vitro or contacting a cell with an RNAi agent in vivo. Such contact can be performed directly or indirectly. Thus, for example, the RNAi agent can be physically contacted with the cell by the person performing the method, or the RNAi agent can be placed in a situation that allows or causes it to contact the cell later.

[0044] In vitro contacting of cells can be achieved, for example, by incubating the cells with an RNAi agent. In vivo contacting of cells can be achieved, for example, by injecting the RNAi agent into or near the tissue where the cells are located, or by injecting the RNAi agent into another region, the bloodstream, or the subcutaneous space so that the RNAi agent will subsequently reach the tissue where the contacted cells are located. For example, the RNAi agent can include and / or be bound to a ligand, such as a GalNAc3 ligand, that directs the RNAi agent to a desired site, for example, the liver. A combination of in vitro and in vivo contacting methods is also possible. In connection with the methods of the present invention, cells can be contacted with an RNAi agent in vitro and then transplanted into a subject.

[0045] As used herein, a "subject" includes a human or a non-human animal, preferably a vertebrate, more preferably a mammal. A subject may include a transgenic organism. Most preferably, a subject is a human, e.g., a human suffering from or susceptible to developing an HAO1-associated disorder.

[0046] 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.

[0047] 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).

[0048] 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., to 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 manner in 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 concurrent therapy (if any), and other personal characteristics of the patient being treated.

[0049] As used herein, a "prophylactically effective amount" is intended to include 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 does not show symptoms of the disease but who may develop the disease. Ameliorating the disease includes slowing 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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 duplexed 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.

[0058] 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.

[0059] In one embodiment, each nucleotide in the overhang region of an RNAi agent can independently be a modified nucleotide, including, but not limited to, a 2'-sugar modification, 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, or can be an unmodified nucleotide. 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.

[0060] 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.

[0061] RNAi agents can only contain 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.

[0062] 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, such as 5'-end modifications (phosphorylation, conjugation, reverse linkage) or 3'-end modifications (conjugation, DNA nucleotide, reverse linkage, etc.); base modifications, such as 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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; 5,4 Nos. 66,677; 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.

[0067] In other embodiments, suitable RNA mimics are contemplated 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.

[0068] Some embodiments featured herein include RNAs with phosphorothioate backbones, as well as oligonucleosides with heteroatom backbones, particularly -CH-NH-CH-, -CH-N(CH)-O-CH- (known as methylene(methylimino) or MMI backbones), -CH-ON(CH)-CH-, -CH-N(CH)-N(CH)-CH-, and N(CH)-CH-CH- (a natural phosphodiester backbone is represented as -OPO-CH-) 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, RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Patent No. 5,034,506.

[0069] 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.

[0070] 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 in 2'-5'-linked dsRNA and the 5' position of the 5'-terminal nucleotide. iRNAs can also have, for example, sugar mimetics, such as cyclobutyl moieties, 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.

[0071] iRNAs may also include 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. These include 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 U.S. 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.

[0072] Representative United States patents that teach the formation of the above specific modified nucleobases and other modified nucleobases include, but are not limited to, the above-mentioned U.S. Pat. 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 ,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.

[0073] The RNA of iRNA can also be modified to contain one or more locked nucleic acids (LNAs). 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).

[0074] 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.

[0075] 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). Disclosure of this modification can be found in PCT Publication WO 2011 / 005861.

[0076] Modified iRNAs containing the motifs of the present invention In certain aspects 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.

[0077] As shown herein and in U.S. 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 an RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of an RNAi agent can be completely modified by other methods. The introduction of these motifs can interrupt the existing modification pattern of the sense strand and / or antisense strand. The RNAi agent can optionally be conjugated to a GalNAc derivative ligand, for example, on the sense strand. The resulting RNAi agent exhibits excellent gene silencing activity.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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; and 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 two-nucleotide overhang. Preferably, the two-nucleotide overhang is at the 3'-end of the antisense strand. When the two-nucleotide overhang is at the 3'-end of the antisense strand, two phosphorothioate internucleotide bonds may be present between the three terminal nucleotides, two of the three nucleotides being overhanging nucleotides, and the third nucleotide being a pairing nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further comprises two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand. In one embodiment, each nucleotide in the sense strand and the antisense strand of the RNAi agent is a modified nucleotide, including a 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).

[0083] 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, the second strand 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 when the RNAi agent is introduced into a mammalian cell, it reduces expression of the target gene, 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.

[0084] 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.

[0085] 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.

[0086] In RNAi agents having a duplex region 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 within 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 RNAi agent at the 5' end.

[0087] 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 the strand; 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 the strand.When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be aligned so that one motif consisting of three nucleotides in the sense strand and one motif consisting of three nucleotides in the antisense strand overlap by at least one nucleotide, that is, at least one of the three nucleotides in the motif of the sense strand forms a base pair with at least one of the three nucleotides in the motif of the antisense strand.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0088] 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 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 that is present 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 near the first motif or separated by at least one or more nucleotides. If the motifs are closer to each other than they are chemically, the motifs are different from each other; if the motifs are separated by one or more nucleotides than they are chemically, 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 at one end of the first motif at or near the cleavage site, or on either side of the lead motif.

[0089] Like the sense strand, the antisense strand of an RNAi agent can contain 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 on the antisense strand. The antisense strand can also contain one or more wing modifications in a sequence similar to the wing modifications that may be present on the sense strand.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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 by one, two, or three nucleotides in the duplex region.

[0094] 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 alterations of one or both of the non-linked phosphate oxygen and / or one or more linking phosphate oxygens; alterations of 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.

[0095] 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 the nucleic acid. In some cases, modifications occur at all desired positions within the nucleic acid, but often not. For example, modifications may occur only at the 3' or 5' terminal position, or only in terminal regions, such as terminal nucleotide positions or the last two, three, four, five, or ten nucleotides of the chain. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in double-stranded regions of the RNA or only in single-stranded regions of the 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 the chain, or in both double-stranded and single-stranded regions, especially at the ends. The 5' end or both ends may be phosphorylated.

[0096] 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.

[0097] In one embodiment, each residue in the sense strand and the 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. The sense strand and the antisense strand may contain two or more modifications. In one embodiment, each residue in the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0098] At least two different modifications are typically present in the sense and antisense strands. These two modifications may be 2'-O-methyl or 2'-fluoro, or others.

[0099] 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.

[0100] 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...".

[0101] 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.

[0102] 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.

[0103] The introduction of one or more motifs consisting of three identical modifications at three consecutive nucleotides into the sense and / or antisense strands interrupts the initial modification pattern present in the sense and / or antisense strands. This interruption of the initial modification pattern present in the sense and / or antisense strands by the introduction of one or more motifs consisting of three identical modifications at three consecutive nucleotides into the sense and / or antisense strands surprisingly enhances gene silencing activity against the target gene.

[0104] 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 modification that is different from 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. a and / or N b may or may not be present if wing modifications are present.

[0105] The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide bond. The phosphorothioate or methylphosphonate internucleotide bond modification may be present at any nucleotide in the sense strand and / or antisense strand, or at any position in both strands. For example, the internucleotide bond modification may be present at any nucleotide in the sense strand and the antisense strand; each internucleotide bond modification may be present in an alternating pattern in the sense strand and / or the antisense strand; or the sense strand or the antisense strand may contain both internucleotide bond modifications in an alternating pattern. The alternating pattern of the internucleotide bond modification in the sense strand may be the same or different from that of the antisense strand, and the alternating pattern of the internucleotide bond modification in the sense strand may have a shift relative to the alternating pattern of the internucleotide bond modification in the antisense strand.

