CN116583602A

CN116583602A - G protein-coupled receptor 75 (GPR 75) iRNA compositions and methods of use thereof

Info

Publication number: CN116583602A
Application number: CN202180077184.XA
Authority: CN
Inventors: J·D·马克辛尼克; B·L·博斯特威克; A·卡斯托雷诺
Original assignee: Regeneron Pharmaceuticals Inc; Alnylam Pharmaceuticals Inc
Current assignee: Regeneron Pharmaceuticals Inc; Alnylam Pharmaceuticals Inc
Priority date: 2020-10-05
Filing date: 2021-10-04
Publication date: 2023-08-11

Abstract

The present application relates to RNAi agents, e.g., dsRNA agents, targeting the G protein-coupled receptor 75 (GPR 75) gene. The application also relates to methods of using such RNAi agents to inhibit GPR75 gene expression, and to methods of treating or preventing GPR 75-related diseases, such as weight disorders, e.g., obesity, in a subject.

Description

G protein-coupled receptor 75 (GPR 75) iRNA compositions and methods of use thereof

RELATED APPLICATIONS

The present application claims priority from U.S. provisional application No. 63/087,342 and U.S. provisional application No. 63/216,629, filed on 5 and 10 and 2021, respectively. The entire contents of each of the foregoing applications are incorporated herein by reference.

Background

G protein-coupled receptor 75 (GPR 75) is a member of the G protein-coupled receptor family. It contains the characteristics of most GPCRs, namely 7 transmembrane domains, an N-glycosylation site at the N-terminus and a number of serine and threonine phosphorylation sites at the C-terminus. Amino acid sequence analysis showed that GPR75 was most closely related to the putative C.elegans (Caenorhabditis elegans) neuropeptide Y receptor (24% homology), the rat galanin receptor type 3 (25% homology) and the porcine growth hormone secretagogue receptor type 1b (25% homology) (Tarttelin et al (1999) Biochem Biophys Res Commun.260:174-180). GPR75 is classified as a Gq-coupled class a orphan receptor, whose activation is associated with increased intracellular calcium and IP-1 accumulation. GPR75 is expressed in many tissues, in the brain, GPR75 is expressed in neocortex, entorhinal cortex, hippocampus, thalamus and hypothalamus.

Cytochrome P450-derived eicosanoids 20-hydroxyeicosatetraenoic acid (20-HETE) have been shown to bind to and activate the GPR75 receptor. 20-HETE is an omega-hydroxylated metabolite of arachidonic acid produced by the cytochrome P450 (CYP) 4A and 4F enzyme families. Clinical studies have shown that in obese and diabetic individuals, the levels of 20-HETE in urine and/or plasma are elevated and 20-HETE stimulates adipogenesis, promoting the onset of diabetes, inducing hyperglycemia and impeding the cellular action of insulin. In addition, mice overexpressing Cyp4a12-20-HETE synthase develop rapidly obesity, hyperglycemia, hyperinsulinemia, and impaired glucose tolerance when fed high fat foods. These animals also develop insulin resistance in skeletal muscle, liver and adipose tissue, manifested by impaired tyrosine phosphorylation of insulin receptors and insulin receptor substrates. In addition, 20-HETE has been shown to interfere with insulin signaling in a GPR75 dependent manner (Gilani, et al (2019) FASEB J.33 (S1): 514.8;Gilani,et al (2018) Am J Physiol Regul Integr Comp Physiol 315:R934-R944).

Weight disorders such as obesity are an increasingly serious health problem in many countries. Weight disorders such as obesity increase the risk of health problems such as insulin resistance, type 2 diabetes, heart disease, osteoarthritis, sleep apnea, and some forms of cancer. Reducing excessive body weight can significantly reduce the risk of these health problems. The main treatments for weight disorders such as obesity are diet and physical exercise, followed by weight loss drugs and surgery. There are some FDA approved weight-reducing drugs on the market, such as orlistat And sibutramine->However, neither of these drugs achieves the FDA-set weight loss goals. Furthermore, several candidate weight loss drugs, also known as appetite suppressants, have been suspended or eliminated at various stages of development due to their serious side effects. Furthermore, while there are many ways to lose initial weight, it is difficult to maintain weight for a long period of time. Many individuals who initially succeed in losing weight will then rebound in weight. In addition, morbid obese patients may require medications to maintain healthy body weight for long periods of time after successful bariatric surgery. However, there is no weight loss maintenance drug on the market.

Thus, there is an unmet need for an effective treatment of obesity, for example, an agent that can selectively and effectively sink the GPR75 gene using the RNAi machinery of the cell itself, has high bioactivity and in vivo stability, and can effectively inhibit the expression of the target GPR75 gene.

Disclosure of Invention

The present disclosure provides RNAi agent compositions that cause RNA-induced silencing complex (RISC) -mediated cleavage of an RNA transcript of a gene encoding G protein-coupled receptor 75 (GPR 75). The GPR75 gene can be located intracellular, e.g., in a subject (e.g., a human). The present disclosure also provides methods of using the RNAi agent compositions of the present disclosure to inhibit expression of the GPR75 gene or to treat a subject that would benefit from inhibiting or reducing expression of the GPR75 gene, e.g., a subject having a GPR 75-related disorder, e.g., a subject having a weight disorder (e.g., obesity), or a subject at risk of developing a weight disorder.

Thus, in one aspect, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting G protein coupled receptor 75 (GPR 75) gene expression, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having at least 90% nucleotide sequence identity to a portion of any one of SEQ ID NO:1 to SEQ ID NO:4 or to a portion of any one of SEQ ID NO:1 to SEQ ID NO:4, and wherein the antisense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0, 1, 2 or 3 mismatches to a nucleotide sequence of any one of SEQ ID NO:5 to SEQ ID NO:8 or to a portion of any one of SEQ ID NO:5 to SEQ ID NO:8, and wherein the antisense strand or the antisense strand comprises a nucleotide sequence comprising at least 90% consecutive nucleotides having at least 90% nucleotide sequence identity to a portion of any one of SEQ ID NO:5 to SEQ ID NO:8, and wherein the antisense strand is conjugated to one or more of the antisense strand.

In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a GPR75 gene in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region of complementarity to a portion of an mRNA encoding the GPR75 gene (any one of SEQ ID NOs: 1 to 4), wherein each strand is independently 14 to 30 nucleotides in length; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

In another aspect, the invention provides a double stranded RNAi agent for inhibiting expression of a GPR75 gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any of the antisense nucleotide sequences in any of tables 2, 3, 5 and 6, wherein each strand is independently 14 to 30 nucleotides in length; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

In one embodiment, the sense strand or the antisense strand is a sense strand or an antisense strand selected from the group consisting of any of the sense strands and antisense strands in any of tables 2, 3, 5, and 6.

In another aspect, the invention provides a double stranded RNAi agent for inhibiting expression of a G protein-coupled receptor 75 (GPR 75) gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence identical to SEQ ID NO:1 38-60, 50-72, 148-181, 153-175, 159-181, 228-250, 240-262, 341-363, 341-368, 346-368, 369-396, 369-391, 374-396, 388-410, 414-436, 424-461, 424-446, 424-451, 434-456, 439-461, 429-451, 457-504, 462-491, 482-504, 469-491, 457-479, 462-584, 475-497, 469-491, 509-537, 509-531, 515-537, 544-576 544-566, 549-571, 580-607, 580-602, 585-607, 595-617, 615-647, 615-637, 620-642, 620-647, 625-647, 773-806, 773-795, 778-800, 784-806, 837-872, 837-859, 843-872, 843-865, 850-872, 860-882, 889-911, 900-936, 900-922, 908-936, 908-930, 914-936, 938-990, 938-960, 943-965, 968-990, 1060-1101, 1060-1082, 1066-1088, 1073-1095, 1079-1101, 1097-1119, 1238-1260, 1268-1290, 1284-1393, 1284-1306, 1292-1393, 1292-1314, 1292-1383, 1292-1314, 1301-1323, 1307-1383, 1307-1342, 1307-1329, 1313-1335, 1371-1393, 1351-1373, 1320-1342, 1336-1358, 1345-1367, 1351-1373, 1361-1383, 1366-1388, 1393-1415, 1422-1463, 1422-1444, 1441-1463, 1487-1526, 1487-1509, 1493-1526, 1493-1515, 1493-1518-1520, 1504-1526, 1515-1557, 1515-1543, 1521-1522, 1535-1555, 1542-1549, and 1551-1549. 1559-1586, 1559-1581, 1564-1586, 1583-1629, 1583-1605, 1588-1610, 1595-1617, 1600-1629, 1600-1622, 1607-1629, 1624-1646, 1635-1657, 1672-1721, 1672-1710, 1677-1699, 1699-1721, 1672-1699, 1688-1710, 1672-1694, 1683-1705, 1693-1714, 1732-1754, 1744-1798, 1751-1773, 1758-1780, 1767-1789, 1776-1798, 1790-1818, 1790-1812, 1796-1818, 1808-1856, 1808-1848, 1808-1836, 1808-1848, 1814-1836, 1819-1841, 1834-1856, 1877-1882, 1882-20882, 1925-1923-1925 Nucleotides 1882-1904, 1887-1693, 1887-1909, 1898-1920, 1903-1925, 1908-1930, 1913-1935, 1913-1950, 1921-1943, 1928-1950, 1933-1955, 1941-1963, 1946-1968, 1953-1985, 1953-2082, 1953-1975, 1938-1985, 1958-1980, 1963-1985, 1968-1990, 1974-1996, 1974-2065, 1974-2082, 1974-2002, 1980-2002, 1985-2007, 1990-2012, 1990-2033, 1999-2021, 2005-2033, 2005-2027, 2011-2033, 2017-2039, 2025-2055, 2025-2057, 2033-2055, 2038-2050, 2043-2055, 2053-2055, 2058-2055, and 2058-2052, and 2054-2052, and the sequence of nucleotides of which is not more than 15 consecutive nucleotides of any one of the nucleotide or 15; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

In one embodiment, both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties.

In one embodiment, the lipophilic moiety has a lipophilicity of greater than 0 as measured by logKow.

In one embodiment, the double stranded RNAi agent has a hydrophobicity of greater than 0.2 as measured by unbound fraction in a plasma protein binding assay of the double stranded RNAi agent.

In one embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin.

In one embodiment, the dsRNA agent comprises at least one modified nucleotide.

In some embodiments, substantially all of the nucleotides of the antisense strand are modified nucleotides.

In another embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand comprise modifications.

In one embodiment, at least one of the modified nucleotides is selected from the group of: deoxynucleotides, 3' -terminal deoxythymine (dT) nucleotides, 2' -O-methyl modified nucleotides, 2' -fluoro modified nucleotides, 2' -deoxymodified nucleotides, locked nucleotides, unlocked nucleotides, conformational nucleotides, restricted ethyl nucleotides, abasic nucleotides, 2' -amino modified nucleotides, 2' -O-allyl modified nucleotides, 2' -C-alkyl modified nucleotides, 2' -hydroxy modified nucleotides, 2' -methoxyethyl modified nucleotides, 2' -O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising a non-natural base, tetrahydropyran modified nucleotides, 1, 5-anhydrohexitol modified nucleotides cyclohexenyl modified nucleotides, nucleotides comprising a 5' -phosphorothioate group, nucleotides comprising a 5' -methylphosphonate group, nucleotides comprising a 5' -phosphate or a nucleotide comprising a 5' -phosphate mimetic, nucleotides comprising a vinylphosphonate, nucleotides comprising an adenosine-diol nucleic acid (GNA), nucleotides comprising a thymine-diol nucleic acid (GNA) S-isomer, nucleotides comprising 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides comprising 2' -deoxythymidine-3 ' -phosphate, nucleotides comprising 2' -deoxyguanosine-3 ' -phosphate, 2' -O-hexadecyl nucleotides, nucleotides comprising 2' -phosphate, cytidine-2 ' -phosphate nucleotides, guanosine-2 ' -phosphate nucleotide, 2' -O-hexadecyl-cytidine-3 ' -phosphate nucleotide, 2' -O-hexadecyl-adenosine-3 ' -phosphate nucleotide, 2' -O-hexadecyl-guanosine-3 ' -phosphate nucleotide, 5' -Vinyl Phosphonate (VP), 2' -deoxyadenosine-3 ' -phosphate nucleotide, 2' -deoxycytidine-3 ' -phosphate nucleotide, 2' -deoxyguanosine-3 ' -phosphate nucleotide, 2' -deoxythymidine-3 ' -phosphate nucleotide, 2' -deoxyuridine nucleotide, and terminal nucleotide attached to cholesterol derivative and a lauric acid didecamide group; and combinations thereof.

In another embodiment, the modified nucleotide is selected from the group consisting of: 2' -deoxy-2 ' -fluoro modified nucleotides, 2' -deoxy modified nucleotides, 3' -terminal deoxy-thymine nucleotides (dT), locked nucleotides, abasic nucleotides, 2' -amino modified nucleotides, 2' -alkyl modified nucleotides, 2' -O-methyl modified nucleotides, nucleotides comprising a diol nucleic acid (GNA), morpholino nucleotides, phosphoramidates and nucleotides comprising an unnatural base.

In another embodiment, the modified nucleotide comprises a short sequence of 3' -terminal deoxythymidines (dT).

In another embodiment, the modifications on the nucleotides are 2' -O-methyl modifications, 2' -deoxy modifications, 2' -fluoro modifications, 5' -Vinyl Phosphonate (VP) modifications, and 2' -O hexadecyl nucleotide modifications.

In certain embodiments, the double stranded RNAi agent does not comprise an inverted abasic nucleotide.

In one embodiment, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage.

In one embodiment, the dsRNA agent comprises 6 to 8 phosphorothioate internucleotide linkages.

In one embodiment, each strand is no more than 30 nucleotides in length.

In one embodiment, at least one strand comprises a 3' overhang of at least 1 nucleotide.

In another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides.

The double-stranded region may be 15 to 30 nucleotide pairs, 17 to 23 nucleotide pairs, 17 to 25 nucleotide pairs, 23 to 27 nucleotide pairs, 19 to 21 nucleotide pairs, or 21 to 23 nucleotide pairs in length.

Each strand of the dsRNA agent may be 15 to 30, 17 to 20, 19 to 30 nucleotides in length; 19 to 23 nucleotides; or 21 to 23 nucleotides, for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

In certain embodiments, the double stranded RNAi agent further comprises a lipophilic ligand, e.g., a C16 ligand, conjugated to the 3' end of the sense strand through a monovalent or branched divalent or trivalent linker.

In one embodiment, the ligand is conjugated to a nucleotide or modified nucleotide 2' -position within the sense strand or the antisense strand. For example, the C16 ligand may be conjugated as shown in the following structure:

wherein represents a bond to an adjacent nucleotide and B is a nucleotide base or nucleotide base analogue, optionally B is adenine, guanine, cytosine, thymine or uracil.

In other embodiments, the agent further comprises a targeting ligand that targets liver tissue, such as one or more GalNAc derivatives, conjugated to the double stranded RNAi agent via a linker or carrier.

In other embodiments, the agent further comprises a lipophilic ligand (e.g., a C16 ligand) conjugated to the 3 'end of the sense strand through a monovalent or branched divalent or trivalent linker, and a targeting ligand (e.g., one or more GalNAc derivatives) that targets liver tissue conjugated to the 3' end of the sense strand through a monovalent or branched divalent or trivalent linker.

In one embodiment, one or more lipophilic moieties are conjugated to one or more internal positions on at least one chain.

In one embodiment, the one or more lipophilic moieties are conjugated to one or more internal positions on at least one chain via a linker or carrier.

In certain embodiments, the lipophilic moiety is not a cholesterol moiety.

In certain embodiments, the agent further comprises a targeting ligand that targets liver tissue, such as one or more GalNAc derivatives, optionally conjugated to the double stranded RNAi agent via a linker or carrier.

In other embodiments, the agent further comprises one or more lipophilic moieties, optionally conjugated to one or more internal nucleotide positions via a linker or carrier, and a targeting ligand (e.g., one or more GalNAc derivatives) that targets liver tissue, optionally conjugated to the double stranded RNAi agent via a linker or carrier.

In one embodiment, the internal locations include all but two locations from the end of each end of the at least one strand.

In another embodiment, the internal locations include all but three locations from the end of each end of the at least one strand.

In another embodiment, the internal position does not include a cleavage site region of the sense strand.

In another embodiment, the internal positions include all positions except positions 9 to 12 counted from the 5' end of the sense strand. In certain embodiments, the sense strand is 21 nucleotides in length.

In one embodiment, the internal positions include all positions except positions 11 to 13 counted from the 3' end of the sense strand. Optionally, the internal position does not include a cleavage site region of the antisense strand. In certain embodiments, the sense strand is 21 nucleotides in length.

In one embodiment, the internal position does not include a cleavage site region of the antisense strand.

In one embodiment, the internal positions include all positions except positions 12 to 14 counted from the 5' end of the antisense strand. In certain embodiments, the antisense strand is 23 nucleotides in length.

In one embodiment, the internal positions include all positions except positions 11 to 13 on the sense strand counted from the 3 'end and positions 12 to 14 on the antisense strand counted from the 5' end. In certain embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In one embodiment, the one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of: positions 4 to 8 and 13 to 18 on the sense strand and positions 6 to 10 and 15 to 18 on the antisense strand counted from the 5' end of each strand.

In one embodiment, the one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of: positions 5, 6, 7, 15 and 17 on the sense strand and positions 15 and 17 on the antisense strand counted from the 5' end of each strand. In certain embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In one embodiment, the position in the double stranded region does not include the cleavage site region of the sense strand.

In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand, or to position 16 of the antisense strand.

In one embodiment, the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1 or position 7 of the sense strand.

In one embodiment, the lipophilic moiety is conjugated to position 21, position 20 or position 15 of the sense strand.

In one embodiment, the lipophilic moiety is conjugated to position 20 or position 15 of the sense strand.

In one embodiment, the lipophilic moiety is conjugated to the antisense strand at position 16.

In one embodiment, the lipophilic moiety is an aliphatic compound, a cycloaliphatic compound, or a multi-cycloaliphatic compound.

In one embodiment, the lipophilic moiety is selected from the group consisting of: lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranyloxy hexanol, hexadecyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) cholanic acid, dimethoxytrityl, or phenoxazine. In certain embodiments, the lipophilic moiety is not cholesterol.

In one embodiment, the lipophilic moiety comprises a saturated C4-C30 hydrocarbon chain or an unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of: hydroxyl, amine, carboxylic acid, sulfonate, phosphoric acid, thiol, azide, and alkyne.

In one embodiment, the lipophilic moiety comprises a saturated C6-C18 hydrocarbon chain or an unsaturated C6-C18 hydrocarbon chain.

In one embodiment, the lipophilic moiety comprises a saturated C16 hydrocarbon chain or an unsaturated C16 hydrocarbon chain.

In one embodiment, the saturated or unsaturated C16 hydrocarbon chain is conjugated to position 6 counted from the 5' end of the chain.

In one embodiment, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides in one or more internal positions or double stranded regions.

In one embodiment, the carrier is selected from the group consisting of cyclic groups consisting of: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl (piperazinyl), [1,3] dioxolyl, oxazolidinyl (oxazolidinyl), isoxazolidinyl (isozolidinyl), morpholinyl, thiazolidinyl (thiazolidinyl), isothiazolidinyl, quinoxalinyl (quinoxalinyl), pyridazinonyl (pyridazinonyl), tetrahydrofuranyl, and decalinyl; or the carrier is an acyclic moiety based on a serinol backbone or a diethanolamine backbone.

In one embodiment, the lipophilic moiety is conjugated to the double stranded iRNA agent through a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, product of a click reaction, or carbamate.