[0106] 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 link the overhang nucleotide 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 linked by phosphorothioate or methylphosphonate internucleotide bonds, and optionally, there can be an additional phosphorothioate or methylphosphonate internucleotide bond linking 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 after 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.

[0107] In one embodiment, the two-nucleotide overhang is at the 3'-end of the antisense strand, and two phosphorothioate internucleotide bonds are present between the terminal three nucleotides, two of which are overhanging nucleotides, and the third nucleotide is a 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.

[0108] In one embodiment, the RNAi agent contains mismatches with the target, mismatches within the duplex, or a combination thereof. Mismatches can occur in overhang regions or duplex regions. Base pairs may be ranked based on their propensity to promote dissociation or melting (e.g., relative to 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). With regard to 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.

[0109] 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.

[0110] In one embodiment, the nucleotide at position 1 in the duplex region from the 5' end of the 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 duplex region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the duplex region from the 5' end of the antisense strand is an AU base pair.

[0111] 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 -N a -nq 3' (I) During the ceremony, i and j are each independently 0 or 1; p and q each independently represent 0 to 6; each N a independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two 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.

[0112] In one embodiment, N a and / or N b includes alternating pattern modifications.

[0113] 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 17 to 23 nucleotides in length, the YYY motif can be located at or near the cleavage site of the sense strand (e.g., 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.

[0114] 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).

[0115] 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 that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0116] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0117] 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 that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0118] Each of X, Y, and Z may be the same as or different from one another.

[0119] 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).

[0120] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0121] 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.

[0122] In one embodiment, N a ' and / or N b ' includes alternating pattern modifications.

[0123] The Y'Y'Y' motif is present at or near the cleavage site of the antisense strand. For example, the RNAi agent has a duplex region 17 to 23 nucleotides in length, and the Y'Y'Y' motif can be present 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 duplex region from the 5' end. Preferably, the Y'Y'Y' motif is present at positions 11, 12, or 13.

[0124] In one embodiment, the Y'Y'Y' motif is a 2'-OMe modified nucleotide.

[0125] In one embodiment, k is 1 and l is 0, k is 0 and l is l, or both k and l are 1.

[0126] 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).

[0127] 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 represents 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 (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 represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0129] 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 N' 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.

[0130] 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).

[0131] When the antisense strand is represented by formula (IIa), each N a' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0132] Each of X', Y', and Z' may be the same as or different from one another.

[0133] Each nucleotide in the sense strand and the 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 in the sense strand and the antisense strand can be independently modified with 2'-O-methyl or 2'-fluoro. In particular, X, Y, Z, X', Y', and Z' can each represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0134] In one embodiment, the sense strand of the RNAi agent can include a YYY motif located 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.

[0135] 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.

[0136] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId).

[0137] 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' each independently represent 0 to 6; each Na 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.

[0138] 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; or k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0139] Exemplary combinations of sense and antisense strands that form RNAi duplexes include the following formulas: 5'n p -N a -YYY-N a -n q 3' 3'n p '-N a '-Y'Y'Y'-N a 'n q '5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'n p '-N a'-Y'Y'Y'-N b '-Z'Z'Z'-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)

[0140] 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.

[0141] When the RNAi agent is represented by formula (IIIb), each N b each independently represents 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.

[0142] When the RNAi agent is represented by formula (IIIc), each N b , N bEach 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 represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0143] When the RNAi agent is represented by formula (IIId), each N b , 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 , N a ' independently represents 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.

[0144] In formulae (III), (IIIa), (IIIb), (IIIc), and (IIId), X, Y, and Z may be the same as or different from each other.

[0145] 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.

[0146] 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, or at least two of the Z nucleotides can be base-paired with a corresponding Z' nucleotide; or all three of the Z nucleotides can be base-paired with a corresponding Z' nucleotide.

[0147] 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.

[0148] 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.

[0149] 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. 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, 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.

[0151] In one embodiment, the RNAi agent is a multimer comprising at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), the duplexes being 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; alternatively, each duplex may target the same gene at two different target sites.

[0152] In some embodiments, the RNAi agent is a multimer comprising three, four, five, six, or more duplexes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are joined 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; alternatively, each duplex may target the same gene at two different target sites.

[0153] 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.

[0154] A variety of publications describe multimeric RNAi agents and can be used in the method of the present invention.Such publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, each of which is incorporated herein by reference in its entirety.

[0155] 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.

[0156] 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.

[0157] The RNAi agent can be conjugated to the ligand by a carrier, which can be 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.

[0158] 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.

[0159] 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.

[0160] Further motifs In certain aspects, 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.

[0161] In certain aspects, 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.

[0162] In certain aspects, 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.

[0163] In certain aspects, 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.

[0164] 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.

[0165] Ligand The double-stranded RNAi agent of the present invention can be optionally conjugated to one or more ligands. The ligand can be attached to the sense strand, the antisense strand, or both strands at the 3'-end, the 5'-end, or both ends. For example, the ligand can be conjugated to the sense strand. In some embodiments, the ligand is conjugated to the 3'-end of the sense strand. In one embodiment, the ligand is a GalNAc ligand. In certain embodiments, the ligand is GalNAc3. The ligand is directly or indirectly bound, preferably covalently bound, via an intervening tether.

[0166] 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.

[0167] Some ligands may have endosomolytic properties. Endosomolytic ligands promote endosome lysis and / or 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 one in which the endosomolytic ligand promotes endosome lysis and / or 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.

[0168] 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.

[0169] Ligands may generally 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.

[0170] 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.

[0171] 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 acids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, biotin, RGD peptide, RGD peptidomimetic, or aptamer.

[0172] 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)cholenic 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.

[0173] 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.

[0174] 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.

[0175] Ligands can increase cellular uptake of oligonucleotides, for example, by activating the inflammatory response. Exemplary ligands that can have such an effect include tumor necrosis factor alpha (TNFα), interleukin-1β, or gamma interferon.

[0176] In one embodiment, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecule preferably binds to serum proteins, such as human serum albumin (HSA). The HSA-binding ligand allows the conjugate to distribute to target tissues, such as non-renal target tissues of the body. For example, the target tissue may be the liver, including liver parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. The lipid or lipid-based ligand can (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) be used to regulate binding to serum proteins, such as HSA.

[0177] 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 the kidney, and therefore is less likely to be removed from the body.The lipid or lipid-based ligand that binds less strongly to HSA can be used to make the conjugate target the kidney.

[0178] 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 preferably distributes to 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 preferably distributes to the kidney. Other moieties that target kidney cells can be used instead of or in addition to the lipid-based ligand.

[0179] In another aspect, the ligand is a moiety, e.g., 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 B vitamins, 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).

[0180] In another embodiment, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the agent is amphipathic. Exemplary agents are peptides, 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.

[0181] 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 translocating sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 3). A hydrophobic MTS-containing RFGF analog (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 4)) 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: 5) from the HIV Tat protein and the sequence (RQIKIWFQNRRMKWKK) (SEQ ID NO: 6) 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 an 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 facilitate 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 facilitate targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic and can be modified, e.g., glycosylated or methylated, to facilitate 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 that target 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.

[0182] 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, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0183] 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 helical 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 contemplated (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.

[0184] Peptide and peptidomimetic ligands include ligands having natural or modified peptides, such as D-peptides or L-peptides; alpha, beta, or gamma peptides; N-methyl peptides; azapeptides; peptides having one or more amide bonds replaced by one or more urea, thiourea, carbamate, or sulfonylurea bonds, i.e., peptidyl bonds; or cyclic peptides.

[0185] 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 the modifications disclosed herein.

[0186] 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.

[0187] A PK modulator refers to a pharmacokinetic modulator. PK modulators include lipophilics, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing multiple phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides containing multiple phosphorothioate linkages in the backbone, e.g., oligonucleotides of about 5, 10, 15, or 20 bases, are also applicable as ligands (e.g., as PK-modulating ligands) in the present invention.

[0188] In addition, aptamers that bind to serum components (eg, serum proteins) are also applicable to the present invention as PK-regulating ligands.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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 attached to a conjugate moiety. Conjugation to a pyrimidine nucleobase or its derivative can 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 attached to a conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be attached 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.

[0194] GalNAc Ligands and Linkers In some embodiments, siRNA targeting the HAO1 gene is conjugated to a carbohydrate, such as a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, or polysaccharide. In some embodiments, siRNA is conjugated to an N-acetylgalactosamine (GalNAc) ligand. This promotes efficient delivery to hepatocytes after subcutaneous administration. Methods for conjugating carbohydrates, such as N-acetylgalactosamine, to siRNA, for example, are well known to those skilled in the art. Examples can be found in U.S. Pat. No. 8,106,022 and WO 2014 / 025805.

[0195] 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.

[0196] 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), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , 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.

[0197] 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 5C represents a monosaccharide, for example, a GalNAc derivative.

[0198] Suitable bivalent and trivalent branched linker group conjugated GalNAc derivatives include, but are not limited to, the following compounds: [ka] [ka] [ka]

[0199] Further Ligands In some embodiments, the ligand is selected from one of the following: [ka] [ka]

[0200] 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 cell of 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 a cell with an iRNA of the invention in vitro or in vivo. In vivo delivery can also be achieved directly by administering a composition comprising 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.

[0201] 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., by 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, iRNAs can be delivered using drug delivery systems, such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate binding of iRNA molecules (which are negatively charged) and also facilitate interaction with the negatively charged cell membrane, allowing for efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can bind to iRNAs or be induced to form vesicles or micelles that surround the iRNA (see, e.g., Kim SH., et al. (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents degradation of iRNAs 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) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (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, iRNAs are complexed with cyclodextrins for systemic administration. Methods of administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, the entire disclosure of which is incorporated herein by reference.

[0202] 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, e.g., 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. Pat. 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).

[0203] Individual strands or multiple strands of iRNA can be transcribed from the promoter of an expression vector. For example, if two separate strands are expressed to form dsRNA, two separate expression vectors can be simultaneously introduced into target cells (e.g., by transfection or injection). Alternatively, each individual strand of dsRNA can be transcribed by a promoter located on the same expression plasmid. In one embodiment, the dsRNA is expressed as an inverted repeat polynucleotide linked by a linker polynucleotide sequence so that the dsRNA has a stem and loop structure.

[0204] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for expressing the iRNAs described herein can be generated using expression vectors compatible with eukaryotic cells, preferably vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from numerous commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of the iRNA expression vector can be systemic, for example, by intravenous or intramuscular administration, by administration to target cells removed from the patient and subsequently reintroduced into the patient, or by other means that allow for introduction into the desired target cells.

[0205] 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.

[0206] 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.

[0207] Vectors useful for delivery of iRNA contain sufficient regulatory elements (promoters, enhancers, etc.) for expression of the iRNA in the desired target cells or tissues. Regulatory elements can be selected to provide for constitutive or regulated / inducible expression.

[0208] iRNA expression can be precisely regulated, for example, by using inducible regulatory sequences that are sensitive to specific physiological regulators, such as circulating blood glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Suitable inducible expression systems for regulating dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, dimerization of chemical inducers, and isopropyl-β-D1-thiogalactopyranoside (IPTG). Those skilled in the art will be able to select appropriate regulatory / promoter sequences based on the intended use of the iRNA transgene.

[0209] 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 facilitate delivery of the nucleic acid to a patient. 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 a retroviral vector 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, 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.

[0210] 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 a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), provide a review 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 Publication 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.

[0211] 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. Pat. 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 WO 94 / 13788; and International Patent Application WO 93 / 24641, the entire disclosures of each of which are incorporated herein by reference.

[0212] Another viral vector suitable for delivery of iRNA of the invention is a poxvirus, such as a vaccinia virus, for example, an attenuated vaccinia, such as Modified Virus Ankara (MVA) or NYVAC, an avipox, such as canarypox or fowlpox.

[0213] The tropism of viral vectors can be altered by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by substituting various viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, and Mokola. 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.

[0214] 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.

[0215] IV. Pharmaceutical Compositions of the Present Invention The present invention also includes pharmaceutical compositions and pharmaceutical formulations comprising the iRNA of the present invention. In one embodiment, provided herein is a pharmaceutical composition comprising the iRNA described herein and a pharmaceutically acceptable carrier. 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.

[0216] Pharmaceutical compositions containing the RNAi agents of the present invention can be, for example, solutions with or without buffers, or compositions containing pharmaceutically acceptable carriers, including, for example, aqueous or crystalline compositions, liposomal formulations, micelle formulations, emulsions, and gene therapy vectors.

[0217] 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.

[0218] In some embodiments, the buffer further comprises an agent for controlling the osmolality of the solution so that the osmolality is maintained at a desired value, e.g., the physiological value of human plasma. Solutes that can be added to the buffer to control 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.

[0219] The pharmaceutical compositions of the present invention can be administered in a dose sufficient to inhibit the expression of the HAO1 gene.

[0220] dose In some embodiments, the siRNA, i.e., ALN-GO1, is administered to the patient at a dose of at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, or at least 5.0 mg of siRNA per kg of patient body weight.

[0221] In general, a suitable dose of an iRNA of the present invention ranges from about 0.001 to about 200.0 mg per kg of recipient body weight per day, and typically ranges from about 0.1 to 10 mg or 1 to 50 mg per kg of body weight per day. For example, a dsRNA 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.

[0222] In some embodiments, the siRNA is administered at a dose of at least 2.0 mg / kg or at least 5.0 mg / kg.

[0223] 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.

[0224] 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 this invention.

[0225] 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 to 0.5 mg / kg, about 0.01 to 0.4 mg / kg, about 0.01 to 0.3 mg / kg, about 0.01 to 0.2 mg / kg, about 0.01 to 0.1 mg / kg, about 0.01 mg / kg to 0.09 mg / kg, or about 0.01 mg / kg. kg~0.08mg / kg, approximately 0.01mg / kg~0.07mg / kg, approximately 0.01mg / kg~0.06mg / kg, approximately 0.01mg / kg~0.05mg / kg, approximately 0.02~0.5mg / kg, approximately 0.02~0.4mg / kg, approximately 0.02~0.3mg / kg, approximately 0.02~0.2mg / kg, approximately 0.02~0.1mg / kg, approximately 0.02mg / kg~0.09mg / kg, approximately 0.02mg / kg~0.08mg / kg, approximately 0.02mg / kg~0.07mg / kg, approximately 0.02mg / kg~0.06mg / kg, approximately 0.02mg / kg~0. 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 to 0.1 mg / kg, about 0.04 mg / kg to 0.09 mg / kg, about 0.04 mg / kg to 0.08 mg / kg, about 0.04 mg / kg to 0.07 mg / kg, about 0.04 mg / kg to 0.06 mg / kg, about 0.05 to 0.5 mg / kg, about 0.05 to 0.4 mg / kg, about 0.05 to 0.3 mg / kg, about 0.05 to 0.2 mg / kg, about 0.05 to 0.1 mg / kg, about 0.05 mg / kg to 0.09 mg / kg, about 0.05 mg / kg to 0.08 mg / kg, or about 0.05 mg / kg to 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.