In one embodiment, the lipophilic moiety is conjugated to a nucleobase, sugar moiety or internucleoside linkage (internucleosidic linkage).

In one embodiment, the double stranded RNAi agent further comprises a phosphate or phosphate mimetic at the 5' end of the antisense strand. Optionally, the phosphate mimic is 5' -Vinyl Phosphonate (VP).

In certain embodiments, the RNAi agent does not comprise an inverted abasic nucleotide.

In certain embodiments, the double stranded RNAi agent does not comprise a targeting ligand.

In certain embodiments, the double stranded RNAi agent further comprises a targeting ligand, e.g., a lipophilic ligand, that targets a receptor that mediates delivery to liver tissue. In certain embodiments, the targeting ligand is a C16 ligand. In certain embodiments, the lipophilic ligand is not a cholesterol moiety.

In one embodiment, the lipophilic moiety or targeting ligand is conjugated through a bio-cleavable linker selected from the group consisting of: DNA, RNA, disulfides, amides, galactosamine, glucosamine, glucose, galactose, mannose, functional mono-or oligosaccharides and combinations thereof.

In one embodiment, the 3' end of the sense strand is protected by an end cap, which is a cyclic group with an amine, selected from the group consisting of: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, decalinyl.

In one embodiment, the dsRNA agent further comprises a targeting ligand that targets liver tissue.

In one embodiment, the targeting ligand is a GalNAc conjugate.

In one embodiment, the dsRNA agent further comprises a terminal chiral modification of a phosphorus atom of the bond having the Sp configuration occurring at the first internucleotide linkage at the 3' end of the antisense strand, a terminal chiral modification of a phosphorus atom of the bond having the Rp configuration occurring at the first internucleotide linkage at the 5' end of the antisense strand, and a terminal chiral modification of a phosphorus atom of the bond having the Rp configuration or the Sp configuration occurring at the first internucleotide linkage at the 5' end of the sense strand.

In one embodiment, the dsRNA agent further comprises a terminal chiral modification of a phosphorus atom of the Sp configuration occurring at the first and second internucleotide linkages of the 3' end of the antisense strand, a terminal chiral modification of a phosphorus atom of the Rp configuration occurring at the first internucleotide linkage of the 5' end of the antisense strand, and a terminal chiral modification of a phosphorus atom of the Rp configuration or of the Sp configuration occurring at the first internucleotide linkage of the 5' end of the sense strand.

In one embodiment, the dsRNA agent further comprises a terminal chiral modification of a phosphorus atom of the Sp configuration occurring at the first, second and third internucleotide linkages of the 3' end of the antisense strand, a terminal chiral modification of a phosphorus atom of the Rp configuration occurring at the first internucleotide linkage of the 5' end of the antisense strand, and a terminal chiral modification of a phosphorus atom of the Rp configuration or the Sp configuration occurring at the first internucleotide linkage of the 5' end of the sense strand.

In one embodiment, the dsRNA agent further comprises a terminal chiral modification of a phosphorus atom of the Sp configuration occurring at the first and second internucleotide linkages of the 3 'end of the antisense strand, a terminal chiral modification of a phosphorus atom of the Rp configuration occurring at the third internucleotide linkage of the 3' end of the antisense strand, a terminal chiral modification of a phosphorus atom of the Rp configuration occurring at the first internucleotide linkage of the 5 'end of the antisense strand, and a terminal chiral modification of a phosphorus atom of the Rp configuration or of the Sp configuration occurring at the first internucleotide linkage of the 5' end of the sense strand.

In one embodiment, the dsRNA agent further comprises a terminal chiral modification of a phosphorus atom of the Sp configuration occurring at the first and second internucleotide linkages of the 3' end of the antisense strand, a terminal chiral modification of a phosphorus atom of the Rp configuration occurring at the first and second internucleotide linkages of the 5' end of the antisense strand, and a terminal chiral modification of a phosphorus atom of the Rp configuration or the Sp configuration occurring at the first internucleotide linkage of the 5' end of the sense strand.

In one embodiment, the dsRNA agent further comprises a phosphate or phosphate mimetic at the 5' end of the antisense strand.

In one embodiment, the phosphate mimic is a 5' -Vinylphosphonate (VP). When the phosphate mimetic is 5 '-Vinylphosphonate (VP), the 5' -terminal nucleotide may have the following structure,

wherein represents the position linked to the 5' position of the adjacent nucleotide;

r is hydrogen, hydroxy, methoxy, fluoro, or another 2' -modification described herein (e.g., hydroxy or methoxy); and is also provided with

B is a nucleotide base or modified nucleotide base, optionally wherein B is adenine, guanine, cytosine, thymine or uracil.

In one embodiment, the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair.

In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

The invention also provides cells, pharmaceutical compositions, and pharmaceutical compositions comprising the dsRNA agents of the invention for inhibiting the expression of the GPR75 gene in cells.

In one aspect, the invention provides methods of inhibiting the expression of the GPR75 gene in a cell. The method comprises contacting the cell with a dsRNA agent of the invention or a pharmaceutical composition of the invention; and maintaining the cells produced in step (a) for a time sufficient to obtain degradation of mRNA transcripts of the GPR75 gene, thereby inhibiting expression of the GPR75 gene in the cells.

In one embodiment, the cell is in a subject.

In one embodiment, the subject is a human.

In one embodiment, the expression of the GPR75 gene is inhibited by at least 50%.

In one aspect, the invention provides a method of treating a subject having a GPR 75-related disorder, e.g., a weight disorder such as obesity, or a subject at risk of developing a weight disorder, e.g., a subject at risk of becoming obese, e.g., a subject that is overweight or obese that is losing weight but fails to maintain weight loss. The method comprises administering to the subject a therapeutically effective amount of a dsRNA agent of the invention or a pharmaceutical composition of the invention, thereby treating the subject.

In one embodiment, the subject is a human.

In one embodiment, the treatment comprises ameliorating at least one sign or symptom of the disease. In some embodiments, administration of the dsRNA agent results in a decrease in BMI of the subject. In some embodiments, administration of the dsRNA agent results in a decrease in blood glucose level of the subject. In other embodiments, administration of the dsRNA agent results in a decrease in blood lipid levels in the subject.

In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01mg/kg to about 50 mg/kg.

In some embodiments, the double stranded RNAi agent is administered intrathecally to the subject.

In some embodiments, the double stranded RNAi agent is administered to the subject subcutaneously.

In one embodiment, the method further comprises administering to the subject an additional agent or therapy suitable for treating or preventing a GPR 75-related disorder.

In one embodiment, the additional therapeutic agent is selected from the group consisting of: a therapeutic agent for diabetes, a therapeutic agent for diabetic complications, a therapeutic agent for cardiovascular disease, an anti-hyperlipidemia agent, a antihypertensive or antihypertensive agent, an anti-obesity agent, a therapeutic agent for non-alcoholic steatohepatitis (NASH), a chemotherapeutic agent, an immunotherapeutic agent, an immunosuppressant, an anti-inflammatory agent, an anti-steatosis agent, an anti-fibrosis agent, an immunomodulator, a tyrosine kinase inhibitor, an anti-fibrosis agent, and a combination of any of the foregoing.

The invention is further illustrated by the following detailed description.

Drawings

Fig. 1 is a graph depicting the relative quantification of Gpr75 mRNA levels normalized to ActB and Gapdh in the brains of diet-induced obese mice 21 days after a single 150 μg dose of the indicated duplex or control injected intraventricularly. * Indicating P <0.05 compared to control siRNA.

Detailed Description

The present invention provides iRNA compositions that cause RNA-induced silencing complex (RISC) -mediated cleavage of RNA transcripts of the GPR75 gene. The GPR75 gene can be intracellular, e.g., in a subject (e.g., human). The use of these irnas enables targeted degradation of mRNA of the corresponding gene (GPR 75 gene) in mammals. The disclosure also provides methods of using the RNAi compositions of the disclosure to inhibit expression of the GPR75 gene for treating a subject suffering from a disorder that would benefit from inhibiting or reducing expression of the GPR75 gene (e.g., a GPR 75-related disorder, such as a weight disorder, e.g., obesity), or a subject at risk of developing a weight disorder, such as obesity, e.g., an overweight subject or an overweight or obese subject who is weight-reducing but fails to maintain weight-loss.

The iRNA of the present invention comprises an RNA strand (antisense strand) having a region of up to about 30 nucleotides or less in length, for example 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or a region of length 21 to 22 nucleotides, which is at least a portion of the gene is substantially complementary to the transcribed mRNA. In certain embodiments, RNAi agents of the present disclosure comprise an RNA strand (antisense strand) having a region of about 21-23 nucleotides in length that is substantially complementary to at least a portion of the mRNA transcript of the GPR75 gene.

In certain embodiments, one or both strands of a double stranded RNAi agent of the invention have a length of up to 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 consecutive nucleotides that is substantially complementary to at least a portion of the mRNA of the GPR75 gene. In some embodiments, such iRNA agents having a longer antisense strand may, for example, comprise a second RNA strand (sense strand) that is 20 to 60 nucleotides in length, wherein the sense strand and the antisense strand form a duplex of 18 to 30 consecutive nucleotides.

The use of the iRNA of the invention is capable of targeted degradation of GPR75 mRNA in mammals. Thus, methods and compositions comprising these irnas are useful for treating a subject suffering from a GPR 75-related disorder (e.g., a weight disorder, such as obesity), or a subject at risk of developing a weight disorder (e.g., obesity), such as an overweight subject or a subject who is weight-reducing but fails to maintain weight-reducing overweight or obesity.

The following detailed description discloses compositions, uses, and methods of how to make and use iRNA-containing compositions to inhibit GPR75 gene expression, as well as for treating subjects (e.g., subjects susceptible to, or diagnosed with, a GPR 75-related disorder) who would benefit from inhibition and/or reduction of GPR75 gene expression.

I. Definition of the definition

In order that the invention may be more readily understood, certain terms are first defined. Furthermore, it should be noted that whenever values or ranges of values for parameters are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of the present invention.

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

The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to.

The term "or" is used herein to mean and be used interchangeably with the term "and/or" unless the context clearly indicates otherwise.

The term "about" is used herein to mean within typical tolerances in the art. For example, "about" may be understood as differing from the average by about 2 standard deviations. In certain embodiments, "about" refers to ± 10%. In certain embodiments, "about" refers to ± 5%. When "about" occurs before a series of numbers or ranges, it is to be understood that "about" can modify each number in the series or range.

The term "at least", "not less than" or more than "preceding a number or a series of numbers is understood to include the number adjacent to the term" at least ", as well as all subsequent numbers or integers that may be logically included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21 nucleotide nucleic acid molecule" means that 18, 19, 20 or 21 nucleotides have the indicated properties. When "at least" occurs before a series of numbers or ranges, it is to be understood that "at least" can modify each number in the series or ranges.

As used herein, "no more than" or less than "is understood to mean logically contiguous values and logically smaller values or integers, from the context, up to zero. For example, a duplex with a "no more than 2 nucleotides" overhang has a 2, 1, or 0 nucleotide overhang. When "no more than" occurs before a series of numbers or ranges, it is to be understood that "no more than" can modify each number in the series or ranges. As used herein, a range includes upper and lower limits.

As used herein, a detection method may include determining that an amount of analyte present is below a detection level of the method.

In the event of a conflict between the nucleotide sequences of the designated target site and the sense or antisense strand, preference is given to the designated sequence.

In the event of a conflict between a sequence and its designated site on a transcript or other sequence, the nucleotide sequences referred to in the specification take precedence.

As used herein, the term "G protein-coupled receptor 75" ("GPR 75") refers to genes and polypeptides that are well known in the art also referred to as "possible G protein-coupled receptors 75", "WI-31133", "GPRchr2" and "WI 31133". GPR75 binds to 20-HETE and interferes with insulin signaling leading to obesity.

The term "GPR75" includes human GPR75, the amino acid and nucleotide sequences of which are found, for example, in GenBank accession No. NM-006794.4 (SEQ ID NO: 1); mouse GPR75, the amino acid and nucleotide sequences of which can be found, for example, in GenBank accession No. NM-175490.4 (SEQ ID NO: 2); and rat GPR75, the amino acid and nucleotide sequences of which are found, for example, in GenBank accession No. NM-001109096.1 (SEQ ID NO: 3).

The term "GPR75" also includes macaque (Macaca mulatta) GPR75, the amino acid and nucleotide sequences of which are found, for example, in GenBank accession No. NM-001204509.2 (SEQ ID NO: 4).

Other examples of GPR75 mRNA sequences are readily available using, for example, genBank, uniProt, OMIM and cynomolgus genome project sites.

Exemplary GPR75 nucleotide sequences can also be found in SEQ ID NO. 1 to SEQ ID NO. 4.SEQ ID NO. 5 to SEQ ID NO. 8 are the reverse complement of SEQ ID NO. 1 to SEQ ID NO. 4, respectively.

More information about GPR75 is provided, for example, in NCBI gene database www.ncbi.nlm.nih.gov/gene/10936.

The entire contents of the GenBank accession numbers and gene database numbers described above are incorporated herein by reference since the date of filing the present application.

As used herein, the terms "G protein-coupled receptor 75" and "GPR75" also refer to DNA sequence variants of the naturally occurring GPR75 gene. Many sequence variants in the GPR75 gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, e.g., https:// www.ncbi.nlm.nih.gov/snp/term = GPR 75), the entire contents of which are incorporated herein by reference since the date of filing the present application.

As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during GPR75 gene transcription, including the RNA processing product mRNA as a primary transcript. In one embodiment, the target portion of the sequence will be at least a portion of the nucleotide sequence of an mRNA molecule that is long enough to be formed during transcription of the GPR75 gene or its vicinity, to serve as a substrate for RNAi-directed cleavage. In one embodiment, the target sequence is within the protein coding region of the GPR75 gene. In another embodiment, the target sequence is within the 3' utr of the GPR75 gene.

The target sequence may be about 9 to 36 nucleotides in length, for example about 15 to 30 nucleotides. For example, the target sequence may be about 15 to 30 nucleotides, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides in length. In some embodiments, the target sequence is about 19 to about 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 21 to about 23 nucleotides in length. Ranges and lengths between those enumerated above are also considered to be part of the present invention.

As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a nucleotide strand, described by reference to a sequence using standard nucleotide nomenclature.

In general, "G", "C", "a", "T" and "U" each represent a nucleotide containing guanine, cytosine, adenine, thymine and uracil as bases, respectively. However, it is understood that the term "ribonucleotide" or "nucleotide" may also refer to a modified nucleotide, as described in further detail below, or a surrogate moiety (surrogate replacement moiety) (see, e.g., table 1). It is well known to the skilled artisan that guanine, cytosine, adenine and uracil can be replaced with other moieties without substantially altering the base pairing properties of oligonucleotides comprising nucleotides with such replacement moieties. It will be appreciated that when a cDNA sequence is provided, the corresponding mRNA or RNAi agent will contain U in place of T. For example, but not limited to, a nucleotide containing inosine as its base may base pair with a nucleotide containing adenine, cytosine, or guanine. Thus, nucleotides containing uracil, guanine or adenine in the nucleotide sequence of the dsRNA characterized in the present invention may be replaced with nucleotides containing, for example, inosine. In another example, adenine and cytosine at any positions in the oligonucleotide may be replaced with guanine and uracil, respectively, to form a G-U Wobble (Wobble) base pairing with the target mRNA. Sequences containing such substitutions are suitable for use in the compositions and methods of the present invention. In addition, in the RNAi agents of the invention, one skilled in the art will typically replace T of the target gene sequence or its reverse complement with U.

As used interchangeably herein, the terms "iRNA," "RNAi agent," "iRNA agent," "RNA interfering agent" refer to an agent comprising RNA as defined herein that mediates targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs sequence-specific degradation of mRNA. RNAi modulates, e.g., inhibits, expression of the GPR75 gene in a cell, e.g., in a cell in a subject (e.g., a mammalian subject).

In one embodiment, the RNAi agents of the invention include single stranded RNAi that interact with a target RNA sequence (e.g., GPR75 mRNA sequence) to direct cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double stranded RNA introduced into the cell is broken down into double stranded short interfering RNA (siRNA) comprising a sense strand and an antisense strand by a type III endonuclease called Dicer (Sharp et al (2001) Genes Dev.15:485). Dicer, a ribonuclease III-like enzyme, processes these dsRNA into 19 to 23 base pair short interfering RNAs with characteristic dibasic 3' overhangs (Bernstein, et al, (2001) Nature 409:363). These siRNAs are then integrated into an RNA-induced silencing complex (RISC), where one or more helices cleave the siRNA duplex, enabling the complementary antisense strand to direct targeted recognition (Nykanen, et al, (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir, et al, (2001) Genes Dev.15:188). Thus, in one aspect, the present disclosure relates to single stranded RNA (ssRNA) (the antisense strand of an siRNA duplex) produced in a cell that promotes the formation of RISC complexes to effectively silence a target gene. Thus, the term "siRNA" is also used herein to refer to RNAi as described above.

In another embodiment, the RNAi agent can be a single-stranded RNA that is introduced into a cell or organism to inhibit a target mRNA. The single stranded RNAi agent binds to RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single stranded siRNA is typically 15 to 30 nucleotides and is chemically modified. The design and testing of single stranded RNAs 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 in its entirety. Any of the antisense nucleotide sequences described herein can be used as a single stranded siRNA described herein or by the method described by Lima et al, (2012) Cell 150:883-894, and then used after chemical modification.

In another embodiment, the "RNAi agent" used in the compositions and methods of the present disclosure 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 referred to as having a "sense" orientation and an "antisense" orientation with respect to a target RNA (i.e., GPR75 mRNA sequence). In some embodiments of the disclosure, double-stranded RNA (dsRNA) triggers degradation of target RNA (e.g., mRNA) by a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).

Typically, the dsRNA molecule may include ribonucleotides, but as described in detail herein, each strand or both strands may also include one or more non-ribonucleotides, e.g., deoxyribonucleotides, modified nucleotides. In addition, "RNAi agent" as used in this specification may include ribonucleotides with chemical modification; RNAi agents can include a number of modifications at multiple nucleotides.

As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase. Thus, the term "modified nucleotide" includes substitution, addition or removal of, for example, functional groups or atoms of internucleotide linkages, sugar moieties or nucleobases. Modifications suitable for use in the agents of the invention include all types of modifications disclosed herein or known in the art. For the purposes of the present specification and claims, any such modification for use in an siRNA-type molecule is included in an "RNAi agent".

In certain embodiments of the present disclosure, inclusion of deoxynucleotides (which are considered naturally occurring forms of nucleotides), if any, in an RNAi agent can be considered to constitute modified nucleotides.

The duplex region may be any length that allows for specific degradation of the desired target RNA via the RISC pathway, and may be about 9 to 36 base pairs in length, such as about 15-30 base pairs in length, such as about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, for example about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs. Ranges and lengths between those enumerated above are also considered to be part of the present invention.

The two strands forming the duplex structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. When two strands are part of one larger molecule and are thus joined by an uninterrupted nucleotide chain between the 3 'end of one strand and the 5' end of the opposite strand, the joined RNA strand is referred to as a "hairpin loop". The hairpin loop may comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop may comprise at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides or nucleotides not directed against a target site of the dsRNA. In some embodiments, the hairpin loop may be 10 nucleotides or less. In some embodiments, the hairpin loop may be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop may be 4 to 10 unpaired nucleotides. In some embodiments, the hairpin loop may be 4 to 8 nucleotides.

In certain embodiments, the two strands of a double-stranded oligomeric compound may be joined together. The two strands may be connected to each other at both ends or may be connected to each other at only one end. By one-terminal ligation is meant that the 5 'end of the first strand is ligated to the 3' end of the second strand, or that the 3 'end of the first strand is ligated to the 5' end of the second strand. When the two strands are linked to each other at both ends, the 5 'end of the first strand is linked to the 3' end of the second strand and the 3 'end of the first strand is linked to the 5' end of the second strand. The two strands may be joined together by an oligonucleotide linker including, but not limited to, (N) N; wherein N is independently a modified nucleotide or an unmodified nucleotide and N is 3 to 23. In some embodiments, n is 3 to 10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G) 4, (U) 4, and (dT) 4, wherein N is a modified nucleotide or an unmodified nucleotide and R is a modified purine nucleotide or an unmodified purine nucleotide. Some nucleotides in the linker may participate in base pair interactions with other nucleotides in the linker. The two strands may also be joined together by a non-nucleoside linker, such as the linkers described herein. Those of skill in the art will appreciate that any of the oligonucleotide chemical modifications or variants described herein may be used for the oligonucleotide adaptors.

Hairpin and dumbbell oligomeric compounds will have duplex regions equal to or at least 14, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24 or 25 nucleotide pairs. The duplex region may have a length equal to or less than 200, 100, or 50. In some embodiments, the duplex region ranges in length from 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs.

The hairpin oligomeric compound may have a single stranded protruding or terminal unpaired region, which in some embodiments is at the 3' end, in some embodiments is on the antisense side of the hairpin. In some embodiments, the length of the protrusions is 1 to 4, more typically 2 to 3 nucleotides. Hairpin oligomeric compounds that induce RNA interference are also referred to herein as "shRNA".

When the two substantially complementary strands of the dsRNA are made up of separate RNA molecules, these molecules need not be, but may be, covalently linked. When two strands are covalently linked by means other than formation of a duplex structure by an uninterrupted nucleotide chain between the 3 'end of one strand and the 5' end of the opposite strand, then the linking structure is referred to as 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 the number of any overhangs present in the duplex. In addition to duplex structure, RNAi can comprise one or more nucleotide overhangs.

In one embodiment, the RNAi agents of the invention are dsRNA, each strand of which is 24 to 30 nucleotides in length, that interact with a target RNA sequence (e.g., GPR75 mRNA sequence) to guide cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into the cell is broken down into siRNA by a type III endonuclease called Dicer (Sharp et al (2001) Genes Dev.15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into 19 to 23 base pair short interfering RNA with a characteristic dibasic 3' overhang (Bernstein, et al, (2001) Nature 409:363). These siRNAs are then integrated into an RNA-induced silencing complex (RISC), where one or more helices cleave the siRNA duplex, enabling the complementary antisense strand to direct targeted recognition (Nykanen, et al, (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir, et al, (2001) Genes Dev.15:188).

In one embodiment, the RNAi agent of the invention is a dsRNA agent comprising 19 to 23 nucleotides per strand that interacts with GPR75 mRNA sequence to guide cleavage of target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into the cell is broken down into siRNA by a type III endonuclease called Dicer (Sharp et al (2001) Genes Dev.15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into 19 to 23 base pairs of short interfering RNAs with characteristic dibasic 3' overhangs (Bernstein, et al, (2001) Nature 409:363). These siRNAs are then integrated into an RNA-induced silencing complex (RISC), where one or more helices cleave the siRNA duplex, enabling the complementary antisense strand to direct targeted recognition (Nykanen, et al, (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir, et al, (2001) Genes Dev.15:188). In one embodiment, the RNAi agent of the invention is a 24 to 30 nucleotide dsRNA that interacts with a GPR75 mRNA sequence to guide cleavage of the target RNA.

As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the duplex structure of an RNAi agent (e.g., dsRNA). For example, nucleotide overhang exists when the 3 'end of one strand of a dsRNA extends beyond the 5' end of the other strand, or when the 3 'end of one strand of a dsRNA extends not beyond the 5' end of the other strand. The dsRNA may comprise a overhang of at least one nucleotide; or the overhang may comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. Nucleotide projections may comprise or consist of nucleotide/nucleoside analogues, including deoxynucleotides/nucleosides. The one or more protrusions may be located on the sense strand, the antisense strand, or any combination thereof. In addition, the protruding nucleotide or nucleotides may be present at the 5 'end, 3' end or both ends of the antisense strand or sense strand of the dsRNA.

In one embodiment of the dsRNA, at least one strand comprises a 3' overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3 'overhang of at least 2 nucleotides, e.g., a 3' overhang of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5 'overhang of at least 2 nucleotides, e.g., a 5' overhang of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, both the 3 'and 5' ends of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

In one embodiment, the antisense strand of the dsRNA has a 1 to 10 nucleotide overhang at the 3 '-end or the 5' -end, for example a 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10 nucleotide overhang, for example a 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide overhang. In one embodiment, the sense strand of the dsRNA has a 1 to 10 nucleotide overhang at the 3 '-end or the 5' -end, for example a 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide overhang. In another embodiment, one or more nucleotides in the overhang are replaced with a nucleoside phosphorothioate.

In certain embodiments, the overhang located in the sense strand or the antisense strand, or both, can include an extension length of greater than 10 nucleotides, for example, a length of 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 15 nucleotides. In certain embodiments, the extended overhang is located on the sense strand of the duplex. In certain embodiments, the extended overhang is present at the 3' end of the sense strand of the duplex. In certain embodiments, the extended overhang is present at the 5' end of the sense strand of the duplex. In certain embodiments, the extended overhang is located on the antisense strand of the duplex. In certain embodiments, the extended overhang is present at the 3' end of the antisense strand of the duplex. In certain embodiments, the extended overhang is present at the 5' end of the antisense strand of the duplex. In certain embodiments, one or more nucleotides in the overhang are replaced with a nucleoside phosphorothioate. In certain embodiments, the overhang includes a self-complementary moiety such that the overhang is capable of forming a hairpin structure that is stable under physiological conditions.

As used herein, the term "blunt" or "blunt-ended" when referring to a dsRNA, refers to the absence of unpaired nucleotides or nucleotide analogs, i.e., no nucleotide overhang, at a given end of the dsRNA. One or both ends of the dsRNA may be blunt. When both ends of a dsRNA are blunt, then the dsRNA is said to be blunt-ended. For clarity, "blunt-ended" dsRNA is dsRNA that is blunt at both ends, i.e., no nucleotide protrudes at either end of the molecule. In most cases, such molecules will be double stranded throughout their length.

The term "antisense strand" or "guide strand" refers to a strand of an iRNA, e.g., dsRNA, that includes a region that is substantially complementary to a target sequence (e.g., GPR75 mRNA sequence).

As used herein, the term "complementarity region" refers to a region on the antisense strand that is substantially complementary to a target sequence (e.g., GPR75 nucleotide sequence) as defined herein. If the region of complementarity is not perfectly complementary to the target sequence, a mismatch may exist in the internal or terminal regions of the molecule. Typically, the most tolerated mismatch is present within the terminal region, e.g., within 5, 4, 3, or 2 nucleotides of the 5 '-end or 3' -end of the RNAi agent.

In some embodiments, the double stranded RNA agent of the invention comprises a nucleotide mismatch in the antisense strand. In some embodiments, the antisense strand of a double-stranded RNA agent of the invention comprises no more than 4 mismatches with a target mRNA, e.g., the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, an antisense strand double-stranded RNA agent of the invention comprises no more than 4 mismatches with the sense strand, e.g., the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, the double stranded RNA agent of the invention comprises a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the invention comprises no more than 4 mismatches with the antisense strand, e.g., the sense strand comprises 4, 3, 2, 1 or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, 3 nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

Thus, RNAi agents described herein can contain one or more mismatches with the target sequence. In one embodiment, an RNAi agent described herein comprises no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent described herein comprises no more than 2 mismatches. In one embodiment, the RNAi agents described herein comprise no more than 1 mismatch. In one embodiment, the RNAi agents described herein comprise 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains a mismatch to the target sequence, the mismatch can optionally be limited to the last 5 nucleotides from the 5 'or 3' end of the complementarity region. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand complementary to the GPR75 gene region typically does not contain any mismatches within the central 13 nucleotides. Methods described herein or known in the art can be used to determine whether RNAi agents containing mismatches to the target sequence are effective in inhibiting expression of the GPR75 gene. It is important to consider the efficacy of RNAi agents with mismatches in inhibiting GPR75 gene expression, particularly if a change in a specific region of complementarity in the GPR75 gene is known.

As used herein, the term "sense strand" or "passenger strand" refers to the strand of an RNAi agent that includes a region substantially complementary to the antisense strand region defined herein.

As used herein, "substantially all nucleotides are modified" is that most, but not all, of the nucleotides are modified and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

As used herein, the term "cleavage region" refers to a region immediately adjacent to a cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases located at either end of the cleavage site and immediately adjacent to the cleavage site. In some embodiments, the cleavage region comprises two bases located at either end of the cleavage site and immediately adjacent to the cleavage site. In some embodiments, cleavage sites occur specifically at the site to which nucleotides 10 and 11 of the antisense strand bind, and the cleavage region comprises nucleotides 11, 12 and 13.

As used herein, unless otherwise indicated, when the term "complementary" is used to describe the relationship of a first nucleotide sequence and a second nucleotide sequence, it refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize under specific conditions to an oligonucleotide or polynucleotide comprising the second nucleotide sequence and form a duplex structure, as understood by the skilled artisan. Such conditions may be, for example, "stringent conditions," where stringent conditions may include: 400mM NaCl,40mM PIPES pH 6.4,1mM EDTA,50 ℃ or 70 ℃ for 12-16 hours and then washed (see, e.g., "Molecular Cloning: ALaboratory Manual, sambrook, et al (1989) Cold Spring Harbor Laboratory Press). Other conditions such as physiologically relevant conditions that may be encountered in an organism may be applied. The skilled person will be able to determine the condition settings most suitable for the complementarity test of the two sequences depending on the end use of the hybridized nucleotides.

Complementary sequences within RNAi agents, such as dsRNA, as described herein include base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the full length of one or both nucleotide sequences. Such sequences may be referred to herein as being "fully complementary" to each other. However, in this context, if a first sequence is said to be "substantially complementary" to a second sequence, the two sequences may be fully complementary, or when hybridized to form a duplex of up to 30 base pairs they may form one or more but typically no more than 5, 4, 3 or 2 mismatched base pairs, while retaining the ability to hybridize under conditions best suited for their end use (e.g., inhibiting gene expression in vitro or in vivo). However, if two oligonucleotides are designed to form one or more single stranded protrusions upon hybridization, such protrusions should not be considered mismatches in terms of determining complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, wherein the longer nucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter nucleotide, which may still be referred to as being "fully complementary" for purposes described herein.

As used herein, a "complementary" sequence may also include base pairs that are not Watson-Crick base pairs or are formed from non-natural nucleotides and modified nucleotides, or base pairs that are formed entirely from non-Watson-Crick base pairs or are formed from non-natural nucleotides and modified nucleotides, so long as the requirements set forth above with respect to their hybridization capabilities are met. Such non-Watson-Crick base pairs include, but are not limited to, G: U wobble or Hoogstein base pairing.

In this context, the terms "complementary", "fully complementary" and "substantially complementary" may be used to describe a base match between the sense strand and the antisense strand of a dsRNA, or between two oligonucleotides or polynucleotides, such as between the antisense strand and a target sequence of an RNAi agent, as will be understood from the context of its use.

As used herein, a polynucleotide that is substantially complementary to "at least a portion of a messenger RNA (mRNA) or target sequence refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA or target sequence of interest (e.g., an mRNA encoding GPR 75). For example, if the polynucleotide sequence is substantially complementary to an uninterrupted portion of the mRNA encoding GPR75, then the polynucleotide is complementary to at least a portion of the GPR75 RNA.

Thus, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target GPR75 sequence.

In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to a target GPR75 sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary (e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or about 99% complementary) over its entire length to the nucleotide sequence of SEQ ID NO:1 to SEQ ID NO:4 of GPR75 or to the equivalent region of a fragment of SEQ ID NO:1 to SEQ ID NO: 4.

In other embodiments, an antisense polynucleotide disclosed herein is substantially complementary to a target GPR75 sequence and comprises a contiguous nucleotide sequence that is at least about 80% complementary (e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary) over its entire length to any of the sense strand nucleotide sequences in any of tables 2, 3, 5, and 6 or to a fragment of any of the sense strand nucleotide sequences in any of tables 2, 3, 5, and 6.

In one embodiment, the RNAi agents of the present disclosure comprise a sense strand that is substantially complementary to an antisense polynucleotide, which in turn is identical to the target GPR75 sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary (e.g., about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary) over its entire length to the nucleotide sequence of SEQ ID NO:5 to SEQ ID NO:8 or the equivalent region of any fragment of SEQ ID NO:5 to SEQ ID NO: 8.

In some embodiments, an iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is in turn complementary to a target GPR75 sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary (e.g., about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary) over its entire length to the antisense strand nucleotide sequence of any one of tables 2, 3, 5, and 6, or a fragment of the antisense strand nucleotide sequence of any one of tables 2, 3, 5, and 6.

In some embodiments, an antisense polynucleotide disclosed herein is substantially complementary to a fragment of a target GPR75 sequence and comprises a contiguous nucleotide sequence that is contiguous with the sequence selected from the group consisting of SEQ ID NOs: 1, 38-60, 50-72, 148-181, 153-175, 159-181, 228-250, 240-262, 341-363, 341-368, 346-368, 369-396, 369-391, 374-396, 388-410, 414-436, 424-461, 424-446, 424-451, 434-456, 439-461, 429-451, 457-504, 462-491, 482-504, 469-491, 457-479, 462-584, 475-497, 469-491, 509-537, 509-531, 515-537, 544-576, 544-566, 549-571 580-607, 580-602, 585-607, 595-617, 615-647, 615-637, 620-642, 620-647, 625-647, 773-806, 773-795, 778-800, 784-806, 837-872, 837-859, 843-872, 843-865, 850-872, 860-882, 889-911, 900-936, 900-922, 908-936, 908-930, 914-936, 938-990, 938-960, 943-965, 968-990, 1060-1101, 1060-1082, 1066-1088, 1073-1095, 1079-1101, 1097-1119, 1238-1260, 1268-1290, 1284-1393, 1284-1306, 1292-1393, 1292-1314, 1292-1383, 1292-1314, 1301-1323, 1307-1383, 1307-1342, 1307-1329, 1313-1335, 1371-1393, 1351-1373, 1320-1342, 1336-1358, 1345-1367, 1351-1373, 1361-1383, 1366-1388, 1393-1415, 1422-1463, 1422-1444, 1441-1463, 1487-1526, 1487-1509, 1493-1526, 1493-1515, 1493-1520, 1504-1526, 1515-1571, 1515-1543, 1515-1537, 1521-1543, 1530-1552, 1545-1557, 1540-1562, 1549-1571, 1559-1586, 1581-1586, 1586-1589, 1583-1526. 1583-1605, 1588-1610, 1595-1617, 1600-1629, 1600-1622, 1607-1629, 1624-1646, 1635-1657, 1672-1721, 1672-1710, 1677-1699, 1699-1721, 1672-1699, 1688-1710, 1672-1694, 1683-1705, 1693-1714, 1732-1754, 1744-1798, 1751-1773, 1758-1780, 177-1789, 1776-1798, 1790-1818, 1790-1812, 1796-1818, 1808-1856, 1808-1848, 1808-1830, 1826-1848, 1814-1836, 1819-1841, 1834-1856, 1877-2082, 1877-1899, 1882-2, 1882-5, 1882-1903, 1882-1908, 1887-19098, 1890-1819, 1929-1929 1903-1925, 1908-1930, 1913-1935, 1913-1950, 1921-1943, 1928-1950, 1933-1955, 1941-1963, 1946-1968, 1953-1985, 1953-2082, 1953-1975, 1938-1985, 1958-1980, 1963-1985, 1968-1990, 1974-1996, 1974-2065, 1974-2082, 1974-2002, 1980-2002, 1985-2007, 1990-2012, 1990-2033, 1999-2021, 2005-2033, 2005-2027, 2011-2033, 2017-2039, 2025-2055, 2025-2047, 2033-2055, 2038-2060, 2033-2055, 2048-2054-2086, and 0 are about 92% of complementary (e.g., about 93%, about 95% of the set of SEQ ID's), about 93%, or about 95% of the complementary SEQ ID (e.g., about 93%).

In some embodiments, the double-stranded region of the double-stranded iRNA agent is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotide pairs in length.

In some embodiments, the antisense chain length of the double stranded iRNA agent is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

In some embodiments, the sense chain length of the double-stranded iRNA agent is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

In one embodiment, the sense strand and the antisense strand of the double-stranded iRNA agent are each independently 15 to 30 nucleotides in length.

In one embodiment, the sense strand and the antisense strand of the double-stranded iRNA agent are each independently 19 to 25 nucleotides in length.

In one embodiment, the sense strand and the antisense strand of the double-stranded iRNA agent are each independently 21 to 23 nucleotides in length.

In one embodiment, the sense strand of the iRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, wherein the strand forms a double-stranded region of 21 consecutive base pairs with a single-stranded overhang of 2 nucleotides in length at the 3' end.

In one aspect of the invention, the agents used in the methods and compositions of the invention are single stranded antisense nucleic acid molecules that inhibit the target mRNA by an antisense inhibition mechanism. The single stranded antisense RNA molecule is complementary to a sequence within the target mRNA. The single stranded antisense oligonucleotide can inhibit translation stoichiometrically by base pairing with mRNA and physically blocking the translation mechanism, see Dias, N.et al, (2002) Mol Cancer Ther 1:347-355. The single stranded antisense RNA molecule can be about 15 to about 30 nucleotides in length and have a sequence complementary to the target sequence. For example, a single stranded antisense RNA molecule can comprise a sequence of at least about 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from any of the antisense sequences described herein.

In one embodiment, at least partial inhibition of GPR75 gene expression is assessed by a decrease in the amount of isolated or detected GPR75 mRNA in a first cell or cell population in which the GPR75 gene is transcribed and which has been treated such that expression of the GPR75 gene is inhibited relative to a second cell or cell population (control cell) that is substantially the same as the first cell or cell population but not treated as such. The extent of inhibition can be expressed as:

In one embodiment, inhibition of expression is determined by a dual luciferase method (wherein RNAi agent is present at 10 nM).

As used herein, the phrase "contacting a cell with an RNAi agent," e.g., dsRNA, includes contacting the cell by any possible means. Contacting the cell with the RNAi agent comprises contacting the cell with the RNAi agent in vitro or contacting the cell with the RNAi agent in vivo. The contacting may be accomplished directly or indirectly. Thus, for example, an RNAi agent can be physically contacted with a cell by a method to effect physical contact therewith, or the RNAi agent can be subjected to conditions that will allow or result in subsequent contact therewith.

In vitro contact with cells may be accomplished, for example, by incubating the cells with an RNAi agent. In vivo contact with cells may be accomplished, for example, by injecting the RNAi agent into or adjacent to the tissue in which the cells are located, or by injecting the RNAi agent into another area, such as the Central Nervous System (CNS) (optionally by intrathecal injection, intravitreal injection, or other injection), or into the blood stream or subcutaneous space, such that the iRNA agent will then reach the tissue in which the cells to be contacted are located. For example, the RNAi agent can comprise or be conjugated to a ligand, e.g., a lipophilic moiety or moieties, as described below and further detailed in, e.g., PCT/US2019/031170 (which is incorporated herein by reference), that directs or stabilizes the RNAi agent at a site of interest, e.g., the CNS. In some embodiments, the RNAi agent can comprise or be conjugated to a ligand, e.g., one or more GalNAc derivatives as described below, that directs or stabilizes the RNAi agent at a site of interest, e.g., the liver. In other embodiments, the RNAi agent can comprise or be coupled to one or more lipophilic moieties and one or more GalNAc derivatives. Combinations of in vitro and in vivo contact methods are also possible. For example, the cells may be contacted with an RNAi agent in vitro and then transplanted into a subject.