[0226] 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 The compound may 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.

[0227] Treatment Plan Pharmaceutical compositions, such as siRNAs, may be administered once daily, or iRNAs may be administered in two or more subdoses at appropriate intervals throughout the day, or continuous infusion or continuous delivery may be used via controlled-release formulations. In this case, the amount of iRNA contained in each subdose must be correspondingly small to achieve the total daily dose. Dose units can also be formulated for delivery over several days, for example, using conventional sustained-release formulations that provide sustained release of 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 dose unit contains a corresponding multiple of the daily dose.

[0228] In other embodiments, a single administration of the pharmaceutical composition can be sustained such that subsequent administrations occur at intervals of not more than 3 days, not more than 4 days, or not more than 5 days, or not more than 1 week, not more than 2 weeks, not more than 3 weeks, or not more than 4 weeks. 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.

[0229] In some embodiments, the pharmaceutical composition, for example, a single dose of siRNA is administered once a month, for example, once a month for 3 months.In some embodiments, the pharmaceutical composition, for example, a single dose of siRNA is administered quarterly, for example, for at least 2 quarters, at least 3 quarters, at least 4 quarters, for example, once a quarter.

[0230] In some embodiments, the siRNA, i.e., ALN-GO1, is administered to the patient quarterly at a dose of at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, or at least 5.0 mg of siRNA per kg of patient body weight.

[0231] Those skilled in the art will understand that certain factors, including but not limited to, the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other diseases present, can affect the dosage and timing required to effectively treat a subject. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimation of the effective dose and in vivo half-life of individual iRNAs encompassed by the present invention can be performed using conventional methodology.

[0232] The effective dose and in vivo half-life of each iRNA encompassed by the present invention can also be estimated based on in vivo testing using an appropriate animal model. For example, advances in mouse genetics have led to the creation of numerous mouse models for the study of various human diseases, such as disorders associated with HAO1 expression. Such models can be used for in vivo testing of iRNAs and for determining therapeutically effective doses. Suitable mouse models are known in the art and include, for example, the animal models described herein.

[0233] Administration method The pharmaceutical compositions of the present invention can be administered in a variety of ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical (e.g., transdermal patch), pulmonary, by inhalation or injection, including by powder or aerosol atomizer; intratracheal, intranasal, epidermal, and transdermal; oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous, e.g., by implantable device; or intracranial, e.g., intraparenchymal, intrathecal, or intraventricular administration. In some embodiments, the method of administration is subcutaneous.

[0234] iRNA can be delivered in a manner that targets a specific tissue, for example, the liver.

[0235] 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.

[0236] The pharmaceutical formulations 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 the step of combining the active ingredient with pharmaceutical carriers or excipients. In general, the formulations are prepared by uniformly and intimately combining the active ingredient with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0237] 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. The suspension can also contain stabilizers.

[0238] The compositions of the present invention can be formulated for oral administration; parenteral, intraparenchymal (intracerebral), intrathecal, intracerebroventricular or intrahepatic administration, and / or topical administration.

[0239] Compositions and formulations for oral administration 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 one in which the dsRNA featured in 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 pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, a combination of penetration enhancers is used, such as a fatty acid / salt combined with a bile acid / salt. An exemplary combination is the sodium salt of lauric acid, capric acid, and UDCA. Additional penetration enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The dsRNA featured in the present invention can be orally delivered in granular form, including spray-dried particles, or complexed to form microparticles or nanoparticles.dsRNA complexing agent includes polyamino acid; polyimine; polyacrylate; polyalkyl acrylate, polyoxetane (polyoxethane), polyalkyl cyanoacrylate; cationized gelatin, albumin, starch, acrylate, polyethylene glycol (PEG) and starch; polyalkyl cyanoacrylate; DEAE-derivatized polyimine, pullulan, cellulose and starch.Suitable complexing agent includes chitosan, N-trimethyl chitosan, 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 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.

[0240] 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.

[0241] 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. Pat. No. 6,747,014, which is incorporated herein by reference.

[0242] 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 membrane is 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 of the liposome with a 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.

[0243] 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. An RNAi agent preparation is then 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.

[0244] If necessary, a carrier compound that aids in condensation can be added during the condensation reaction, for example, by controlled addition. For example, the carrier compound can be a polymer other than nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to suit condensation.

[0245] 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 The present invention may also include one or more embodiments of the exemplary methods 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 readily adapted for packaging RNAi agent preparations into liposomes.

[0246] 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).

[0247] 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 acids are 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).

[0248] 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.

[0249] 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.

[0250] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have also been tested to determine their usefulness in delivering drugs to the skin. Nonionic liposomal formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-steryl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-steryl ether) were used to deliver cyclosporine A to the dermis of mouse skin. The results showed that such nonionic liposomal systems were effective in promoting the deposition of cyclosporine A into various layers of the skin (Hu et al. STP Pharma. Sci., 1994, 4(6)466).

[0251] 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).

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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).

[0256] 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.

[0257] Other reported cationic lipid compounds include those conjugated to one of two types of lipids and conjugated to various moieties, including carboxyspermine containing compounds, such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

[0258] 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.

[0259] Liposome preparations are particularly suitable for topical administration, and liposomes have several advantages over other preparations.These advantages include the reduction of 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 the 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).

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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-activators, 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.

[0264] 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 the 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).

[0265] 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.

[0266] If surfactant molecule has negative charge when dissolved or dispersed in water, this surfactant molecule is classified as anionic.Anionic surfactants include carboxylates such as soaps, acyl lactates, acyl amides of amino acids, sulfuric acid esters such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.The most important members of anionic surfactant class are alkyl sulfates and soaps.

[0267] 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.

[0268] 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.

[0269] 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).

[0270] 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.

[0271] Mixed micelle formulations suitable for transdermal delivery are prepared by combining an aqueous solution of the siRNA composition with alkali metal ions (C8-C8). 22 They can be prepared by mixing alkyl sulfates with micelle-forming compounds. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, 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 ethers and their analogs, polidocanol alkyl ethers and their analogs, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle-forming compounds can be added simultaneously with or after the addition of alkali metal alkyl sulfates. Mixed micelles can be formed by mixing virtually any type of components, but vigorous mixing is required to produce smaller micelles.

[0272] In one method, a first micelle composition containing an siRNA composition and at least an alkali metal alkyl sulfate is prepared. The first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition. In another method, the micelle composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulfate, and at least one micelle-forming compound, and then adding the remaining micelle-forming compounds and vigorously mixing.

[0273] 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.

[0274] 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.

[0275] 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.

[0276] The specific concentrations of the essential ingredients 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.

[0277] 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.

[0278] 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 lifetimes 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.

[0279] 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, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or from about 6:1 to about 9:1. Ranges intermediate to the above-listed ranges are also considered part of the invention.

[0280] 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 analogs thereof , (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 from about 20 mol% to about 50 mol%, or about 40 mol% of the total lipid present in the particle.

[0281] 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.

[0282] 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.

[0283] 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-phosphatidylethanolamine (D ... The non-cationic lipid may be an anionic lipid or neutral lipid, 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 a mixture thereof. When cholesterol is included, the non-cationic lipid may comprise about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the particle.

[0284] 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 inhibits particle aggregation can be 0 mol% to about 20 mol%, or about 2 mol%, of the total lipid present in the particle.

[0285] 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.

[0286] 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.