In one embodiment, contacting the cell with an RNAi agent comprises: by promoting or causing uptake or uptake into the cell, "introduction of RNAi agent" or "delivery of RNAi agent into the cell. The uptake or uptake of the RNAi agent can occur by unassisted diffusion or active cellular processes, or by adjuvants or auxiliary devices. The introduction of the RNAi agent into the cell can be in vitro or in vivo. For example, for in vivo introduction, the RNAi agent can be injected to the tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Further methods are described below or are known in the art.

The term "lipophilic" or "lipophilic moiety" refers broadly to any compound or chemical moiety having an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by octanol-water partition coefficient log K _ow Wherein K is _ow Is the ratio of the concentration of the chemical in the octanol phase to its concentration in the aqueous phase at equilibrium. Octanol-water partition coefficient is a laboratory measured property of a substance. However, it can also be predicted by coefficients belonging to the structural components of the chemical substance calculated using first principles or empirical methods (see, e.g., tetko et al, J.chem. Inf. Comput. Sci.41:1407-21 (2001), the entire contents of which are incorporated herein by reference). It provides a thermodynamic measure that a substance tends to choose a non-aqueous or oily environment rather than water (i.e., its hydrophilic/lipophilic balance). In principle, when the logK of a chemical substance _ow When the value exceeds 0, it is lipophilic. Typically, the lipophilic moiety has a log k of more than 1, more than 1.5, more than 2, more than 3, more than 4, more than 5 or more than 10 _ow . For example, logK of 6-amino hexanol _ow About 0.7 is expected. Using the same method, the log K of cholesterol N- (hex-6-ol) carbamate _ow 10.7 is expected.

The lipophilicity of a molecule may vary depending on the functional group it carries. For example, addition of hydroxyl or amine groups at the end of the lipophilic moiety can increase or decrease the partition coefficient of the lipophilic moiety (e.g., log K _ow ) Values.

Alternatively, the hydrophobicity of double stranded RNAi agents conjugated to one or more lipophilic moieties can be measured by their protein binding properties. For example, in certain embodiments, the unbound fraction in a plasma protein binding assay of a double stranded RNAi agent can be determined to be positively correlated with the relative hydrophobicity of the double stranded RNAi agent, which can then be positively correlated with the silencing activity of the double stranded RNAi agent.

In one embodiment, the plasma protein binding assay that is determined is an Electrophoretic Mobility Shift Assay (EMSA) using human serum albumin. An exemplary protocol for such a binding assay is described in detail in, for example, PCT publication No. WO 2019/217459. The hydrophobicity of the double stranded RNAi agent measured by the fraction of unbound siRNA in the binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 for enhanced in vivo delivery of the siRNA.

Thus, conjugation of the lipophilic moiety to the internal location of the double stranded RNAi agent provides optimal hydrophobicity for enhancing in vivo delivery of siRNA.

The term "lipid nanoparticle" or "LNP" is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, e.g. a nucleic acid molecule, e.g. an RNAi agent or a plasmid transcribing an RNAi agent. LNP is described, for example, in U.S. patent nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

As used herein, a "subject" is an animal, such as a mammal, that endogenously or heterologously expresses a target gene, including primates (e.g., humans, non-human primates such as monkeys and chimpanzees), or non-primates (such as cows, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, or mice), or birds. In one embodiment, the subject is a human, e.g., a human who is receiving treatment or being evaluated for a disease, disorder, or condition that would benefit from reduced GPR75 expression; a person at risk of a disease, disorder or condition that would benefit from reduced GPR75 expression; a human suffering from a disease, disorder or condition that would benefit from reduced GPR75 expression; or a person undergoing treatment for a disease, disorder, or condition that would benefit from reduced GPR75 expression as described herein. In some embodiments, the subject is a female. In other embodiments, the subject is a male. In one embodiment, the subject is an adult subject. In another embodiment, the subject is a pediatric subject.

As used herein, the term "treatment" or "treatment" refers to a beneficial or desired outcome, including, but not limited to, alleviation or amelioration of one or more signs or symptoms associated with GPR75 expression or GPR75 protein production, for example, a GPR 75-related disease such as obesity, or a symptom associated with undesired GPR75 expression; reducing the extent of undesired GPR75 activation or stabilization; improving or reducing undesired GPR75 activation or stabilization. "treatment" may also refer to an increase in survival compared to the expected survival when untreated.

In the context of a subject's GPR75 level or disease marker or symptom, the term "lower" refers to a statistically significant decrease in that level. The decrease may be, for example, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In certain embodiments, the decrease is at least 20%. In certain embodiments, the decrease in the level of expression of a disease marker, such as a protein or gene, is at least 50%. In the context of GPR75 levels in a subject, "lower" refers to a level as low as acceptable within the normal range for individuals not suffering from such disorders. In certain embodiments, expression of the target is normalized, i.e., reduced toward or to levels acceptable in the normal range for individuals not suffering from such disorders, e.g., BMI, blood glucose levels, blood lipid levels, blood oxygen levels, white blood cell count, kidney function, spleen function, liver function. As used herein, "lower" in a subject may refer to a decrease in gene expression or protein production in a subject's cells, without requiring a decrease in expression in all cells or tissues of the subject. For example, as used herein, a decrease in a subject may include a decrease in gene expression or protein production in the subject.

The term "lower" may also be used to normalize the symptoms of a disease or disorder, i.e., reduce the difference between the level of a subject with a GPR 75-related disease and the level of a normal subject not with a GPR 75-related disease. As used herein, a "normal" is considered to be the upper limit of normal if the disease is associated with an elevated value of symptoms. If the disease is associated with a reduced value of symptoms, "normal" is considered to be the lower limit of normal.

As used herein, "preventing" or "prevention" when used in reference to a disease, disorder, or condition thereof that would benefit from a reduction in GPR75 gene expression or GPR75 protein production refers to a reduction in the likelihood that a subject will develop symptoms associated with such disease, disorder, or condition (e.g., symptoms of a GPR 75-related disease, e.g., weight disorders such as obesity, e.g., diabetes, or lipid metabolism disorders). The manifestation of a disease, disorder or condition not developing, or a reduction in symptom development or symptoms associated with such a disease, disorder or condition (e.g., at least about 10% reduction in a clinically acceptable level of the disease or disorder), or a delay in symptoms (e.g., a delay of days, weeks, months or years) is considered effective prevention.

As used herein, the term "GPR 75-related disease" is a disease or disorder that would benefit from reduced GPR75 expression or activity. The term "GPR 75-related disease" is a disease or disorder caused by or associated with GPR75 expression or GPR75 protein production. The term "GPR 75-related disease" includes diseases, disorders or conditions that would benefit from GPR75 expression or decreased GPR75 protein activity. Non-limiting examples of GPR75 related diseases include, for example, weight disorders such as obesity.

As used herein, a "weight disorder" is a condition associated with abnormal or excessive fat accumulation and weight. Such conditions may include obesity, metabolic syndrome including separate components of metabolic syndrome (e.g., central obesity, FBG/pre-diabetes/diabetes, hypercholesterolemia, hypertriglyceridemia and hypertension), hypometabolic status, hypothyroidism, uremia and other conditions associated with a risk of weight gain (including rapid weight gain), maintenance of weight loss or weight regain following weight loss.

Body weight can be estimated by Body Mass Index (BMI), i.e., the body weight (in kilograms) of a person divided by the square of his or her height (in meters). BMI less than about 18.5 indicates that the subject is overweight; a BMI of about 18.5 to about <25 indicates that the subject has normal body weight; a BMI of about 25.0 to about <30 indicates that the subject is overweight, and a BMI of about 30.0 or higher indicates that the subject is obese.

Other diseases or conditions associated with weight disorders will be apparent to the skilled artisan and are within the scope of the disclosure.

Symptoms of GPR 75-related diseases (e.g., weight disorders such as obesity) include, for example, excessive fat mass, BMI of about 25 or higher, increased body mass index, lower metabolic rate, central obesity, FBG/pre-diabetes/diabetes, hypercholesterolemia, hypertriglyceridemia and hypertension, insulin resistance, lack of glycemic control, high blood glucose levels, diabetes, and/or excessive weight gain. Further details regarding signs and symptoms of various diseases or conditions are provided herein, and are well known in the art

As used herein, "therapeutically effective amount" is intended to include an amount of RNAi agent sufficient to effectively treat a GPR 75-related disease (e.g., by alleviating, ameliorating, or maintaining one or more symptoms of the existing disease or disease) when the RNAi agent is administered to a subject having the disease. The "therapeutically effective amount" may vary depending on the RNAi agent, the mode of administration of the agent, the disease and its severity and the subject's history, age, weight, family history, genetic constitution, the type of past treatment or concomitant treatment (if any), and other individual characteristics to be treated.

As used herein, "prophylactically effective amount" is intended to include an amount of RNAi agent sufficient to prevent or ameliorate a GPR 75-related disease (e.g., a body weight disorder such as obesity) or one or more symptoms of the disease when the RNAi agent is administered to a subject suffering from the disease. Remission of a disease includes slowing the progression of the disease or lessening the severity of a subsequent disease. The "prophylactically effective amount" may vary depending on the RNAi agent, the mode of administration of the agent, the degree of risk of the disease and the history, age, weight, family history, genetic constitution, type of past treatment or concomitant treatment (if any), and other individual characteristics of the subject to be treated.

"therapeutically effective amount" or "prophylactically effective amount" also includes the amount of RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. The RNAi agents used in the methods of the present disclosure can be administered in amounts sufficient to produce a reasonable benefit/risk ratio suitable for such treatment.

As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle (vehicle), such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate, zinc stearate, or stearic acid), or solvent encapsulating material, involved in carrying or transporting the test compound from one organ or body part to another organ or body part. Each carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the formulation and does not harm the subject being treated. Some examples of materials that may be used as pharmaceutically acceptable carriers include (1) sugars such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) Cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) Lubricants, such as magnesium sulfate, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository waxes; (9) Oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) Polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) ringer's solution; (19) ethanol; (20) a pH buffer solution; (21) a polyester, polycarbonate or polyanhydride; (22) Fillers, such as polypeptides and amino acid (23) serum components, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances used in pharmaceutical formulations. Pharmaceutically acceptable carriers for pulmonary delivery are known in the art and will vary depending on the desired location of agent deposition (e.g., upper or lower respiratory system) and the type of device used for delivery (e.g., nebulizer, dry powder inhaler).

As used herein, the term "sample" includes similar bodily fluids, cells, or tissues isolated from a subject, as well as collections of bodily fluids, cells, or tissues present in a subject. Examples of biological fluids include blood, serum, serosal fluid, plasma, bronchial fluid, sputum, cerebrospinal fluid, ocular fluid, lymph fluid, urine, saliva, sputum, and the like. Tissue samples may include samples derived from tissues, organs, or localized areas. For example, the sample may originate from a particular organ, a portion of an organ, or body fluids or cells within those organs. In certain embodiments, the sample may be derived from the brain (e.g., whole brain or parts of the brain such as the striatum, or certain types of cells in the brain such as neurons and glial cells (astrocytes, oligodendrocytes, microglia)). In other embodiments, a "subject-derived sample" refers to liver tissue (or a sub-component thereof) derived from a subject. In some embodiments, a "subject-derived sample" refers to blood drawn from a subject or plasma or serum derived from a subject. In other embodiments, a "subject-derived sample" refers to brain tissue (or a sub-component thereof) or retinal tissue (or a sub-component thereof) derived from a subject.

RNAi agents of the present disclosure

Described herein are RNAi agents that inhibit GPR75 gene expression. In one embodiment, the RNAi agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of the GPR75 gene in a cell, e.g., a cell in a subject, e.g., a mammal, e.g., a human, e.g., a subject having a GPR 75-related disorder, e.g., body weight (e.g., obesity), or at risk for developing a GPR 75-related disease.

The dsRNA comprises an antisense strand having a complementarity region that is complementary to at least a portion of a target RNA (e.g., mRNA formed in GPR75 gene expression). The length of the complementarity region is about 15 to 30 nucleotides or less. When contacted with cells expressing the GPR75 gene, the RNAi agent inhibits expression of the GPR75 gene (e.g., human gene, primate gene, non-primate gene) by at least 50% as determined by, for example, PCR or branched DNA (bDNA) based methods, or by protein based methods such as by immunofluorescence analysis using, for example, western blotting or flow cytometry. In certain embodiments, expression is inhibited by at least 50% as determined by the dual luciferase assay in example 1 (where the concentration of siRNA is 10 nM).

The dsRNA comprises two complementary RNA strands that hybridize under conditions in which the dsRNA will be used to form a duplex structure. One strand (the antisense strand) of the dsRNA comprises a region of complementarity that is substantially complementary, and typically fully complementary, to a target sequence. For example, the target sequence may be derived from an mRNA sequence formed during GPR75 gene expression. The other strand (the sense strand) includes a region complementary to the antisense strand such that the two strands hybridize and form a duplex structure when bound under suitable conditions. As described elsewhere herein and known in the art, the complementary sequence of a dsRNA may also be contained as a self-complementary region of a single nucleic acid molecule, rather than on separate oligonucleotides.

Typically, duplex structures are 15 to 30 base pairs in length, such as 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, 21 to 22, or 21 to 22 base pairs. In certain embodiments, the duplex structure is 18 to 25 base pairs in length, e.g., 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 25, 21 to 24, 21 to 23, 21 to 22, 22 to 25, 22 to 24, 22 to 23, 23 to 25, 23 to 24, or 24 to 25 base pairs in length, e.g., 19 to 21 base pairs in length. Ranges and lengths between those enumerated above are also considered to be part of this disclosure.

Similarly, the region of complementarity which is complementary to the target sequence is 15 to 30 nucleotides in length, e.g., 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 23, 21 to 21, 21 to 23, or 21 to 22 nucleotides in length, e.g., 23 to 23 nucleotides. Ranges and lengths between those enumerated above are also considered to be part of this disclosure.

In some embodiments, the dsRNA is 15 to 23 nucleotides in length, or 25 to 30 nucleotides in length. Typically, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, dsrnas greater than about 21 to 23 nucleotides in length may be used as substrates for Dicer, as is well known in the art. Those skilled in the art will also recognize that the RNA region targeted for cleavage is typically part of a larger RNA molecule (typically an mRNA molecule). In related cases, a "portion" of an mRNA target is a contiguous sequence of mRNA target of sufficient length to allow it to become a substrate for RNAi-directed cleavage (i.e., cleavage by the RISC pathway).

Those skilled in the art will also recognize that duplex regions are the major functional portion of dsRNA, e.g., duplex regions of about 15 to 36 base pairs, e.g., 15 to 36, 15 to 35, 15 to 34, 15 to 33, 15 to 32, 15 to 31, 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24,20 to 22, 20 to 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 21, 21 to 25, 21 to 21, 21 to 22, or 21 to 21 pairs, for example. Thus, in one embodiment, the RNA molecule or complex of RNA molecules having a duplex region of more than 30 base pairs is a dsRNA in terms of its functional duplex, e.g., 15 to 30 base pairs, that is processed to target the desired RNA for cleavage. Thus, one of ordinary skill in the art will recognize that in one embodiment, the miRNA is dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, RNAi agents useful for targeting GPR75 expression are not generated in the target cell by cleavage of larger dsRNA.

The dsRNA described herein may also include one or more single-stranded nucleotide overhangs having, for example, 1, 2, 3, or 4 nucleotides. Nucleotide projections may comprise or consist of nucleotide/nucleoside analogues, including deoxynucleotides/nucleosides. The one or more protrusions may be located on the sense strand, the antisense strand, or any combination thereof. In addition, the protruding nucleotide or nucleotides may be present at the 5 '-end, 3' -end or both ends of the antisense strand or sense strand of the dsRNA. In certain embodiments, longer, extended protrusions are possible.

dsRNA can be synthesized by standard methods known in the art, e.g., by using an automated DNA synthesizer, such as those available from, e.g., biosearch, applied Biosystems, inc, as discussed further below.

The iRNA compounds of the invention can be prepared in a two-step process. First, each strand of a double-stranded RNA molecule is prepared separately. Subsequently, the constituent chains are annealed. The individual strands of the siRNA compound may be prepared using solution phase organic synthesis, solid phase organic synthesis, or both. The organic synthesis has the advantages that: oligonucleotide chains comprising non-natural or modified nucleotides can be readily prepared. The single stranded oligonucleotides of the invention may be prepared using solution phase organic synthesis, solid phase organic synthesis, or both.

siRNA can be mass produced by a variety of methods. An exemplary method includes: organic synthesis and RNA cleavage, e.g. in vitro cleavage.

siRNA can be prepared by synthesizing each respective strand of a single-stranded RNA molecule or a double-stranded RNA molecule separately, and then the constituent strands can be annealed.

Large bioreactors, such as OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), can be used to produce large numbers of specific RNA strands for a given siRNA. The OligoPilot II reactor can efficiently couple nucleotides using only a 1.5 molar excess of phosphoramidite nucleotides. To prepare the RNA strand, ribonucleotide amides are used. Standard cycles of monomer addition can be used to synthesize 21 to 23 nucleotide strands of siRNA. Typically, two complementary strands are produced separately and then annealed, e.g., after release from the solid support and deprotection.

Organic synthesis can be used to generate isolated siRNA species. The complementarity of this species to the GPR75 gene can be precisely determined. For example, the species may be complementary to a region that includes a polymorphism (e.g., a single nucleotide polymorphism). Furthermore, the position of the polymorphism can be precisely determined. In some embodiments, the polymorphism is located in an internal region, e.g., at least 4, 5, 7, or 9 nucleotides from one or both ends.

In one embodiment, the resulting RNA is carefully purified to remove the ends, and the iRNA is cleaved in vitro into siRNA, e.g., using Dicer or equivalent RNAse III-based activity. For example, dsiRNA may be cultured in vitro extracts from Drosophila (Drosophila) or using purified components, such as purified RNAse or RISC complexes (RNA-induced silencing complexes). See, e.g., ketting et al genes Dev 2001Oct15;15 (20) 2654-9 and Hammond Science 2001Aug 10;293 (5532):1146-50.

dsiRNA cleavage typically produces multiple sirnas, each of which is a specific 21 to 23 nucleotide fragment of the source dsiRNA molecule. For example, there may be an siRNA comprising sequences complementary to overlapping and adjacent regions of the source dsiRNA molecule.

Regardless of the method of synthesis employed, the siRNA preparation can be prepared in a solution (e.g., an aqueous or organic solution) suitable for formulation. For example, the siRNA preparation can be precipitated and redissolved in pure double distilled water, and then lyophilized. The dried siRNA can then be resuspended in a solution suitable for the intended formulation process.

In one aspect, the dsRNA of the present disclosure comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand sequence for GPR75 may be selected from the group of sequences provided in any of tables 2, 3, 5 and 6, and the nucleotide sequence of the corresponding antisense strand of the sense strand may be selected from the group of sequences in any of tables 2, 3, 5 and 6. In this regard, one of the two sequences is complementary to the other of the two sequences, wherein one of the sequences is substantially complementary to the mRNA sequence produced in the expression of the GPR75 gene. Thus, in this regard, a dsRNA will comprise two oligonucleotides, one of which is described in any of tables 2, 3, 5 and 6 as the sense strand (passenger strand) and the second lattice oligonucleotide is described in any of tables 2, 3, 5 and 6 as the corresponding antisense strand (guide strand) to the sense strand of GPR 75.

In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequence of the dsRNA is contained on a single oligonucleotide.

It should be understood that although the sequences provided herein are described as modified or conjugated sequences, the RNAs of the RNAi agents of the present disclosure, e.g., dsRNA of the application, may comprise any of the unmodified, unconjugated, or modified or conjugated sequences listed in any of tables 2, 3, 5, and 6, or different therefrom. One or more lipophilic ligands or one or more GalNAc ligands may be included at any position of the RNAi agents provided herein.

Dsrnas having duplex structures of about 20 to 23 base pairs (e.g., 21 base pairs) are known to the skilled artisan to be particularly effective in inducing RNA interference (Elbashir et al, (2001) EMBO j., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures may also be effective (Chu and Rana (2007) RNA 14:1714-1719; kim et al (2005) Nat Biotech 23:222-226). In the above embodiments, due to the nature of the oligonucleotide sequences provided herein, the dsRNA described herein may comprise at least one strand of at least 21 nucleotides in length. It is reasonably expected that shorter duplexes, minus only a few nucleotides at one or both ends, are equally effective compared to the dsRNA described above. Thus, dsRNA having a sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides derived from one of the sequences provided herein, and which differs by no more than 10%, 15%, 20%, 25% or 30% in its ability to inhibit GPR75 gene expression in an in vitro assay using an RNA agent having a concentration of Cos7 and 10nM as compared to dsRNA comprising the complete sequence in the PCR assay provided in the examples herein, are contemplated to be included within the scope of the present disclosure.

In addition, the RNAs described herein are capable of recognizing one or more sites in GPR75 transcript that are susceptible to RISC-mediated cleavage. Thus, the disclosure also describes RNAi agents targeting these sites. As used herein, an RNAi agent is said to target a particular site if the RNAi agent promotes cleavage of transcripts anywhere within the site. Such RNAi agents typically comprise at least about 15 contiguous nucleotides, e.g., at least 19 nucleotides, from one of the sequences provided herein coupled to other nucleotide sequences taken from the region adjacent to the selected sequence in the GPR75 gene.

RNAi agents described herein can contain one or more mismatches with the target sequence. In one embodiment, an RNAi agent described herein comprises no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent described herein comprises no more than 2 mismatches. In one embodiment, the RNAi agents described herein comprise no more than 1 mismatch. In one embodiment, the RNAi agents described herein comprise 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains a mismatch to the target sequence, the mismatch can optionally be limited to the last 5 nucleotides from the 5 'or 3' end of the complementarity region. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand complementary to the GPR75 gene region typically does not contain any mismatches within the central 13 nucleotides. Methods described herein or known in the art can be used to determine whether RNAi agents containing mismatches to the target sequence are effective in inhibiting expression of the GPR75 gene. Considering the efficacy of RNAi agents with mismatches in inhibiting GPR75 gene expression is important, particularly if a mutation in a specific region of complementarity in the GPR75 gene is known.

III modified RNAi agents of the present disclosure

In one embodiment, the RNA, e.g., dsRNA, of the RNAi agents of the present disclosure is unmodified and does not comprise chemical modifications or conjugation, e.g., as known in the art and described herein. In some embodiments, the RNA, e.g., dsRNA, of the RNAi agents of the invention is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the present disclosure, substantially all of the nucleotides of the RNAi agents of the present disclosure are modified. In other embodiments of the disclosure, all nucleotides of the RNAi agents of the disclosure are modified. The "substantially all nucleotides are modified" in RNAi agents of the present disclosure are most but not all nucleotides are modified, and can include no more than 5, 4, 3, 2, or 1 unmodified nucleotides. In other embodiments of the disclosure, RNAi agents of the disclosure can include no more than 5, 4, 3, 2, or 1 modified nucleotide.

The nucleic acids of the present disclosure may be synthesized or modified by well-established methods in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, s.l. et al (edrs.), john Wiley & Sons, inc., new York, NY, USA, incorporated herein by reference. The modification comprises the following steps: for example, terminal modifications, e.g., 5 '-terminal modifications (phosphorylation, conjugation, reverse ligation) or 3' -terminal modifications (conjugation, DNA nucleotides, reverse ligation, etc.); base modification, e.g., substitution with the following bases: stabilizing bases, destabilizing bases, or bases that base pair with an extended pool of partners, removing bases (abasic nucleotides), or conjugating bases; sugar modifications (e.g., at the 2 '-position or the 4' -position) or sugar substitutions; or backbone modification, including modification or substitution of phosphodiester bonds. Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs that contain a modified backbone or do not contain natural internucleoside linkages. RNA having a modified backbone includes, in addition, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs that do not have phosphorus atoms in their internucleoside backbones can also be considered oligonucleotides. In some embodiments, the modified RNAi agent will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methylphosphonates and other alkylphosphates including 3 '-alkylene phosphates and chiral phosphates, phosphonites, phosphoramidates including 3' -phosphoramidates and aminoalkyl phosphoramidates, thiocarbonylphosphoramidates, thiocarbonylalkylphosphonates, and borophosphate, 2'-5' linked analogs of these, and those with reverse polarity wherein adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5 '-2'. Various salts (e.g., sodium salts, mixed salts) and free acid forms may also be included.

Representative U.S. patents teaching the preparation of the above-described phosphorus-containing linkages 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, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,316, 5,550,111, 5,563,253, 5,571,799, 5,587,361, and 5,587,361, U.S. Pat. RE 5,587,361, the entire contents of each patent are incorporated herein by reference.

Wherein the modified RNA backbone excluding phosphorus atoms has a backbone formed by internucleoside linkages of short chain alkyl or cycloalkyl groups, internucleoside linkages of mixed heteroatoms and alkyl or cycloalkyl groups, or internucleoside linkages of one or more short chain heteroatoms or heterocycles. These include those backbones having morpholino linkages (formed in part from the sugar moiety of the nucleoside); a siloxane backbone; sulfide, sulfoxide, and sulfone backbones; formylacetyl (formacetyl) and thioformylacetyl backbones; a methylene methylacetyl and a thioformylacetyl backbone; a backbone comprising olefins; a sulfamate backbone; methylene imino and methylene hydrazino backbones; sulfonate and sulfonamide backbones; an amide backbone; with a mixture of N, O, S and CH ₂ Other backbones of the constituent components.

Representative U.S. patents teaching the preparation of such oligonucleotides include, but are not limited to, U.S. Pat. nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 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, the entire contents of each of which are incorporated herein by reference.

In other embodiments, suitable RNA mimics are contemplated for use in RNAi agents in which both the sugar and internucleoside linkages, i.e., the backbone, of the nucleotide units are replaced with new groups. The base unit remains unchanged so as to hybridize to the appropriate nucleic acid target compound. One such oligomeric compound is known as Peptide Nucleic Acid (PNA), in which RNA mimics have been shown to have excellent hybridization properties. In PNA compounds, the sugar backbone of RNA is replaced with an amide containing backbone, especially an aminoethylglycine backbone. Nucleobases are retained and are bound directly or indirectly to the nitrogen heteroatoms of the amide moiety of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. patent nos. 5,539,082, 5,714,331, and 5,719,262, each of which is incorporated by reference in its entirety. Other PNA compounds suitable for use in RNAi agents of the present disclosure are described, for example, in Nielsen et al, science,1991,254,1497-1500.

Some embodiments presented in this disclosure include RNAs with phosphorothioate backbones and oligonucleotides with heteroatom backbones, particularly the —ch of U.S. patent No. 5,489,677 cited above ₂ --NH--CH ₂ --、--CH ₂ --N(CH ₃ )--O--CH ₂ - - [ is called methylene (methylimino) or MMI backbone ] ]、--CH ₂ --O--N(CH ₃ )--CH ₂ --、--CH ₂ --N(CH ₃ )--N(CH ₃ )--CH ₂ -and-N (CH) ₃ )--CH ₂ --CH ₂ - - - -, and the amide backbone of the above-mentioned U.S. Pat. No. 5,602,240. In some embodiments, the RNAs set forth herein have morpholinyl backbone structures of U.S. patent No. 5,034,506 referred to above. The natural phosphodiester backbone may be represented as O-P (O) (OH) -OCH ₂ -。

The modified RNA may also comprise one or moreA substituted sugar moiety. RNAi agents, e.g., dsRNA, presented herein can comprise one of the following at the 2' -position: OH; f, performing the process; o-alkyl, S-alkyl or N-alkyl; o-alkenyl, S-alkenyl or N-alkenyl; o-alkynyl, S-alkynyl or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C ₁ To C ₁₀ Alkyl or C ₂ To C ₁₀ Alkenyl and alkynyl groups. Exemplary suitable modifications include O [ (CH) ₂ ) _n O] _m CH ₃ 、O(CH ₂ ). _n OCH ₃ 、O(CH ₂ ) _n NH ₂ 、O(CH ₂ ) _n CH ₃ 、O(CH ₂ ) _n ONH ₂ And O (CH) ₂ ) _n ON[(CH ₂ ) _n CH ₃ )] ₂ Wherein n and m are from 1 to about 10. In other embodiments, the dsRNA comprises one of the following at the 2' position: c (C) ₁ To C ₁₀ Lower alkyl, substituted lower alkyl, alkylaryl, arylalkyl, O-alkylaryl or O-arylalkyl, SH, SCH ₃ 、OCN、Cl、Br、CN、CF ₃ 、OCF ₃ 、SOCH ₃ 、SO ₂ CH ₃ 、ONO ₂ 、NO ₂ 、N ₃ 、NH ₂ A heterocycloalkyl group, a heterocycloalkyl aryl group, an aminoalkylamino group, a polyalkylamino group, a substituted silyl group, an RNA cleavage group, a reporter group, an intercalator, a group for improving the pharmacokinetics of an RNAi agent, or a group for improving the pharmacokinetics of an RNAi agent, as well as other substituents having similar properties. In some embodiments, the modification comprises 2 '-methoxyethoxy (2' -O- -CH) ₂ CH ₂ OCH ₃ 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 2' -dimethylaminooxyethoxy, i.e., O (CH) ₂ ) ₂ ON(CH ₃ ) ₂ Groups, also known as 2' -DMAEE, as described in the examples below, and 2' -dimethylaminoethoxyethoxy (also known in the art as 2' -O-dimethylaminoethoxyethyl or 2' -DMAEOE), i.e., 2' -O- -CH ₂ --O--CH ₂ --N(CH ₃ ) ₂ . Further exemplary modifications include: 5'-Me-2' -F nucleotide, 5'-Me-2' -OMe nucleotide, 5'-Me-2' -deoxynucleotide (R isomer and S isomer in these three families); 2' -alkoxyalkyl; and 2' -NMA (N-methylacetamide).

Other modifications include 2 '-methoxy (2' -OCH) ₃ ) 2 '-aminopropoxy (2' -OCH) ₂ CH ₂ CH ₂ NH ₂ ) 2' -O-hexadecyl and 2' -fluoro (2 ' -F). Similar modifications can also be made at other positions on the RNA of the RNAi agent, particularly in the 3 'position of the sugar of the 3' terminal nucleotide or in the 2'-5' linked dsRNA, as well as in the 5 'position of the 5' terminal nucleotide. RNAi agents can also have a glycomimetic such as a cyclobutyl moiety in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. patent nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,576,427, 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 commonly owned with the present application, the entire contents of each of which are incorporated herein by reference.

RNAi agents of the invention can also include modification or substitution of nucleobases (commonly referred to in the art simply as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (a) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6-azocytosine and 6-azothymine, 5-uracil (pseudouracil), 4-thiourea, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-aza and 8-aza adenine and 7-deaza and 3-deaza. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, modified Nucleosides in Biochemistry, biotechnology and Medicine, herdewijn, P.ed.Wiley-VCH,2008, the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz,J.L,ed.John Wiley&Sons,1990, englisch et al, (1991) Angewandte Chemie, international Edition,30:613, and Sanghvi, Y.S., chapter 15,dsRNA Research and Applications,pages 289-302,Crooke,S.T.and Lebleu,B, ed., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds proposed in the present disclosure. These nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyl adenine, 5-propyl uracil, and 5-propynyl cytosine. 5-methylcytosine substitutions have been shown to increase the stability of nucleic acid duplex by 0.6 ℃ to 1.2 ℃ (Sanghvi, y.s., rooke, s.t. and Lebleu, b., eds., dsRNA Research and Applications, CRC Press, boca Raton,1993, pp.276-278) and are exemplary base substitutions, especially when combined with 2' -O-methoxyethyl sugar modifications.

Representative U.S. patents teaching the preparation of some of the above-described modified nucleobases, as well as others, include, but are not limited to, 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, 5,552,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088, the entire contents of each of which are incorporated herein by reference.

RNAi agents of the present disclosure can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a ring-modified furanosyl ring formed by bridging two carbons (either adjacent or non-adjacent atoms). A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety comprising a ring formed by bridging two carbon atoms of a sugar ring, the ring comprising a bridge connecting two carbons (whether adjacent or not), thereby forming a bicyclic ring system. In certain embodiments, the bridge optionally connects the 4' -carbon and the 2' -carbon of the sugar ring through a 2' -acyclic oxygen atom. Thus, in some embodiments, an agent of the present disclosure may include one or more Locked Nucleic Acids (LNAs). Locked nucleic acids are nucleotides with a modified ribose moiety, where the ribose moiety contains an additional bridge linking the 2 'carbon and the 4' carbon. In other words, LNA is a nucleotide comprising a bicyclic sugar moiety comprising 4' -CH ₂ -O-2' bridge. This structure effectively "locks" the ribose in the 3' -inward facing structural conformation. The addition of locked nucleic acids to siRNA has been shown to increase stability of siRNA in serum and reduce off-target effects (Elmen, j.et., (2005) Nucleic Acids Research 33 (1): 439-447;Mook,OR.et al., (2007) Mol Canc Ther6 (3): 833-843;Grunweller,A.et al., (2003) Nucleic Acids Research31 (12): 3185-3193). Examples of bicyclic nucleosides for polynucleotides of the present disclosure include, but are not limited to, nucleosides comprising a bridge between a 4 'ribosyl ring atom and a 2' ribosyl ring atom. In certain embodiments, antisense polynucleotide agents of the present disclosure include one or more bicyclic nucleosides comprising a 4 'to 2' bridge.

The locked nucleoside can be represented by the following structure (stereochemistry omitted),

wherein B is a nucleobase or modified nucleobase and L is a linking group that links the 2 '-carbon of the ribose ring to the 4' -carbon.

Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4' - (CH) ₂ )—O-2'(LNA)；4'-(CH ₂ )—S-2′；4′-(CH ₂ ) ₂ —O-2′(ENA)；4'-CH(CH ₃ ) -O-2 '(also referred to as "restricted ethyl" or "cEt") and 4' -CH (CH) ₂ OCH ₃ ) -O-2' (and analogues thereof; see, for example, U.S. patent No. 7,399,845); 4' -C (CH) ₃ )(CH ₃ ) -O-2' (and analogues thereof; see, for example, U.S. patent No. 8,278,283); 4' -CH ₂ —N(OCH ₃ ) -2' (and analogues thereof; see, for example, U.S. patent No. 8,278,425); 4' -CH ₂ —O—N(CH ₃ ) -2' (see, e.g., U.S. patent publication No. 2004/0171570); 4' -CH ₂ -N (R) -O-2', wherein R is H, C1-C12 alkyl or a nitrogen protecting group (see e.g. us patent 7,427,672); 4' -CH ₂ —C(H)(CH ₃ ) -2' (see e.g. Chattopadhyaya et al, j.org. chem.,2009,74,118-134); and 4' -CH ₂ —C(＝CH ₂ ) -2' (and analogues thereof; see, for example, U.S. patent No. 8,278,426). The entire contents of the foregoing are incorporated herein by reference.

Other representative U.S. patents and U.S. patent publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,133, 7,084,125, 7,399,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, US 2008/0039618, and US 2009/0012281, the entire contents of each of which are incorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations, including, for example, α -L-ribofuranose and β -D-ribofuranose (see WO 99/14226).

RNAi agents of the present disclosure can also be modified to include one or more restriction ethyl nucleotides. As used herein, a "restriction ethyl nucleotide" or "cEt" is a locked nucleic acid comprising a bicyclic sugar moiety comprising 4' -CH (CH ₃ ) An O-2' bridge (i.e., L in the foregoing structure). In one embodiment, the restriction ethyl nucleotide is in the S conformation, referred to herein as "S-cEt".

RNAi agents of the present disclosure can also include one or more "conformational restriction nucleotides" ("CRNs"). CRNs are nucleotide analogs having a linker linking the C2 'carbon and the C4' carbon of ribose or the-C3 'carbon and the-C5' carbon of ribose. CRN locks the ribose ring in a stable conformation and increases its hybridization affinity to mRNA. The length of the linker is sufficient to place the oxygen in an optimal position for stability and affinity, thereby reducing wrinkling of the ribose ring (puckering).

Representative patent publications that teach the preparation of the CRNs described above include, but are not limited to, US 2013/0190383 and WO 2013/036868, the respective contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, RNAi agents of the present disclosure comprise one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid in which any bonds of the sugar have been removed, forming an unlocked "sugar" residue. In one example, the UNA further includes a C1'-C4' bond (i.e., a carbon-oxygen-carbon covalent bond between the C1 'and C4' carbons) that has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon covalent bond between the C2 'and C3' carbons) has been removed (see nuc.acids symp. Series,52,133-134 (2008) and fluidizer et al., mol. Biosystem., 2009,10,1039, incorporated herein by reference).

Representative U.S. patent publications that teach the preparation of UNA include, but are not limited to, US 8,314,227 and U.S. patent publication nos. 2013/0096289, 2013/0011922, and 2011/0313020, each of which is incorporated herein by reference in its entirety.

Potential stabilizing modifications to the ends of the RNA molecule may include N- (acetylaminohexanoyl) -4-hydroxyproline (Hyp-C6-NHAc), N- (hexanoyl-4-hydroxyproline (Hyp-C6), N- (acetyl-4-hydroxyproline (Hyp-NHAc), thymine-2 '-O-deoxythymine (ether), N- (aminohexanoyl) -4-hydroxyproline (Hyp-C6-amino), 2-behenyl-uridine-3' -phosphate, inverted base dT (idT), etc. such modifications are disclosed in WO 2011/005861.

Other modifications of the RNAi agents of the present disclosure include 5' phosphates or 5' phosphate mimics, e.g., 5' terminal phosphates or phosphate mimics on the antisense strand of the RNAi agent. Suitable phosphate mimetics are disclosed, for example, in US 2012/0157511, the entire contents of which are incorporated herein by reference.

A. Modified RNAi agents comprising motifs of the present disclosure

In certain aspects of the present disclosure, double stranded RNAi agents of the present disclosure include agents with chemical modifications as disclosed, for example, in WO2013/075035 (the entire contents of which are incorporated herein by reference). As shown herein and in WO2013/075035, one or more motifs of three identical modifications located on three consecutive nucleotides can be introduced into the sense or antisense strand of an RNAi agent, in particular at or near the cleavage site. In some embodiments, the sense and antisense strands of the RNAi agent can be fully modified in other ways. The introduction of these motifs interrupts the modification pattern of the sense strand or antisense strand, if present. The RNAi agent can optionally be conjugated to a lipophilic ligand, e.g., a C16 ligand, e.g., on the sense strand. RNAi agents can optionally be modified with (S) -diol nucleic acid (GNA) modifications, e.g., on one or more residues of the antisense strand.

Thus, the present disclosure provides double stranded RNAi agents capable of inhibiting expression of a target genome or gene (i.e., GPR75 gene) in vivo. RNAi agents include a sense strand and an antisense strand. Each strand of the RNAi agent can be 15 to 30 nucleotides in length. For example, each strand may be 16 to 30 nucleotides, 17 to 30 nucleotides, 25 to 30 nucleotides, 27 to 30 nucleotides, 17 to 23 nucleotides, 17 to 21 nucleotides, 17 to 19 nucleotides, 19 to 25 nucleotides, 19 to 23 nucleotides, 19 to 21 nucleotides, 21 to 25 nucleotides, or 21 to 23 nucleotides in length. In certain embodiments, each strand is 19 to 23 nucleotides in length.