[0287] Further exemplary lipid-dsRNA formulations are shown in Table A.

[0288] [Table 1]

[0289] [Table 2]

[0290] Abbreviations in Table A include the following: DSPC: distearoylphosphatidylcholine; DPPC: dipalmitoylphosphatidylcholine; PEG-DMG: PEG-dimyristoylglycerol (C14-PEG, or PEG-C14) (average 2000 molecular weight PEG); PEG-DSG: PEG-distyrylglycerol (C18-PEG, or PEG-C18) (average 2000 molecular weight PEG); PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (average 2000 molecular weight PEG).

[0291] 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.

[0292] 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.

[0293] 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.

[0294] 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.

[0295] 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.

[0296] 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.

[0297] Emulsions are characterized by little or no thermodynamic stability.In many cases, the dispersed or discontinuous phase of an emulsion is well dispersed in the external or continuous phase, and is maintained in this form by the viscosity of the emulsifier or 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).

[0298] Synthetic surfactants, also known as surface active agents, find widespread application in the formulation of emulsions and are 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; 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), which is a useful tool for 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).

[0299] 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.

[0300] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions, including 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).

[0301] 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, increasing the viscosity of the external phase and thereby stabilizing the emulsion.

[0302] Emulsions often contain 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 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 the preparation.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.

[0303] 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.

[0304] ii. Microemulsions In one embodiment of the present invention, iRNA and nucleic acid compositions are formulated as microemulsions. A microemulsion can be defined as a system of water, oil, and an amphiphile that is a single optically isotropic and thermodynamically stable solution (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Typically, microemulsions are systems prepared by first dispersing oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, typically a medium-chain alcohol, to form a clear system. Therefore, microemulsions are also described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of 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).

[0305] 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.

[0306] 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, typically 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.

[0307] Microemulsions are particularly important in terms of drug solubilization and enhanced 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. Pat. 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 advantages of improved drug solubilization, protection of drugs from enzymatic hydrolysis, potential enhanced drug absorption due to surfactant-mediated alterations of 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 form spontaneously 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.

[0308] 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.

[0309] iii. Particulates The RNAi agents of the present invention can be contained within particles, such as 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.

[0310] 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 readily cross cell membranes. It has been found that non-lipophilic drugs can also cross cell membranes when the membrane 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.

[0311] 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.

[0312] 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 mucosal 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).

[0313] 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, acylcarnitines, acylcholines, 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).

[0314] The physiological role of bile includes facilitating the dispersion and absorption of lipids and fat-soluble vitamins (see, e.g., 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 derivatives 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, Vol. 1, No. 1, pp. 111-114, 2002). 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).

[0315] 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).

[0316] As used herein, a non-chelating, non-surfactant penetration enhancer can be defined as a compound that exhibits minimal activity as a chelator or surfactant, yet still enhances the absorption of iRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancer includes, for example, unsaturated cyclic ureas, 1-alkyl-alkanone derivatives, and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 92); and nonsteroidal anti-inflammatory drugs, such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0317] Agents that promote cellular uptake of iRNA can also be added to the pharmaceutical 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 WO 97 / 30731), are known to promote 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), and HiFect™ (B-Bridge International, Mountain View, CA, USA).

[0318] 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.

[0319] v. Carrier Certain compositions of the invention also include a carrier compound in the formulation. As used herein, "carrier compound" or "carrier" can refer to nucleic acids or analogs thereof that 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).

[0320] 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).

[0321] Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not adversely react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0322] Formulations for topical administration of nucleic acids include, but are not limited to, sterile and non-sterile aqueous solutions, common solvents, non-aqueous solutions in alcohol, or solutions of nucleic acids in liquid or solid oil bases. These solutions may also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not adversely react with nucleic acids can be used.

[0323] 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.

[0324] vii. Other ingredients The compositions of the present invention may further comprise other adjuvant components conventionally found in pharmaceutical compositions at the use levels established in the art.Thus, for example, the compositions may comprise additional compatible pharmaceutically active substances, such as antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents, or may comprise additional substances useful for the physical formulation of various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers.However, such substances should not excessively inhibit the biological activity of the components of the compositions of the present invention when added.These formulations can be stabilized, and if desired, can be mixed with auxiliary substances that do not adversely interact with the nucleic acid of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, coloring agents, flavoring agents, and / or aromatic substances.

[0325] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0326] 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.

[0327] 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.

[0328] 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.

[0329] In addition to these administrations, as noted 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.

[0330] V. Methods of Inhibiting Expression of HAO1 The present invention provides a method for inhibiting expression of HAO1 (hydroxyacid oxidase 1, e.g., GO) in a cell, 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.

[0331] Contacting a cell with a double-stranded RNAi agent can be performed in vitro or in vivo. Contacting a cell with an RNAi agent in vivo includes contacting a cell or group of cells within the body of a subject, e.g., a human subject, with the RNAi agent. A combination of in vitro and in vivo contacting methods is also possible. Contacting can be performed directly or indirectly, as described above. Furthermore, contacting a cell can be accomplished by a targeting ligand, including any ligand described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc3 ligand, or other ligand that directs the RNAi agent to a site of interest, e.g., the liver of a subject.

[0332] As used herein, the term "inhibit" is used interchangeably with "reduce," "silencing," "downregulate," and other similar terms, and includes any level of inhibition.

[0333] 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.

[0334] "Inhibiting expression of the HAO1 gene" includes inhibition of any level of the HAO1 gene, for example, at least partial suppression of expression of the HAO1 gene. Expression of the HAO1 gene can be assessed based on the level of any variable associated with 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 assessed, for example, in an individual cell or a group of cells, including a sample derived from a subject.

[0335] 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).

[0336] 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%.

[0337] Inhibition of 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 (e.g., by contacting one or more cells with an RNAi agent of the invention, or by administering an RNAi agent of the invention to a subject in which the cells are present or were present) such that the expression of the HAO1 gene is inhibited compared to a second cell or group of cells that are substantially identical to the first cell or group of cells but are not treated (control cells). 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

[0338] Alternatively, inhibition of HAO1 gene expression can be assessed in terms of a parameter functionally linked to HAO1 gene expression, such as a decrease in HAO1 protein expression. HAO1 gene silencing can be determined by any assay known in the art in any cell that constitutively or genomically manipulated expresses HAO1. The liver is the primary site of HAO1 expression. Other important sites of expression include the kidney and uterus.

[0339] 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 a treated cell or group of cells can similarly be expressed as a percentage of the protein levels in a control cell or group of cells.

[0340] Control cells or control cell groups that can be used to evaluate the inhibition of HAO1 gene expression include cells or cell groups that have not been contacted with the RNAi agent of the present invention. For example, control cells or control cell groups can be derived from an individual subject (e.g., a human or animal subject) before the subject is treated with the RNAi agent.

[0341] 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, e.g., 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 utilizing 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.

[0342] 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 may be synthesized by those skilled in the art or derived from an appropriate biological specimen. Probes may 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.

[0343] 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. One method for determining mRNA levels involves contacting the isolated mRNA 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.

[0344] 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 (an experimental embodiment described in Mullis, 1987, 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).

[0345] The expression level of HAO1 mRNA can be monitored using membrane blots (such as those used in hybridization analyses, 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 involve using a nucleic acid probe in solution.

[0346] 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 provided herein.

[0347] 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 assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence analysis, and electrochemiluminescence analysis.

[0348] As used herein, the term "sample" 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 region. 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.

[0349] 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 a 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.

[0350] 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 expression of the HAO1 gene. For example, the compositions described herein can be used to treat any disorder associated with PH1.