The sense and antisense strands typically form duplex double-stranded RNAs ("dsRNA"), also referred to herein as "RNAi agents. The duplex region of the RNAi agent can be 15 to 30 nucleotide pairs in length. For example, the duplex region can be 16 to 30 nucleotide pairs, 17 to 30 nucleotide pairs, 27 to 30 nucleotide pairs, 17 to 23 nucleotide pairs, 17 to 21 nucleotide pairs, 17 to 19 nucleotide pairs, 19 to 25 nucleotide pairs, 19 to 23 nucleotide pairs, 19 to 21 nucleotide pairs, 21 to 25 nucleotide pairs, or 21 to 23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length. In some embodiments, the duplex region is 19 to 21 nucleotide pairs in length.

In one embodiment, the RNAi agent can contain one or more protruding regions or end-capping groups at the 3 '-terminus, 5' -terminus, or both of one or both strands. The overhang may be 1 to 6 nucleotides in length, for example, 2 to 6 nucleotides in length, 1 to 5 nucleotides in length, 2 to 5 nucleotides in length, 1 to 4 nucleotides in length, 2 to 4 nucleotides in length, 1 to 3 nucleotides in length, 2 to 3 nucleotides in length, or 1 to 2 nucleotides in length. In some embodiments, the nucleotide overhang region is 2 nucleotides in length. The overhang may be the result of one strand being offset from the other, or the result of two strands of the same length being offset. The overhang may form a mismatch with the target mRNA, or it may be complementary to the gene sequence being targeted or may be another sequence. The first strand and the second strand may also be joined, for example, by additional bases to form a hairpin, or by other non-base linkers.

In one embodiment, the nucleotides in the RNAi agent protruding region can each independently be a modified or unmodified nucleotide, including, but not limited to, 2 '-sugar modified (e.g., 2-F, 2' -O-methyl) thymidine (T), and any combination thereof.

For example, TT may be an overhang sequence at either end of either strand. The overhang may form a mismatch with the target mRNA, or it may be complementary to the gene sequence being targeted, or it may be another sequence.

The 5 '-overhang or 3' -overhang on the sense strand, antisense strand, or both strands of the RNAi agent can be phosphorylated. In some embodiments, one or more of the protruding regions comprises two nucleotides with a phosphorothioate therebetween, wherein the two nucleotides may be the same or different. 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 in the antisense strand. In one embodiment, the 3' -overhang is present in the sense strand.

RNAi agents may comprise only a single protrusion that may enhance the interfering activity of RNAi without affecting its overall stability. For example, the single stranded overhang may be located at the 3 'end of the sense strand, or at the 3' end of the antisense strand. RNAi may also have a blunt end located 5 'of the antisense strand (e.g., 3' of the sense strand), and vice versa. Typically, the antisense strand of an RNAi has a nucleotide overhang at the 3 'end and a blunt end at the 5' end. While not wishing to be bound by theory, the asymmetric blunt end of the 5 'end of the antisense strand and the 3' overhang of the antisense strand facilitate loading of the guide strand into the RISC process.

In one embodiment, the RNAi agent is a double blunt end 19 nucleotides in length, wherein the sense strand contains at least one motif modified with three 2'-F at three consecutive nucleotides at positions 7, 8, and 9 from the 5' end. The antisense strand contains at least one motif modified with three 2 '-O-methyl groups located at three consecutive nucleotides from positions 11, 12 and 13 from the 5' end.

In another embodiment, the RNAi agent is a double-blunt end 20 nucleotides in length, wherein the sense strand contains at least one motif with three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand contains at least one motif modified with three 2 '-O-methyl groups located at three consecutive nucleotides from positions 11, 12 and 13 from the 5' end.

In another embodiment, the RNAi agent is a double-blunt end 21 nucleotides in length, wherein the sense strand contains at least one motif with three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand contains at least one motif modified with three 2 '-O-methyl groups located at three consecutive nucleotides from positions 11, 12 and 13 from the 5' end.

In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif with three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; the antisense strand contains at least one motif modified with three 2 '-O-methyl groups located at three consecutive nucleotides from positions 11, 12 and 13 from the 5' end, wherein one end of the RNAi agent is blunt-ended and the other end comprises a protrusion with 2 nucleotides. In one embodiment, the overhang having 2 nucleotides is located at the 3' end of the antisense strand. When a 2 nucleotide overhang is located at the 3' end of the antisense strand, there may be two thiosulfate internucleotide linkages between the terminal three nucleotides, two of which are the overhang nucleotides and the third is the pairing nucleotide immediately adjacent to the overhang nucleotide. In one embodiment, the RNAi agent has two additional phosphorothioate internucleotide linkages between the terminal three nucleotides of both the 5 'end of the sense strand and the 5' end of the antisense strand. In one embodiment, each nucleotide in the sense and antisense strands of the RNAi agent, including the nucleotide as part of the motif, is a modified nucleotide. In one embodiment, each residue is independently modified with a 2 '-O-methyl or 2' -fluoro group, e.g., in an alternating motif. Optionally, the RNAi agent further comprises a ligand (e.g., a lipophilic ligand, optionally, a C16 ligand).

In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5' terminal nucleotide (position 1), positions 1-23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length and, starting from the 3' terminal nucleotide, those positions paired with positions 1 to 23 of the sense strand to form a duplex comprise at least 8 ribonucleotides; wherein at least the 3' terminal nucleotide of the antisense strand is not paired with the sense strand and up to 6 consecutive 3' terminal nucleotides are not paired with the sense strand, thereby forming a 3' single stranded overhang having 1 to 6 nucleotides; wherein the 5 'end of the antisense strand comprises 10 to 30 consecutive nucleotides that are not paired with the sense strand, thereby forming a single-stranded 5' overhang of 10 to 30 nucleotides; wherein, when the sense strand is aligned with the antisense strand to achieve maximum complementarity, at least the 5 'terminal nucleotide and the 3' terminal nucleotide of the sense strand base pair with the nucleotides of the antisense strand, thereby forming a region of substantially duplex between the sense strand and the antisense strand; and, the antisense strand is sufficiently complementary to the target RNA over a length of at least 19 nucleotides along the antisense strand to reduce expression of the target gene upon introduction of the double stranded nucleic acid into a mammalian cell; and wherein the sense strand contains at least one motif of three 2' -F modifications located on three consecutive nucleotides, wherein at least one motif occurs at or adjacent to the cleavage site. The antisense strand contains at least one motif modified with three 2' -O-methyl groups at or adjacent to the cleavage site at three consecutive nucleotides.

In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the RNAi agent comprises a first strand of at least 25 and up to 29 nucleotides in length and a second strand of up to 30 nucleotides in length and having at least one motif of three 2 '-O-methyl modifications located on three consecutive nucleotides at positions 11, 12 and 13 from the 5' end; wherein the 3 'end of the first strand and the 5' end of the second strand form blunt ends and the second strand is 1 to 4 nucleotides longer than the first strand at its 3 'end, wherein the duplex region is at least 25 nucleotides in length and the second strand is sufficiently complementary to the target mRNA over at least 19 nucleotides in length along the second strand to reduce expression of the target gene upon introduction of the RNAi agent into a mammalian cell, and wherein Dicer cleavage of the RNAi agent produces an siRNA comprising the 3' -end of the second strand, thereby reducing expression of the target gene in the mammalian body. Optionally, the RNAi agent further comprises a ligand.

In one embodiment, the sense strand of the RNAi agent comprises at least one motif located on three identical modifications on three consecutive nucleotides, with one motif occurring at the cleavage site of the sense strand.

In one embodiment, the antisense strand of the RNAi agent can further comprise at least one motif located at three identical modifications on three consecutive nucleotides, with one motif occurring at or adjacent to the cleavage site of the antisense strand.

For RNAi agents having duplex regions of 17 to 23 nucleotides in length, the cleavage site of the antisense strand is typically located near positions 10, 11, and 12 from the 5' -end. Thus, motifs with three identical modifications may occur at positions 9, 10 and 11, positions 10, 11 and 12, positions 11, 12 and 13, positions 12, 13 and 14, or positions 13, 14 and 15 of the antisense strand, counting from the first nucleotide at the 5 '-end of the antisense strand, or counting from the first paired nucleotide at the 5' -end of the antisense strand within the duplex region. The cleavage site of the antisense strand may also vary depending on the length of the duplex region from the 5' -end of the RNAi.

The sense strand of an RNAi agent can comprise at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand can have at least one motif located at or adjacent to the cleavage site of the strand with three identical modifications at three consecutive nucleotides. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand may be aligned such that one trinucleotide motif located on the sense strand overlaps with one trinucleotide motif located on the antisense strand by at least one nucleotide, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pairing with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of an RNAi agent can comprise more than one motif with three identical modifications located on three consecutive nucleotides. The first motif may occur at or adjacent to the cleavage site of the strand, while the other motif may be a wing modification. Herein, the term "wing modification (wing modification)" refers to a motif that is present in another part of the strand that is separated from a motif located at or adjacent to the cleavage site of the strand. The wing modification is either adjacent to the first motif or separated from the first motif by at least one or more nucleotides. When the motifs are immediately adjacent to each other, then the chemical nature of the motifs is quite different from each other; and when motifs are separated by one or more nucleotides, then the chemical properties may be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may occur at one end relative to the first motif located at or adjacent to the cleavage site, or at either side of the main motif (lead motif).

Like the sense strand, the antisense strand of an RNAi agent can comprise more than one motif of the same modification located on three consecutive nucleotides, with at least one motif occurring at or adjacent to the cleavage site of the strand. The antisense strand may also contain one or more wing modifications aligned in a manner similar to the wing modifications that may be present in the sense strand.

In one embodiment, the wing modification on the sense or antisense strand of an RNAi agent generally does not include the first or first two terminal nucleotides at the 3', 5', or both ends of the strand.

In another embodiment, the flanking modification on the sense or antisense strand of the RNAi agent generally does not include the first or first two paired nucleotides located within the duplex region at the 3', 5', or both ends of the strand.

When the sense and antisense strands of an RNAi agent each comprise at least one winged modification, the winged modifications can fall on the same end of the duplex region and have one, two, or three nucleotide overlap.

When the sense and antisense strands of an RNAi agent each comprise at least two flanking modifications, the sense and antisense strands can be aligned such that the two modifications from one strand each fall on one end of the duplex region and have an overlap of one, two, or three nucleotides; such that two modifications from one strand each fall on the other end of the duplex region and have an overlap of one, two, or three nucleotides; such that two modifications of one strand fall on each end of the main motif, respectively, and have one, two or three nucleotide overlaps within the duplex region.

In one embodiment, the RNAi agent comprises one or more mismatches with the target, one or more mismatches in the duplex, or a combination thereof. Mismatches may occur in the overhang region or in the duplex region. Base pairs may be ordered based on their propensity to promote dissociation or melting (e.g., the binding or dissociation free energy of a particular pairing is the simplest means to examine the pairing based on each pairing, although adjacent or similar assays may also be used). In terms of promoting dissociation: a is better than G and C; g is better than G and C; and I: C is better than G: C (i=inosine). Mismatches, such as atypical pairings or pairings other than typical pairings (as described elsewhere herein) are preferred over typical (A: T, A: U, G: C) pairings; and pairing involving universal bases is preferred over typical pairing.

In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs in the duplex region from the 5' end of the antisense strand, said base pairs being independently selected from the group consisting of: a U, G: U, I:C, and mismatched pairs such as atypical pairing or pairing other than typical pairing or pairing including universal bases to facilitate dissociation of the antisense strand at the 5' end of the duplex.

In one embodiment, the nucleotide at position 1 within 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 1, 2 or 3 base pairs located within the duplex region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair located within the duplex region from the 5' end of the antisense strand is an AU base pair.

In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymidine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymidine (dT). In one embodiment, for example, there is a short sequence of deoxythymidines, e.g., two dT nucleotides, on the 3' end of the sense strand or antisense strand.

In one embodiment, the sense strand sequence may be represented by formula (I):

5′n _p -N _a -(XXX) _i -N _b -YYY-N _b -(ZZZ) _j -N _a -n _q 3′ (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0 to 6;

each N _a Independently represent oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N _b Independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides;

Each n _p And n _q Independently represent a protruding nucleotide;

wherein N is _b And Y does not have the same modification; and is also provided with

XXX, YYY and ZZZ each independently represents three identical modified motifs located on three consecutive nucleotides. In one embodiment, YYY is an all 2' -F modified nucleotide.

In one embodiment, N _a Or N _b Including alternating patterns of modifications.

In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif occurs at or near the cleavage site of the sense strand (e.g., can occur at positions 6, 7, 8;7, 8, 9;8, 9, 10;9, 10, 11;10, 11, 12; or 11, 12, 13), counting from nucleotide 1 of the 5' end; or optionally, counting from the 1 st paired nucleotide in the duplex region at the 5' end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand can be represented by the following 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 alternatively

5′n _p -N _a -XXX-N _b -YYY-N _b -ZZZ-N _a -n _q 3′ (Id)。

When the sense strand is represented by formula (Ib), N _b Represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides.

Each N _a An oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides may be represented independently.

When the sense strand is represented by (Ic), N _b Represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Each N _a An oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides may be represented independently.

When the sense strand is represented by formula (Id), each N _b Independently represent an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. In some embodiments, N _b Is 0, 1, 2, 3, 4, 5 or 6. Each N _a An oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides may be represented independently. Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and 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)。

when the sense strand is represented by formula (Ia), each N _a Independently represent an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides.

In one embodiment, the antisense strand sequence of RNAi 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)，

Wherein:

k and l are each independently 0 or 1;

p 'and q' are each independently 0 to 6;

each N _a ' independently represents oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N _b ' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides;

each n _p ' and n _q ' independently represents a protruding nucleotide;

wherein N is _b 'and Y' do not have the same modification; and is also provided with

X ' X ' X ', Y ' Y ' Y ' and Z ' Z ' Z ' each independently represent three identical modified motifs located on three consecutive nucleotides.

In one embodiment, N _a ' or N _b ' comprising an alternating pattern of modifications.

The Y ' Y ' Y ' motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the Y' motif occurs at positions 9, 10, 11 of the antisense strand; 10. 11, 12; 11. 12, 13; 12. 13, 14; or 13, 14, 15, counting from nucleotide 1 at the 5' end; or optionally, counting from the first paired nucleotide within the duplex region at the 5' end. In some embodiments, the Y ' Y ' Y ' motif occurs at positions 11, 12, 13.

In one embodiment, the Y 'Y' Y 'motif is an all 2' -OMe modified nucleotide.

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

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 alternatively

5′n _q′ -N _a ′-Z′Z′Z′-N _b ′-Y′Y′Y′-N _b ′-X′X′X′-N _a ′-n _p′ 3′ (IId)。

When the antisense strand is represented by formula (IIb), N _b ' means an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Each N _a ' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides.

When the antisense strand is represented by formula (IIc), N _b ' means an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Each N _a ' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides.

When the antisense strand is represented by formula (IId), each N _b ' independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Each N _a ' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides. In some embodiments, N _b Is 0, 1, 2, 3, 4, 5 or 6.

In other embodiments, k is 0,l 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)。

when the antisense strand is represented by formula (IIa), each N _a ' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides.

Each of X ', Y ' and Z ' may be the same or different from each other.

Each nucleotide of the sense and antisense strands may be independently modified by LNA, ethylene Glycol Nucleic Acid (GNA), hexitol Nucleic Acid (HNA), 2 '-methoxyethyl, 2' -O-methyl, 2 '-O-allyl, 2' -C-allyl, 2 '-hydroxy, or 2' -fluoro. For example, each nucleotide of the sense strand and the antisense strand is independently modified by a 2 '-O-methyl group or a 2' -fluoro group. Specifically, each X, Y, Z, X ', Y ' and Z ' may represent a 2' -O-methyl modification or a 2' -fluoro modification.

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

In one embodiment, the antisense strand may comprise a Y ' motif present at positions 11, 12, 13 of the strand, counting from the first nucleotide at the 5' end, or optionally counting from the first paired nucleotide within the duplex region at the 5' end; y 'represents a 2' -O-methyl modification. The antisense strand may also contain a wing modification of either the X 'X' X 'motif or the Z' Z 'Z' motif as opposite ends of the duplex region; and X 'X' X 'and Z' Z 'Z' each independently represent a 2'-OMe modification or a 2' -F modification.

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

Thus, RNAi agents useful in the methods of the present disclosure can comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex being represented by formula (III):

sense strand: 5'n _p -N _a -(XXX) _i -N _b -YYY-N _b -(ZZZ) _j -N _a -n _q 3′

Antisense strand: 3' n _p ′-N _a ′-(X′X′X′) _k -N _b ′-Y′Y′Y′-N _b ′-(Z′Z′Z′) _l -N′ _a -n _q ′ 5′(III)

Wherein:

i. j, k and l are each independently 0 or 1;

p, p ', q and q' are each independently 0 to 6;

each N _a And N _a ' independently represents oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

Each N _b And N _b ' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides;

wherein the method comprises the steps of

Each n _p ′、n _p 、n _q ' and n _q May be present or absent and independently represent a protruding nucleotide; and is also provided with

XXX, YYY, ZZZ, X ' X ' X ', Y ' Y ' Y ' and Z ' Z ' Z ' each independently represent three identical modified motifs located on three consecutive nucleotides.

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

Exemplary combinations of sense and antisense strands forming an RNAi duplex 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 -Z Z Z-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)

when the RNAi agent is represented by formula (IIIa), each N _a Independently represent an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N _b Independently represent oligonucleotide sequences comprising 1 to 10, 1 to 7, 1 to 5 or 1 to 4 modified nucleotides. Each N _a Independently represent an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides.

When the RNAi agent is represented by formula (IIIc), each N _b 、N _b ' independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Each N _a Independently represent an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides.

When the RNAi agent is represented by formula (IIId), each N _b 、N _b ' independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Each N _a 、N _a ' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides. N (N) _a 、N _a '、N _b And N _b Each of the' independently contains an alternating pattern of modifications.

In one embodiment, when the RNAi agent is represented by formula (IIId), N _a The modification is a 2 '-O-methyl modification or a 2' -fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), N _a The modification is a 2 '-O-methyl modification or a 2' -fluoro modification, and n _p '>0, and at least one n _p ' linkage to adjacent nucleotides via phosphorothioate linkages. In another embodiment, when the RNAi agent is represented by formula (IIId), N _a The modification is 2 '-O-methyl modification or 2' -fluoro modification, n _p ′>0, and at least one n _p ' through phosphorothioate linkages to adjacent nucleotides, and the sense strand is conjugated to one or more C16 (or related) moieties attached through a divalent or trivalent branching linker (as described below). In another embodiment, when the RNAi agent is represented by formula (IIId), N _a The modification is 2 '-O-methyl modification or 2' -fluoro modification, n _p ′>0, and at least one n _p ' is linked to adjacent nucleotides by phosphorothioate linkages and the sense strand comprises at least one phosphorothioate linkage and the sense strand is conjugated to one or more lipophilic moieties such as C16 (or related) moieties, optionally attached by a divalent or trivalent branching linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa), N _a The modification is 2 '-O-methyl modification or 2' -fluoro modification, n _p ′>0, and at least one n _p ' being linked to adjacent nucleotides by phosphorothioate linkages, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic moieties such as a C16 (or related) moiety attached by a divalent or trivalent branching linker.

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

In one embodiment, the RNAi agent is a multimer comprising three, four, five, six, or more duplex represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplex is connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each duplex may target the same gene or two different genes; or each duplex may target two different target sites of the same gene.

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

A number of publications describe multimeric RNAi agents useful in the methods of the present disclosure. Such publications include WO2007/091269, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520; and US 7858769, the entire contents of each of which are incorporated herein by reference.