[0351] 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.

[0352] The effectiveness of treatment or prevention is evidenced by a statistically significant improvement in one or more parameters of the disease state, or by the absence of a worsening or onset of symptoms that would normally be expected. As an example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, or 50% or more in a measurable parameter of the disease may indicate effective treatment. The effectiveness of a given iRNA drug or formulation of this drug 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 evidenced by the observation of a statistically significant decrease in a marker or symptom.

[0353] 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.

[0354] 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.

[0355] In some embodiments, efficacy is measured by plasma glycolate levels and / or urinary oxalate excretion and / or GO enzyme inhibition.

[0356] In some embodiments, plasma glycolate levels are increased by 20-40 days after administration, or by 29 days after administration; or are increased and sustained by 75 days, 85 days, or 95 days after administration. In some embodiments, plasma glycolate levels are increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold.

[0357] In some embodiments, the subject's urinary oxalate excretion is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least one-, two-, three-, four-, five-, six-, seven-, eight-, nine-, or ten-fold.

[0358] In some embodiments, the GO enzyme is inhibited by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold.

[0359] Administration The RNAi agents of the present invention can be administered by any administration method known in the art, including, for example, 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.

[0360] In some embodiments, the administration is by depot injection. Depot injections can consistently release the RNAi agent over an extended period of time. Thus, depot injections can reduce the frequency of administration required to achieve a desired effect, such as the desired inhibition of HAO1, or a therapeutic or prophylactic effect. Depot injections can also achieve a more consistent serum concentration. Depot injections can include subcutaneous or intramuscular injections. In some embodiments, the depot injection is a subcutaneous injection.

[0361] In some embodiments, the administration is via 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. Infusion pumps can be used for intravenous, subcutaneous, arterial, or epidural injections. 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.

[0362] Other administration methods include epidural administration, intracerebral administration, intraventricular administration, intranasal administration, intraarterial administration, intracardiac administration, intraosseous injection, intrathecal administration, intravitreal administration, and intrapulmonary administration. The administration method can be selected based on whether local or systemic treatment is desired and the area to be treated. The route and site of administration can be selected to improve targeting.

[0363] 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.

[0364] 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.

[0365] Generally, an iRNA agent does not activate the immune system, e.g., does not increase cytokine levels, e.g., TNF-α or IFN-α levels, e.g., increases TNF-α or IFN-α levels by less than 30%, less than 20%, or less than 10% of control cells treated with a control dsRNA, e.g., a dsRNA that does not target HAO1, as measured by an assay, e.g., an in vitro PBMC assay as described herein.

[0366] For example, a subject can be administered a therapeutic amount of an iRNA agent, e.g., 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, e.g., 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes. Administration can be repeated, for example, periodically, e.g., every two weeks (i.e., every two weeks) for one, two, three, four, or more months. After the initial treatment regimen, treatment can be administered less frequently. For example, after three months of biweekly administration, administration can be repeated once a month for six months, 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.

[0367] 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 in 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).

[0368] Patients who need HAO1 RNAi agents can be identified by taking a family history. A healthcare provider, such as a doctor, a nurse, or a family member, can take a family history before prescribing or administering HAO1 dsRNA. Before the HAO1 RNAi agent is administered to a patient, DNA testing can also be performed on the patient to identify mutations in the AGT1 gene. The diagnosis of PH1 can be confirmed by any test known to those skilled in the art.

[0369] A therapeutic or prophylactic effect is evidenced by a statistically significant improvement in one or more parameters of the disease state, or by the absence of a worsening or onset of symptoms that would normally be expected. By way of example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, or 50% or more in a measurable parameter of the disease, may indicate effective treatment. The efficacy of a given iRNA agent of the invention, or a formulation of this iRNA agent, can also be assessed using an experimental animal model of a given disease known in the art. When using an experimental animal model, the efficacy of the treatment is evidenced by the observation of a statistically significant reduction in a marker or symptom.

[0370] 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.

[0371] In some embodiments, the RNAi agent is administered in two or more separate doses. Where repeated or frequent infusions are desired, a delivery device, e.g., a pump, implantation of a semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal, or intracisternal), or a reservoir may be desirable. In some embodiments, the number or amount of subsequent administrations depends on achieving a 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 per week, twice per week, three times per week, four times per week, or five times per 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 set of administrations administered at relatively short intervals (e.g., about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, or about every 72 hours), followed by 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 later 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 later 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 a 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.

[0372] 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.

[0373] Any of these schedules can optionally be repeated for one or more iterations, the number of iterations 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.

[0374] 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.

[0375] VII. Kit The present invention also provides kits for using any of the iRNA agents and / or for performing any of the methods of the present invention. Such kits include one or more RNAi agents and instructions for use, e.g., for inhibiting expression of HAO1 in a cell by contacting the cell with an amount of the RNAi agent effective to inhibit expression of HAO1. The kits may optionally further include a means for contacting the cell with the RNAi agent (e.g., an injection device) or a means for measuring inhibition of HAO1 (e.g., a means for measuring inhibition of HAO1 mRNA or protein). Such a means for measuring inhibition of HAO1 may include a means for obtaining a sample from the subject, e.g., a plasma sample. The kits of the present invention may optionally further include a means for administering the RNAi agent to a subject or a means for determining a therapeutically or prophylactically effective amount.

[0376] 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.

[0377] In another aspect, the present invention relates to a method for treating a subject with a PH1 condition (lithiasis, particularly PH1). The diagnostic method comprises: (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 inhibition of the glycolate oxidase enzyme, which prevents the production of oxalate caused by the PH1 condition.

[0378] 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.

[0379] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one 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 practice or test the iRNAs 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, will control. Additionally, the materials, methods, and examples are merely illustrative and are not intended to be limiting. [Example]

[0380] Materials and Methods The following materials and methods were used in the examples: As used herein, "HAO" and "GO" are used interchangeably.

[0381] Synthesis of siRNA Single-stranded RNA was produced by solid-phase synthesis at the 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).

[0382] 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).

[0383] 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.

[0384] In some cases, duplexes (dsRNA) were synthesized more than once, with different batches having different extensions, for example, AD-62933.1 and AD-62933.2 are different batches of the same duplex.

[0385] 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 μg 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.

[0386] 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.

[0387] cDNA synthesis Synthesis of cDNA was performed using the ABI High Performance cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat #4368813).

[0388] 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.

[0389] 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: 7), R-TGGGTGTCGCTGTTGAAGTC (SEQ ID NO: 8), probe-CCAGGTGGTCTCCTCC (SEQ ID NO: 9)), 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.

[0390] 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.

[0391] The sense and antisense sequences of AD-1955 are: SENSE: 5'-cuuAcGcuGAGuAcuucGAdTsdT-3' (SEQ ID NO: 10); and ANTISENSE: 5'-UCGAAGuACUcAGCGuAAGdTsdT-3' (SEQ ID NO: 11).

[0392] [Table 3]

[0393] [Table 4]

[0394] [Table 5]

[0395] Example 1: ALN-65585 AD-65585 is a double-stranded siRNA targeting nucleotides 1341 to 1363 of the human HAO1 gene. "ALN-GO1" refers to the modified GalNAc version of AD-65585. Nucleotide monomer abbreviations are shown in Table B.

[0396] The sequence of each strand is as follows:

[0397] [Table 6]

[0398] Example 2. Pharmacological studies using ALN-65585 HAO1 inhibition in hepatocytes 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 3.

[0399] In vitro transfection of AD-65585 demonstrates an ED50 of approximately 10 pM in primary cynomolgus monkey hepatocytes.

[0400] Single-dose pharmacology in mice The pharmacology of ALN-GO1 was evaluated in mice by quantifying liver HAO1 mRNA and serum glycolate levels (Figure 4). 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 4, 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 mean of three or four animals, and error bars indicate standard deviation.

[0401] Duration of single administration in mice GO1 silencing was sustained and reversible after a single SC administration (Figure 5). 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 5: 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.

[0402] Single-dose pharmacology in rats The pharmacology of ALN-GO1 was also evaluated in rats by quantifying hepatic HAO1 mRNA levels (Figure 6). 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 6: 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.

[0403] 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 7: 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).

[0404] 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.

[0405] Single-dose pharmacology in the PH1-induced rat model ALN-GO1 was evaluated in a second rodent model of PH1 by inhibiting hepatic AGXT in rats using siRNA and stimulating oxalate levels with ethylene glycol (Figures 8A and 8B). Hepatic 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 8A and 8B: Hepatic 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.

[0406] A single dose of ALN-GO1 in this model demonstrated a dose-responsive reduction in mRNA and urinary oxalate, with a maximum reduction of approximately 85% in mRNA and approximately 90% in urinary oxalate observed at the highest dose of ALN-GO1 (Figures 8A and 8B). In this rat model of PH1 induction, the reduction in mRNA and urinary oxalate showed a 1:1 correlation.

[0407] 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 9: Hepatic HAO1 mRNA levels in a rat induced model of PH1 after 28 days of 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 mean of two or three animals, and error bars indicate standard deviation.

[0408] 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.

[0409] 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 shows a summary of the NHP pharmacology studies detailing the dose levels and dosing schedules.

[0410] [Table 7]

[0411] The results are shown in Figure 10. 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.

[0412] After the first month of treatment (day 29), dose-responsive mRNA silencing was observed in all groups, with up to 99% mRNA silencing in Groups 6 and 7, which received 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, which received 4 mg / kg monthly.

[0413] Example 3: ALN-GO1 Phase 1 / 2 Study in Human Subjects A single ascending dose (SAD) study was conducted in 32 healthy adult human volunteers using subcutaneously administered AD-65585 (ALN-65585, "ALN-GO1") or placebo. The dosing schedule was as follows: 0.3mg / kg×1 SC, N=8 1.0mg / kg×1 SC, N=8 3.0mg / kg×1 SC, N=8 6.0 mg / kg x 1 SC, N = 8

[0414] Safety, pharmacokinetics, and pharmacodynamics were assessed. No serious adverse events or discontinuations due to adverse events occurred. No other clinically significant changes in liver function, renal function, or hematological parameters were observed.

[0415] Serum glycolate levels were determined using methods described herein and / or methods known to those skilled in the art. The results are shown in Figures 11 and 12. Serum glycolate levels increased in a dose-dependent manner, with minimal activity at higher doses evident by day 29 post-dose and persisting through day 85. The lowest dose at which an increase in glycolate was observed was 1 mg / kg.

[0416] The results demonstrate a method for increasing plasma glycolate levels in a subject, comprising administering to the subject an effective amount of ALN-GO1 siRNA, thereby increasing the subject's plasma glycolate levels. The effective amount can be 1.0 mg / kg, 3.0 mg / kg, or 6.0 mg / kg. The ALN-GO1 siRNA can be administered subcutaneously.

[0417] Example 4: ALN-GO1 Phase 1 / 2 Study in PH1 Patients A multiple ascending dose (MAD) study was conducted in patients with PH1 using subcutaneously administered lumasiran, e.g., AD-65585 (ALN-65585, "ALN-GO1"), or placebo in a randomized 3:1, single-blind, placebo-controlled study.

[0418] The administration schedule was as follows: 1.0mg / kg, q28d×3 SC, N=4 3.0mg / kg, q28d×3 SC, N=4 3.0mg / kg, q84d×2 SC, N=4

[0419] PH1 patients were 6 to 64 years of age; had an eGFR (estimated glomerular filtration rate) greater than 45 ml / min / 1.73 m2; and a urinary oxalate excretion of 0.70 mmol / 24 h / 1.73 m2. 2 That was all.

[0420] Cohorts 1 and 2 have the following demographic composition:

[0421] [Table 8]

[0422] Safety was assessed. No serious adverse events or discontinuations due to adverse events occurred.

[0423] Cohort 1 was administered ALN-GO1 using the following dosing schedule: 1 mg / kg q28d x 3 doses. Urinary oxalate excretion levels were determined using methods described herein and / or known to those skilled in the art. The results are shown in Figure 13. Administration of ALN-GO1 reduced urinary oxalate excretion by more than 50%.

[0424] Cohort 2 received ALN-GO1 using the following dosing schedule: 3 mg / kg q28d x 3 doses. Urinary oxalate excretion levels were determined using methods described herein and / or known to those skilled in the art. The results are shown in Figure 14. After the first dose of ALN-GO1 or placebo, mean urinary oxalate excretion on day 29 decreased by an average of more than 50%. Placebo was included in the abstract because patients remain blinded in the ongoing study.

[0425] In all patients, administration of ALN-GO1 resulted in a urinary oxalate level of 1.6 mmol / 1.73 m 2 / 1.1mmol / 1.73m over 24 hours of baseline excretion 2In patients with PH who did not have ESRD at diagnosis, renal survival estimates were lower in patients with the highest levels of urinary oxalate excretion (Zhao et al. CJASN 2016;11:119-126).

[0426] This study demonstrated that multiple doses of AD-65585 (ALN-65585, "ALN-GO1") were well tolerated by PH1 patients without any drug-related SAEs or study discontinuations. Drug treatment significantly reduced urinary oxalate levels in all treated patients, highlighting the potential of substrate reduction therapy through RNAi-mediated glycolate oxidase inhibition.

[0427] First results part B: ALN-GO1 Phase 1 / 2 study in patients with PH1 Part B was a randomized (3:1 drug:placebo), single-blind, placebo-controlled evaluation of lumasiran in patients with PH1. Cohorts 1 and 2 received lumasiran (ALN-65585, "ALN-GOl") at 1 mg / kg or 3 mg / kg once every three months, respectively; Cohort 3 received 3 mg / kg twice quarterly. Eight additional patients received open-label lumasiran in each expansion of the first two cohorts, enrolling a total of 20 patients. Patients randomized to the placebo group also received subcutaneous lumasiran following placebo administration. The mean age of patients was 14.9 years (range: 6-43 years), and the mean estimated glomerular filtration rate (eGFR) was 77 mL / min / 1.73 m2 (range: 42-131 mL / min / 1.73 m2).

[0428] The inclusion criteria for patients were as follows: PH1; age 6–64 years; eGFR 45 ml / min / 1.73 m 2 Urinary oxalate excretion: 0.70 mmol / 24 hours / 1.73 m 2 That's all. The patient demographics were as follows:

[0429] [Table 9]

[0430] Results: Lumasiran demonstrated a mean maximum reduction of 65% in urinary oxalate in patients enrolled in cohorts 1-3 (N=12), all of whom achieved a mean reduction of 0.7 mmol / 24 h / 1.73 m Oxalate, a threshold associated with a slower rate of progression to end-stage renal disease (data not shown). 2 At day 85, patients receiving lumasiran with available data (N=9) maintained a mean reduction in urinary oxalate of 63% (range: 49-73%).