In certain embodiments, the compositions and methods of the present disclosure comprise Vinyl Phosphonate (VP) modifications to RNAi agents described herein. In exemplary embodiments, the 5' -vinylphosphonate modified nucleotide of the present disclosure has the following structure:

Wherein X is O or S;

r is hydrogen, hydroxy, fluoro or C _1-20 Alkoxy (e.g., methoxy or n-hexadecyloxy);

R ⁵ ' is =c (H) -P (O) (OH) ₂ And C5' carbon and R ⁵ The double bond between' is in the E-or Z-orientation (e.g., E-orientation); and

b is a nucleobase or modified nucleobase, optionally wherein B is adenine, guanine, cytosine, thymine or uracil.

The vinyl phosphonates of the present disclosure may be linked to the antisense strand or sense strand of the dsRNA of the present disclosure. In certain embodiments, a vinylphosphonate of the present disclosure is linked to the antisense strand of a dsRNA, optionally at the 5' end of the antisense strand of a dsRNA.

Vinyl phosphate modifications are also contemplated for use in the compositions and methods of the present disclosure. Exemplary vinyl phosphate structures include the foregoing structures, wherein R ⁵ ' is =c (H) -OP (O) (OH) ₂ And C5' carbon and R ⁵ The double bond between' is in the E-or Z-orientation (e.g., E-orientation).

E. Thermally labile modifications

In certain embodiments, RNA interference of dsRNA molecules can be optimized by incorporating a thermally labile modification (theramally destabilizing modification) in the seed region of the antisense strand. As used herein, "seed region" refers to positions 2 to 9 of the 5' end of the reference chain. For example, thermally labile modifications can be incorporated into the seed region of the antisense strand to reduce or inhibit off-target gene silencing.

The term "thermally labile modification" includes a modification that results in a lower overall melting temperature (Tm) of the dsRNA than the Tm of a dsRNA without such modification. For example, the thermally labile modification can reduce the Tm of the dsRNA by 1 ℃ to 4 ℃, such as 1, 2, 3, or 4 ℃. And the term "thermally labile nucleotide" refers to a nucleotide containing one or more thermally labile modifications.

It has been found that dsRNA having an antisense strand comprising at least one thermostable modification of the duplex within the first 9 nucleotide positions counted from the 5' end of the antisense strand has reduced off-target gene silencing activity. Thus, in some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five, or more) thermostable modification of the duplex within the first 9 nucleotide positions of its 5' region. In some embodiments, one or more thermally labile modifications of the duplex are located at positions 2 to 9, e.g., positions 4 to 8, from the 5' -end of the antisense strand. In some other embodiments, the thermostable modification of the duplex is located at position 6, 7 or 8 from the 5' -end of the antisense strand. In still other embodiments, the thermostable modification of the duplex is located at position 7 from the 5' end of the antisense strand. In some embodiments, the thermostable modification of the duplex is located at position 2, 3, 4, 5 or 9 from the 5' end of the antisense strand.

The thermally labile modification may include, but is not limited to: no base modification; mismatches with the opposite nucleotide in the opposite strand; and sugar modifications such as 2' -deoxy modifications or acyclic nucleotides such as Unlocked Nucleic Acids (UNA) or diol nucleic acids (GNA).

Exemplary abasic modifications include, but are not limited to, the following:

wherein r=h, me, et or OMe; r' =h, me, et or OMe; r "=h, me, et or OMe

Wherein B is a modified or unmodified nucleobase.

Exemplary sugar modifications include, but are not limited to, the following:

wherein B is a modified or unmodified nucleobase.

In some embodiments, the thermally labile modification of the duplex is selected from the group consisting of:

wherein B is a modified or unmodified nucleobase and each structurally asterisk represents R, S or racemic.

The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose, e.g., where any bond between ribose carbons (e.g., C1' -C2', C2' -C3', C3' -C4', C4' -O4', or C1' -O4 ') is absent, or at least one of ribose carbons or riboses oxygen (e.g., C1', C2', C3', C4', or O4 ') is absent in the nucleotide, either independently or in combination. In one placeIn some embodiments, the acyclic nucleotide is Wherein B is a modified or unmodified nucleobase, R ¹ And R is ² Independently H, halogen, OR ₃ Or alkyl; r is as follows ₃ Is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar). The term "UNA" refers to an unlocked acyclic nucleic acid in which any bond of a sugar has been removed, forming an unlocked "sugar" residue. In one example, the UNA further includes monomers from which bonds between C1'-C4' (i.e., carbon-oxygen-carbon covalent bonds between C1 'carbon and C4' carbon) have been removed. In another example, the C2' -C3 bond of the sugar (i.e., the carbon-carbon covalent bond between the C2' carbon and the C3' carbon) has been removed (see Mikhailov et al, tetrahedron Letters,26 (17): 2059 (1985); and fluidier et al, mol. Biosystem, 10:1039 (2009), the entire contents of which are incorporated herein by reference). Acyclic derivatives provide greater backbone flexibility without affecting Watson-Crick pairing. The acyclic nucleotides may be linked by a 2'-5' linkage or a 3'-5' linkage.

The term "GNA" refers to a diol nucleic acid which is a polymer similar to DNA or RNA, but which has a different composition of the "backbone" which is composed of repeating glycerol units linked by phosphodiester bonds:

the thermally labile modification of the duplex may be a mismatch (i.e., a non-complementary base pair) between a thermally labile nucleotide in the dsRNA duplex and an opposing nucleotide on the opposing strand. Exemplary mismatched base pairs include G: G, G: A, G: U, G: T, A: A, A: C, C: C, C: U, C: T, U: U, T: T, U:T or combinations thereof. Other mismatched base pairing known in the art are also suitable for use in the present invention. Mismatches may occur between naturally occurring nucleotides or modified nucleotides, i.e., mismatched base pairing may occur between nucleobases from the respective nucleotides, independent of modification on the ribose of the nucleotide. In certain embodiments, the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2' -deoxynucleobase; for example, 2' -deoxynucleobases in the sense strand.

In some embodiments, the thermally labile modification of the duplex in the antisense strand seed region includes Watson-Crick hydrogen bonding (W-C H-binding) compromised nucleotides to complementary bases on the target mRNA, such as modified nucleobases:

further examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA) and mismatch modifications have been described in detail in WO 2011/133876, which is incorporated herein by reference in its entirety.

Thermally labile modifications can also include universal bases with reduced or lost ability to form hydrogen bonds with the opposite base, as well as phosphate modifications.

In some embodiments, the thermally labile modification of the duplex includes a nucleotide having an atypical base, such as, but not limited to, a nucleobase modification with impaired or complete loss of the ability to form hydrogen bonds with bases in the opposite strand. Destabilization of the central region of the dsRNA duplex by these nucleobase modifications has been evaluated as described in WO 2010/0011895, which is incorporated herein by reference in its entirety. Exemplary nucleobase modifications are:

in some embodiments, the thermally labile modification of the duplex in the antisense strand seed region comprises one or more α -nucleotides complementary to bases on the target mRNA, for example:

Wherein R is H, OH, OCH ₃ 、F、NH ₂ 、NHMe、NMe ₂ Or O-alkyl.

Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplex compared to natural phosphodiester linkages are:

the alkyl group of the R group may be C ₁ -C ₆ An alkyl group. Specific alkyl groups for the R group include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

As will be appreciated by those of skill in the art, given that the functional role of nucleobases defines the specificity of RNAi agents of the present disclosure, nucleobase modifications can be made in a variety of ways described herein, e.g., to introduce labile modifications into RNAi agents of the present disclosure, e.g., for the purpose of enhancing the off-target (on-target) effect relative to the off-target effect, the range of modifications available to non-nucleobase modifications and typically present on RNAi agents of the present disclosure is often much greater, e.g., modifications to the glycosyl or phosphate backbone of a polyribonucleotide. Such modifications are described in more detail elsewhere in this disclosure, and are specifically contemplated for use in RNAi agents of this disclosure having a natural nucleobase or modified nucleobase as described above or elsewhere herein.

In addition to the antisense strand comprising a thermostable modification, the dsRNA may also comprise one or more stabilizing modifications. For example, a dsRNA can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, the stabilizing modifications may all be present in one strand. In some embodiments, both the sense strand and the antisense strand comprise at least two stabilizing modifications. Stabilization modifications may occur on any nucleotide of the sense strand or the antisense strand. For example, the stabilizing modification may occur on each nucleotide on the sense strand or the antisense strand; each stabilizing modification may occur in an alternating pattern on the sense strand or the antisense strand; or the sense strand or the antisense strand comprises an alternating pattern of stabilizing modifications. The alternating pattern of stabilizing modifications on the sense strand may be the same as or different from the alternating pattern on the antisense strand, and the alternating pattern of stabilizing modifications on the sense strand may be offset relative to the alternating pattern of stabilizing modifications on the antisense strand.

In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, the stabilizing modification in the antisense strand may be present at any position. In some embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 8, 9, 14 and 16 from the 5' end. In some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 14 and 16 from the 5' end. In some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 14 and 16 from the 5' end.

In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to a destabilizing modification. For example, the stabilizing modification may be a nucleotide located at the 5 'or 3' end of the destabilizing modification (i.e., at the-1 or +1 position of the destabilizing modification). In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5 'and 3' ends of the destabilizing modification (i.e., at the-1 and +1 positions of the destabilizing modification).

In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3' end of the destabilizing modification (i.e., at the +1 and +2 positions of the destabilizing modification).

In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, the stabilizing modification in the sense strand may be present at any position. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10 and 11 from the 5' end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10 and 11 from the 5' end. In some embodiments, the sense strand comprises stabilizing modifications at positions 11, 12 and 15 of the antisense strand (counting from the 5' end of the antisense strand) relative or complementary to each other. In some other embodiments, the sense strand comprises stabilizing modifications at opposite or complementary positions of positions 11, 12, 13 and 15 of the antisense strand (counting from the 5' end of the antisense strand). In some embodiments, the sense strand comprises two, three, or four stabilizing modified segments (blocks).

In some embodiments, the sense strand does not comprise a stabilizing modification at a position opposite or complementary to the thermostable modification of the duplex in the antisense strand.

Exemplary heat stabilization modifications include, but are not limited to, 2' -fluoro modifications. Other heat stabilization modifications include, but are not limited to, LNA.

In some embodiments, the dsRNA of the disclosure comprises at least four (e.g., four, five, six, seven, eight, nine, ten, or more) 2' -fluoro nucleotides. Without limitation, the 2' -fluoro nucleotides may all be present in one strand. In some embodiments, both the sense and antisense strands comprise at least two 2' -fluoro nucleotides. The 2' -fluoro modification may occur on any nucleotide of the sense strand or the antisense strand. For example, the 2' -fluoro modification may occur on each nucleotide on the sense strand or the antisense strand; each 2' -fluoro modification may occur in an alternating pattern on the sense strand or the antisense strand; or the sense or antisense strand comprises an alternating pattern of 2' -fluoro modifications. The alternating pattern of 2' -fluoro modifications on the sense strand may be the same as or different from the antisense strand, and the alternating pattern of 2' -fluoro modifications on the sense strand may be offset relative to the alternating pattern of 2' -fluoro modifications on the antisense strand.

In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2' -fluoro nucleotides. Without limitation, the 2' -fluoro modification in the antisense strand may be present at any position. In some embodiments, the antisense strand comprises 2 '-fluoro nucleotides at positions 2, 6, 8, 9, 14 and 16 from the 5' end. In some other embodiments, the antisense strand comprises 2 '-fluoro nucleotides at positions 2, 6, 14 and 16 from the 5' end. In some other embodiments, the antisense strand comprises 2 '-fluoro nucleotides at positions 2, 14 and 16 from the 5' end.

In some embodiments, the antisense strand comprises at least one 2' -fluoro nucleotide adjacent to a destabilizing modification. For example, the 2' -fluoro nucleotide may be a nucleotide located at the 5' or 3' end of the destabilization modification (i.e., at the-1 or +1 position of the destabilization modification position). In some embodiments, the antisense strand comprises 2' -fluoro nucleotides at each of the 5' and 3' ends of the destabilization modification (i.e., at the-1 and +1 positions of the destabilization modification).

In some embodiments, the antisense strand comprises at least two 2 '-fluoro nucleotides at the 3' end of the destabilization modification (i.e., at the +1 and +2 positions of the destabilization modification).

In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2' -fluoro nucleotides. Without limitation, the 2' -fluoro modification in the sense strand may be present at any position. In some embodiments, the antisense strand comprises 2 '-fluoro nucleotides at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand comprises 2 '-fluoro nucleotides at positions 7, 9, 10 and 11 from the 5' end. In some embodiments, the sense strand comprises 2 '-fluoro nucleotides located at positions opposite or complementary to positions 11, 12 and 15 of the antisense strand (counting from the 5' end of the antisense strand). In some other embodiments, the sense strand comprises 2 '-fluoro nucleotides at opposite or complementary positions of positions 11, 12, 13 and 15 of the antisense strand (counting from the 5' end of the antisense strand). In some embodiments, the sense strand comprises a segment of two, three, or four 2' -fluoro nucleotides.

In some embodiments, the sense strand does not comprise a 2' -fluoro nucleotide at a position opposite or complementary to the thermally labile modification of the duplex in the antisense strand.

In some embodiments, a dsRNA molecule of the present disclosure comprises a sense strand of 21 nucleotides (nt) and an antisense strand of 23 nucleotides (nt), wherein the antisense strand comprises at least one thermally labile nucleotide, wherein the at least one thermally labile nucleotide occurs in a seed region of the antisense strand (i.e., positions 2 to 9 of the 5' end of the antisense strand), wherein one end of the dsRNA is blunt-ended and the other end comprises a protrusion of two nucleotides, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, or all seven) of the following characteristics: (i) The antisense strand comprises 2, 3, 4, 5 or 6 2' -fluoro modifications; (ii) The antisense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) conjugation of the sense strand to the ligand; (iv) the sense strand comprises 2, 3, 4 or 5 2' -fluoro modifications; (v) The sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2' -fluoro modifications; and (vii) the dsRNA comprises a blunt end at the 5' end of the antisense strand. In one embodiment, the two nucleotide overhang is located at the 3' end of the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure comprise a sense strand and an antisense strand, wherein: the sense strand is 25 to 30 nucleotide residues in length, wherein starting from the 5' terminal nucleotide (position 1), positions 1 to 23 of the sense strand comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, starting from the 3' terminal nucleotide, comprising at least 8 ribonucleotides at positions that pair with positions 1 to 23 of the sense strand to form a duplex; wherein at least the 3' terminal nucleotide of the antisense strand is not paired with the sense strand and up to 6 consecutive 3' terminal nucleotides are not paired with the sense strand, thereby forming a 1 to 6 nucleotide 3' single stranded overhang; wherein the 5 'end of the antisense strand comprises 10 to 30 consecutive nucleotides that are not paired with the sense strand, thereby forming a single-stranded 5' overhang of 10 to 30 nucleotides; wherein, when the sense strand is aligned with the antisense strand to achieve maximum complementarity, at least the 5 'terminal nucleotide and the 3' terminal nucleotide of the sense strand base pair with the nucleotides of the antisense strand, thereby forming a region of substantially duplex between the sense strand and the antisense strand; and, the antisense strand is sufficiently complementary to the target RNA over a length of at least 19 nucleotides along the antisense strand to reduce expression of the target gene upon introduction of the double stranded nucleic acid into a mammalian cell; and wherein the sense strand comprises at least one thermally labile nucleotide, wherein the at least one thermally labile nucleotide is located in a seed region of the antisense strand (i.e., positions 2 to 9 of the 5' end of the antisense strand). For example, a thermally labile nucleotide occurs between positions 14 to 17 opposite or complementary to the 5' end of the sense strand, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) The antisense strand comprises 2, 3, 4, 5 or 6 2' -fluoro modifications; (ii) The antisense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) conjugation of the sense strand to the ligand; (iv) the sense strand comprises 2, 3, 4 or 5 2' -fluoro modifications; (v) The sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2' -fluoro modifications; and (vii) the dsRNA comprises a duplex region of 12 to 30 nucleotide pairs in length.

In some embodiments, the dsRNA molecule of the present disclosure comprises a sense strand and an antisense strand, wherein the dsRNA molecule comprises a sense strand of at least 25 and at most 29 nucleotides in length and an antisense strand of at most 30 nucleotides in length, wherein the sense strand comprises modified nucleotides susceptible to enzymatic degradation at position 11 from the 5' end, wherein the 3' end of the sense strand and the 5' end of the antisense strand form blunt ends, and the antisense strand comprises at least one nucleotide at its 3' end than the sense strand by 1 to 4 nucleotides in length, wherein the duplex region is at least 25 nucleotides in length, and the antisense strand is sufficiently complementary to the target RNA along the length of at least 19nt of the antisense strand to reduce expression of the target gene upon introduction of the dsRNA molecule into a mammalian cell, and wherein the dicer cleavage produces an siRNA comprising the 3' end of the antisense strand, thereby reducing expression of the target gene in a mammal, wherein the antisense strand comprises at least one nucleotide, wherein the antisense strand is not at least one nucleotide, and wherein the antisense strand is at least one, is located at one, five, and optionally three, thermally stable (e.g., at least one, 5, five, and three, or more) stable dsRNA strands: (i) The antisense strand comprises 2, 3, 4, 5 or 6 2' -fluoro modifications; (ii) The antisense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) conjugation of the sense strand to the ligand; (iv) The sense strand comprises 2, 3, 4 or 5 2' -fluoro modifications; (v) The sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2' -fluoro modifications; and (vii) the dsRNA comprises a duplex region of 12 to 29 nucleotide pairs in length.

In some embodiments, each nucleotide in the sense and antisense strands of the dsRNA molecule may be modified. Each nucleotide may be modified with the same or different modifications, which may include: one or more changes in one or both of the non-linked oxygen phosphates or one or more changes in one or more of the linked oxygen phosphates; a change in ribose moiety, such as a change in the 2' hydroxyl group on ribose; batch replacement of the phosphate moiety with a "dephosphorylation" linker; modification or substitution of natural bases; substitution or modification of the ribose-phosphate backbone.

Since nucleic acids are polymers of subunits, many modifications occur at repetitive positions within the nucleic acid, such as modification of bases or phosphate moieties or non-linked O of phosphate moieties. In some cases, the modification will occur at all target positions of the nucleic acid, but in many cases will not. For example, the modification may occur only at the 3 'end or 5' end position, may occur only at a terminal region, e.g., at a position on a terminal nucleotide or at a position in the last 2, 3, 4, 5, or 10 nucleotides of the strand. Modification may occur in the double stranded region, the single stranded region, or both. Modification may occur only in the double-stranded region of RNA or only in the single-stranded region of RNA. For example, phosphorothioate modifications at the non-linked O positions may occur at only one or both ends, may occur at only the terminal region, e.g., at a position on the terminal nucleotide or in the last 2, 3, 4, 5 or 10 nucleotides of the strand, or may occur at both the double-stranded region and the single-stranded region, particularly at the ends. One or more of the 5' ends may be phosphorylated.

For example, to enhance stability, a particular base may be included in the overhang, or a modified nucleotide or nucleotide substitute may be included in a single stranded overhang such as a 5 'overhang or a 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the protrusions. In some embodiments, all or some of the bases in the 3 'or 5' projections may be modified, for example, using the modifications described herein. Modifications may include, for example, modifications at the 2' position of the ribose using modifications known in the art, such as deoxyribonucleotides modified with 2' -deoxy-2 ' -fluoro (2 ' -F) or 2' -O-methyl groups, to replace nucleobases, and modifications using phosphate groups such as phosphorothioate modifications. The highlighting need not be homologous to the target sequence.