[0431] As shown in Figures 15 and 16, lumasiran demonstrated a baseline excretion of 1.6 mmol / 24 hours / 1.73 m 2 In all patients with the above, UOx (urinary oxalate) was 1.1 mmol / 24 h / 1.73 m 2 Renal survival was assessed by the urinary oxalate (UOx) excretion (mmol / 24 h / 1.73 m) at diagnosis. 2 Among PH patients without ESRD at diagnosis, renal survival estimates were lower in those with the highest levels of urinary oxalate excretion.

[0432] The following inclusion criteria will also be used: treatment of young patients includes those under 6 years of age; those 12 months of age or older with an eGFR of 45 mL / min / 1.73 m 2 >12 months of age or no renal dysfunction if there is evidence of systemic oxalosis. Treatment of patients with advanced renal disease includes all ages, and for those 12 months of age and older, an eGFR of 45 mL / min / 1.73 m 2 or less, and have renal dysfunction if they are less than 12 months old or have clinical evidence of systemic oxalosis.

[0433] Lumaciran (ALN-GO1) is an investigational subcutaneously administered RNAi therapeutic that reduces hepatic oxalate production in patients with primary hyperoxaluria type 1 (PH1). Multiple doses of lumaciran were well tolerated by PH1 patients without any drug-related SAEs or study discontinuations. Patients treated with lumaciran experienced substantial and sustained reductions in urinary oxalate, supporting RNAi-mediated inhibition of glycolate oxidase as a potent therapeutic for alleviating pathological overproduction of oxalate in this devastating disease. The potent and sustained reduction in urinary oxalate supports a quarterly subcutaneous dosing regimen. GO inhibition reduces and normalizes hepatic oxalate production levels, halting PH1 disease progression.

[0434] Example 5: Pharmacokinetic-Pharmacodynamic (PK-PD) Model of ALN-GO1, an Investigational RNAi Therapeutic for Primary Hyperoxaluria Type 1 (PH1) The objectives of this study were to predict ALN-GO1 and RISC concentration-time profiles in human liver and to quantify the ALN-GO1 dose-response for plasma glycolate elevation in healthy volunteers and urinary oxalate reduction in PH1 patients.

[0435] The glycolate response profile in healthy volunteers was provided by the results of the Phase I study (described above) and was adequately described by the PK-PD model, as shown in Figure 17. A dose-dependent increase in plasma glycolate levels was observed in healthy volunteers. At the 6 mg / kg dose, a 6.5-fold increase in glycolate from baseline was predicted, with a corresponding predicted GO suppression of approximately 85%.

[0436] The oxalate response of PH1 patients provided by the results of the Phase I study (described above) was adequately described by the PK-PD model, as shown in Figure 18. All patients treated with ALN-GO1 showed a decrease in urinary oxalate levels. After three monthly doses of 1 mg / kg, the peak urinary oxalate decrease is predicted to occur 2 months after the last dose, followed by a slow recovery to baseline. After three monthly doses of 1 mg / kg ALN-GO1, the model predicted a median maximum decrease in urinary oxalate of 56%.

[0437] The PK-PD model predicted the relationship between dose and steady-state GO enzyme inhibition in PH1 patients. As shown in Figure 19, monthly doses of 2 mg / kg or more and quarterly doses of 5 mg / kg or more are predicted to result in greater than 90% inhibition of the GO enzyme.

[0438] The PK-PD model predicted the relationship between dose and steady-state urinary oxalate reduction in PH1 patients. As shown in Figure 20, monthly doses of 2 mg / kg or more and quarterly doses of 5 mg / kg or more are expected to result in near-maximal suppression of urinary oxalate in PH1 patients. The simulation predicted a median baseline urinary oxalate of 2.0 (mmol / 24 h) / 1.73 m 2 ) was assumed.

[0439] The PK-PD model adequately explained the observed time course and interindividual variability of the increase in plasma glycolate in healthy volunteers and the decrease in urinary oxalate in patients with PH1. Administering ALN-GO1 at 2 mg / kg monthly or 5 mg / kg quarterly could inhibit the GO enzyme by more than 90%, resulting in a near-maximal decrease in urinary oxalate.

Claims

1. 1. A method of treating a human subject with primary hyperoxaluria type 1 (PH1), comprising subcutaneously administering to the subject quarterly a dose of at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, or at least 5.0 mg of ALN-GO1 per kg of the subject's body weight, thereby treating the subject, wherein the subject's urinary oxalate excretion is reduced by at least 50% after treatment, and / or the subject's plasma glycolate levels are increased and maintained for at least 75 days after treatment, and / or the subject's GO enzyme is inhibited by at least 90% after treatment compared to before treatment.

2. A method of treating a human subject having an HAO1-associated disorder, comprising administering to the subject an effective amount of ALN-GO1, thereby treating the subject, wherein the subject's urinary oxalate excretion is reduced by at least 50% after treatment, and / or the subject's plasma glycolate levels are increased and maintained for at least 75 days after treatment, and / or the subject's GO enzyme is inhibited by at least 90% after treatment compared to before treatment.

3. 3. The method of claim 2, wherein the HAO1-associated disorder is primary hyperoxaluria type 1 (PH1).

4. 1. A method for increasing plasma glycolate levels and / or decreasing urinary oxalate excretion and / or inhibiting GO enzymes in a human subject, comprising administering to the subject an effective amount of ALN-GO1, thereby increasing plasma glycolate levels and / or decreasing urinary oxalate excretion and / or inhibiting GO enzymes in the subject.

5. The method of claim 4, wherein the human subject has an HAO1-associated disorder.

6. 6. The method of claim 5, wherein the HAO1-associated disorder is primary hyperoxaluria type 1 (PH1).

7. the plasma glycolate level is a. increases by 20-40 days after administration, or by 29 days after administration; or b) The method of any one of claims 4 to 6, wherein the increase is maintained up to 75 days, 85 days or 95 days after administration.

8. 8. The method of any one of claims 4 to 7, wherein the subject's urinary oxalate excretion is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least one-, two-, three-, four-, five-, six-, seven-, eight-, nine-, or ten-fold.

9. 9. The method of any one of claims 4 to 8, wherein the GO enzyme is inhibited by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold.

10. The effective amount is a. about 0.01 mg ALN-GO1 / kg body weight to about 10 mg ALN-GO1 / kg body weight; or b. about 1 mg ALN-GO1 / kg body weight to about 10 mg ALN-GO1 / kg body weight; or c. 0.3 mg ALN-GO1 / kg body weight, 1.0 mg ALN-GO1 / kg body weight, 3.0 mg ALN-GO1 / kg body weight; or d. The method of any one of claims 2 to 9, wherein the ALN-GO1 is at least 2.0 mg / kg body weight, at least 5.0 mg / kg body weight, or at least 6.0 mg / kg body weight.

11. 11. The method of any one of claims 2 to 10, wherein the ALN-GO1 is administered monthly in three doses per month or quarterly in two doses per quarter.

12. The ALN-GO1 a. A monthly dose of at least 2.0 mg ALN-GO1 per kg of body weight; or b. The method of any one of claims 2 to 9, wherein the subject is administered a quarterly dose of at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, or at least 5.0 mg of ALN-GO1 per kg of body weight.

13. The method of any one of claims 2 to 12, wherein the administration is subcutaneous.

14. 14. The method of any one of claims 1 to 13, wherein the plasma glycolate level is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold.

15. 15. The method of any one of claims 1 to 14, further comprising determining the subject's plasma glycolate level using ion chromatography optionally coupled to mass spectrometry.

16. 16. The method of any one of claims 1 to 15, further comprising determining urinary oxalate excretion in the subject.

17. 17. The method of any one of claims 1 to 16, further comprising determining said GO enzyme inhibition in said subject.

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