In some embodiments, each residue of the sense and antisense strands is independently modified by LNA, ethylene Glycol Nucleic Acid (GNA), hexitol Nucleic Acid (HNA), 2 '-methoxyethyl, 2' -O-methyl, 2 '-O-allyl, 2' -C-allyl, 2 '-deoxy, or 2' -fluoro. These chains may contain more than one modification. In some embodiments, each residue of the sense strand and the antisense strand is independently modified by a 2 '-O-methyl or 2' -fluoro group. It is understood that these modifications are in addition to at least one heat labile modification of the duplex present in the antisense strand.

There are typically at least two different modifications on the sense and antisense strands. The two modifications may be 2' -deoxy modifications, 2' -O-methyl modifications or 2' -fluoro modifications, acyclic nucleotides or others. In some embodiments, the sense strand and the antisense strand each comprise two different modified nucleotides selected from 2 '-O-methyl or 2' -deoxy. In some embodiments, each residue of the sense strand and the antisense strand is independently modified by a 2' -O-methyl nucleotide, a 2' -deoxy-2 ' -fluoro nucleotide, a 2' -O-N-methylacetamido (2 ' -O-NMA, a 2' O-CH2C (O) N (Me) H) nucleotide, a 2' -O-dimethylaminoethoxyethyl (2 ' -O-DMAEOE) nucleotide, a 2' -O-aminopropyl (2 ' -O-AP) nucleotide, or a 2' -ara-F nucleotide. Also, it is understood that these modifications are in addition to at least one thermally labile modification of the duplex present in the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure comprise an alternating pattern of modification. As used herein, the term "alternating motif" or "alternating pattern" refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. Alternate nucleotides may refer to one every other nucleotide or one every three nucleotides, or similar patterns. For example, if A, B and C represent one type of modification to a nucleotide, respectively, the alternating motifs may be "ababababababab …", "AABBAABBAABB …", "aabababaab …", "AAABAAABAAAB …", "AAABBBAAABBB …" or "abccabcabc …", etc.

The types of modifications contained in the alternating motifs may be the same or different. For example, if A, B, C, D represents one type of modification on a nucleotide, respectively, the alternating motif, i.e., the modification on every other nucleotide, may be identical, but the sense strand or antisense strand may each be selected from several possible modifications within the alternating motif (e.g., "ABABAB …", "ACACAC …", "BDBDBD …" or "cdcd …", etc.).

In some embodiments, the dsRNA molecules of the present disclosure comprise a pattern of modification of alternating motifs on the sense strand that is offset relative to the pattern of modification of alternating motifs on the antisense strand. Such an offset may be such that the modifying group of the nucleotide of the sense strand corresponds to a differently modified group of the nucleotide of the antisense strand, and vice versa. For example, when the sense strand and the antisense strand pair in a dsRNA duplex, within the duplex region, the alternating motif of the sense strand may start with "abababa" from 5 'to 3' of the strand, and the alternating motif of the antisense strand may start with "BABABA" from 3 'to 5' of the strand. As another example, within a duplex region, the alternating motif of the sense strand may start with "AABBAABB" from 5 'to 3' of the strand, and the alternating motif of the antisense strand may start with "BBAABBAA" from 3 'to 5' of the strand, so there is a complete or partial shift in modification pattern between the sense and antisense strands.

The dsRNA molecules of the present disclosure may also comprise at least one phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage. Phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkage modifications may occur on any nucleotide at any position on the sense or antisense strand or strands of both strands. For example, internucleotide linkage modifications may occur on each nucleotide of the sense or antisense strand; each internucleotide linkage modification may occur in alternating patterns on either the sense strand or the antisense strand; or the sense or antisense strand may comprise an alternating pattern of two internucleotide linkage modifications. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same as or different from the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may be offset relative to the alternating pattern of internucleotide linkage modifications on the antisense strand.

In some embodiments, the dsRNA molecule comprises phosphorothioate internucleotide or methylphosphonate internucleotide linkage modifications located within the overhanging region. For example, the overhang region can contain two nucleotides with a phosphorothioate internucleotide linkage or a methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications may also link the overhanging nucleotides to terminal pairing nucleotides within the duplex region. For example, at least 2, 3, 4, or all of the protruding nucleotides can be linked by phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages, and optionally, there can be additional phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages linking the protruding nucleotide to the paired nucleotide immediately adjacent to the protruding nucleotide. For example, there may be at least two thiosulfate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhanging nucleotides and the third nucleotide is the pairing nucleotide immediately adjacent to the overhanging nucleotide. In one embodiment, these terminal three nucleotides may be located at the 3' end of the antisense strand.

In some embodiments, the sense strand of the dsRNA molecule comprises from 1 to 10 segments of 2 to 10 phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphointernucleotide linkages, wherein one of the phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages is located anywhere in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages or phosphointernucleotide linkages or with an antisense strand comprising phosphorothioate linkages or methylphosphonate linkages or phospholinkages.

In some embodiments, the antisense strand of the dsRNA molecule comprises two segments of two phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphointernucleotide linkages, wherein one of the phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages is located anywhere in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate internucleotide linkages, methylphosphonate internucleotide linkages, and phosphointernucleotide linkages, or with an antisense strand comprising phosphorothioate linkages or methylphosphonate linkages or phospholinkages.

In some embodiments, the antisense strand of the dsRNA molecule comprises two segments of three phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphointernucleotide linkages, wherein one of the phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate internucleotide linkages, methylphosphonate internucleotide linkages, and phosphointernucleotide linkages, or with an antisense strand comprising phosphorothioate linkages or methylphosphonate linkages or phospholinkages.

In some embodiments, the antisense strand of the dsRNA molecule comprises two segments of four phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphointernucleotide linkages, wherein one of the phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate internucleotide linkages, methylphosphonate internucleotide linkages, and phosphointernucleotide linkages, or with an antisense strand comprising phosphorothioate linkages or methylphosphonate linkages or phospholinkages.

In some embodiments, the antisense strand of the dsRNA molecule comprises two segments of five phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphointernucleotide linkages, wherein one of the phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate internucleotide linkages, methylphosphonate internucleotide linkages, and phosphointernucleotide linkages, or with an antisense strand comprising phosphorothioate linkages or methylphosphonate linkages or phospholinkages.

In some embodiments, the antisense strand of the dsRNA molecule comprises two segments of six phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphointernucleotide linkages, wherein one of the phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate internucleotide linkages, methylphosphonate internucleotide linkages, and phosphointernucleotide linkages, or with an antisense strand comprising phosphorothioate linkages or methylphosphonate linkages or phospholinkages.

In some embodiments, the antisense strand of the dsRNA molecule comprises two segments of seven phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphointernucleotide linkages, wherein one of the phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate internucleotide linkages, methylphosphonate internucleotide linkages, and phosphointernucleotide linkages, or with an antisense strand comprising phosphorothioate linkages or methylphosphonate linkages or phospholinkages.

In some embodiments, the antisense strand of the dsRNA molecule comprises two segments of eight phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphointernucleotide linkages, wherein one of the phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with the sense strand comprising any combination of phosphorothioate internucleotide linkages, methylphosphonate internucleotide linkages, and phosphointernucleotide linkages, or with the antisense strand comprising phosphorothioate linkages or methylphosphonate linkages or phospholinkages.

In some embodiments, the antisense strand of the dsRNA molecule comprises two segments of nine phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages separated by 1, 2, 3, or 4 phosphointernucleotide linkages, wherein one of the phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate internucleotide linkages, methylphosphonate internucleotide linkages, and phosphointernucleotide linkages, or with an antisense strand comprising phosphorothioate linkages or methylphosphonate linkages or phospholinkages.

In some embodiments, the dsRNA molecules of the present disclosure further comprise one or more phosphorothioate internucleotide or methylphosphonate internucleotide linkage modifications within 1 to 10 nucleotides of the terminal position of the sense strand or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked at one or both ends of the sense strand or antisense strand by phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages.

In some embodiments, the dsRNA molecules of the present disclosure further comprise one or more phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkage modifications within 1 to 10 nucleotides of the interior region of the duplex of each sense strand or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked at positions 8 to 16 of the double-stranded region (counted from the 5' end of the sense strand) by phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages; the dsRNA molecule may also optionally comprise one or more phosphorothioate internucleotide or methylphosphonate internucleotide linkage modifications within 1 to 10 terminal positions.

In some embodiments, the dsRNA molecules of the present disclosure further comprise 1 to 5 phosphorothioate internucleotide or methylphosphonate internucleotide modifications in positions 1 to 5 and 1 to 5 phosphorothioate internucleotide or methylphosphonate internucleotide modifications in positions 18 to 23 of the sense strand (counting from the 5 'end), and 1 to 2 phosphorothioate internucleotide or methylphosphonate internucleotide modifications in positions 1 to 2 and 1 to 5 phosphorothioate internucleotide or methylphosphonate internucleotide modifications in positions 18 to 23 of the antisense strand (counting from the 5' end).

In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1 to 5 and one phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage modification within positions 18 to 23 of the sense strand (counting from the 5 'end), and one phosphorothioate internucleotide linkage modification at position 1 or 2 and two phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end).

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications in positions 1 to 5 and one phosphorothioate internucleotide linkage modification in positions 18 to 23 of the sense strand (counting from the 5 'end), and one phosphorothioate internucleotide linkage modification in positions 1 and 2 and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 of the antisense strand (counting from the 5' end).

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1 to 5 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the sense strand (counting from the 5 'end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end).

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications in positions 1 to 5 and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 of the sense strand (counting from the 5 'end), and one phosphorothioate internucleotide linkage modification in positions 1 and 2 and one phosphorothioate internucleotide linkage modification in positions 18 to 23 of the antisense strand (counting from the 5' end).

In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification in positions 1 to 5 and one phosphorothioate internucleotide linkage modification in positions 18 to 23 of the sense strand (counting from the 5 'end), and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 of the antisense strand (counting from the 5' end).

In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification in positions 1 to 5 and one phosphorothioate internucleotide linkage modification in positions 18 to 23 of the sense strand (counting from the 5 'end), and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 and one phosphorothioate internucleotide linkage modification in positions 18 to 23 of the antisense strand (counting from the 5' end).

In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1 to 5 of the sense strand (counting from the 5 'end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification at positions 18 to 23 of the antisense strand (counting from the 5' end).

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide modifications within positions 1 to 5 of the sense strand (counted from the 5 'end), as well as one phosphorothioate internucleotide modification at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide modifications at positions 18 to 23 (counted from the 5' end).

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications in positions 1 to 5 and one phosphorothioate internucleotide linkage modification in positions 18 to 23 of the sense strand (counting from the 5 'end), and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 and one phosphorothioate internucleotide linkage modification in positions 18 to 23 of the antisense strand (counting from the 5' end).

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications in positions 1 to 5 and one phosphorothioate internucleotide linkage modification in positions 18 to 23 of the sense strand (counting from the 5 'end), and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 of the antisense strand (counting from the 5' end).

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 of the sense strand (counting from the 5 'end), and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5' end).

In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5 'end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end of the antisense strand).

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 of the sense strand (counting from the 5 'end), and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5' end).

In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5 'end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 of the antisense strand (counting from the 5' end).

In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 of the sense strand (counting from the 5 'end), and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5' end).

In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5 'end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 of the antisense strand (counting from the 5' end).

In some embodiments, the compounds of the present disclosure comprise a pattern of backbone chiral centers. In some embodiments, the common pattern of backbone chiral centers comprises at least 5 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 6 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 7 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 8 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 9 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 19 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 8 internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 7 internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 6 internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 5 internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 4 internucleotide linkages in Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 3 internucleotide linkages in Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 2 internucleotide linkages in Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 1 internucleotide linkage in Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 8 achiral internucleotide linkages (as a non-limiting example, phosphodiester). In some embodiments, the common pattern of backbone chiral centers comprises no more than 7 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises no more than 6 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises no more than 5 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises no more than 4 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises no more than 3 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises no more than 2 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises no more than 1 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration and no more than 8 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration and no more than 7 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration and no more than 6 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration and no more than 6 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration and no more than 5 achiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration and no more than 4 achiral internucleotide linkages. In some embodiments, the internucleotide linkages in the Sp configuration are optionally continuous or discontinuous. In some embodiments, the internucleotide linkage in the Rp configuration is optionally continuous or discontinuous. In some embodiments, achiral internucleotide linkages are optionally continuous or discontinuous.

In some embodiments, the compounds of the present disclosure comprise segments that are stereochemical segments (blocks). In some embodiments, the segment is an Rp segment, as each internucleotide linkage of the segment is Rp. In some embodiments, the 5' -segment is the Rp segment. In some embodiments, the 3' -segment is the Rp segment. In some embodiments, the segment is an Sp segment in that each internucleotide linkage of the segment is Sp. In some embodiments, the 5' -segment is an Sp segment. In some embodiments, the 3' -segment is an Sp segment. In some embodiments, the provided oligonucleotides comprise both Rp and Sp segments. In some embodiments, provided oligonucleotides comprise one or more Rp segments but do not comprise an Sp segment. In some embodiments, provided oligonucleotides comprise one or more Sp segments but no Rp segments. In some embodiments, provided oligonucleotides comprise one or more PO segments, wherein each internucleotide linkage is a natural phosphate linkage.

In some embodiments, the 5 '-segment comprised by the compounds of the present disclosure is an Sp segment, wherein each sugar moiety comprises a 2' -F modification. In some embodiments, the 5 '-segment is an Sp segment, wherein each internucleotide linkage is a modified internucleotide linkage, and each sugar moiety comprises a 2' -F modification. In some embodiments, the 5 '-segment is an Sp segment, wherein each internucleotide linkage is a phosphorothioate linkage, and each sugar moiety comprises a 2' -F modification. In some embodiments, the 5' -segment comprises 4 or more nucleoside units. In some embodiments, the 5' -segment comprises 5 or more nucleoside units. In some embodiments, the 5' -segment comprises 6 or more nucleoside units. In some embodiments, the 5' -segment comprises 7 or more nucleoside units. In some embodiments, the 3 '-segment is an Sp segment, wherein each sugar moiety comprises a 2' -F modification. In some embodiments, the 3 '-segment is an Sp segment, wherein each internucleotide linkage is a modified internucleotide linkage, and each sugar moiety comprises a 2' -F modification. In some embodiments, the 3 '-segment is an Sp segment, wherein each internucleotide linkage is a phosphorothioate linkage, and each sugar moiety comprises a 2' -F modification. In some embodiments, the 3' -segment comprises 4 or more nucleoside units. In some embodiments, the 3' -segment comprises 5 or more nucleoside units. In some embodiments, the 3' -segment comprises 6 or more nucleoside units. In some embodiments, the 3' -segment comprises 7 or more nucleoside units.

In some embodiments, the compounds of the present disclosure comprise a nucleoside type or oligonucleotide in one region, followed by a specific type of internucleotide linkage, e.g., a natural phosphate linkage, a modified internucleotide linkage, an Rp chiral internucleotide linkage, an Sp chiral internucleotide linkage, and the like. In some embodiments, a is followed by Sp. In some embodiments, a is followed by Rp. In some embodiments, a is followed by a natural phosphate bond (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by a natural phosphate bond (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a natural phosphate bond (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a natural phosphate bond (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by a natural phosphate bond (PO). In some embodiments, a and G are followed by Sp. In some embodiments, a and G are followed by Rp.

In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, wherein the antisense strand comprises at least one thermostable modification of the duplex located in the seed region of the antisense strand (i.e., positions 2 to 9 of the 5' end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) The antisense strand comprises 2, 3, 4, 5 or 6 2' -fluoro modifications; (ii) The antisense strand comprises 3, 4 or 5 phosphorothioate internucleotide linkages; (iii) conjugation of the sense strand to the ligand; (iv) The sense strand comprises 2, 3, 4 or 5 2' -fluoro modifications; (v) The sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2' -fluoro modifications; (vii) The dsRNA comprises a duplex region of 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand comprises at least one thermostable modification of the duplex located in the seed region of the antisense strand (i.e., positions 2 to 9 of the 5' end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) The antisense strand comprises 2, 3, 4, 5 or 6 2' -fluoro modifications; (ii) conjugation of the sense strand to a ligand; (iii) the sense strand comprises 2, 3, 4 or 5 2' -fluoro modifications; (iv) The sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2' -fluoro modifications; (vi) The dsRNA comprises a duplex region of 12 to 40 nucleotide pairs in length; (vii) The dsRNA comprises a duplex region of 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, wherein the antisense strand comprises at least one thermostable modification of the duplex located in the seed region of the antisense strand (i.e., positions 2 to 9 of the 5' end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) The antisense strand comprises 2, 3, 4, 5 or 6 2' -fluoro modifications; (ii) The antisense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) conjugation of the sense strand to the ligand; (iv) the sense strand comprises 2, 3, 4 or 5 2' -fluoro modifications; (v) The sense strand comprises 3, 4 or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2' -fluoro modifications; (vii) The dsRNA comprises a duplex region of 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

In some embodiments, the sense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand comprises at least one thermally labile modification of a duplex located in the seed region of the antisense strand (i.e., positions 2 to 9 5' of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) The antisense strand comprises 2, 3, 4, 5 or 6 2' -fluoro modifications; (ii) conjugation of the sense strand to a ligand; (iii) the sense strand comprises 2, 3, 4 or 5 2' -fluoro modifications; (iv) The sense strand comprises 3, 4 or 5 phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2' -fluoro modifications; (vi) The dsRNA comprises a duplex region of 12 to 40 nucleotide pairs in length; and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand.

In some embodiments, the dsRNA molecules of the present disclosure comprise one or more mismatches with the target, mismatches in one or more duplexes, or a combination thereof. Mismatches may occur in the overhang region or duplex region. Base pairs may be ordered based on their propensity to promote dissociation or melting (e.g., based on the binding or dissociation free energy of a particular pairing, the simplest approach is to examine the pairing on a single pairing basis, although adjacent or similar assays may also be used). In terms of promoting dissociation: a is better than G and C; g is better than G and C; and I: C is better than G: C (i=inosine). Mismatches, such as atypical pairs or pairs other than typical pairs (as described elsewhere herein), are preferred over typical (A: T, A: U, G: C) pairs; and pairing involving universal bases is preferred over typical pairing.

In some embodiments, the dsRNA molecules of the present disclosure comprise at least one of the first 1, 2, 3, 4, or 5 base pairs located within the duplex region from the 5' end of the antisense strand, said base pairs being independently selected from the group consisting of: a U, G: U, I:C, and mismatched pairing, such as atypical pairing or pairing other than typical pairing or pairing involving universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

In some embodiments, the nucleotide at position 1 within 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 1, 2 or 3 base pairs located within the duplex region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair located within the duplex region from the 5' end of the antisense strand is an AU base pair.

It has been found that the introduction of a 4' -modified nucleotide or a 5' -modified nucleotide at the 3' -end of a Phosphodiester (PO), phosphorothioate (PS) or phosphorodithioate (PS 2) linkage of a nucleotide at any position of a single-or double-stranded oligonucleotide can produce a steric effect on the internucleotide linkage, thereby protecting or stabilizing it from the influence of nucleases.

In some embodiments, a 5 '-modified nucleotide is introduced at the 3' end of the dinucleotide at any position of the single-stranded or double-stranded siRNA. For example, a 5 '-alkylated nucleotide may be introduced at the 3' end of a dinucleotide at any position of a single-stranded or double-stranded siRNA. The alkyl group at the 5' position of ribose may be a racemic or chirally pure R or S isomer. An exemplary 5 '-alkylated nucleotide is a 5' -methyl nucleotide. The 5' -methyl group may be the racemic or chirally pure R or S isomer.

In some embodiments, a 4 '-modified nucleotide is introduced at the 3' end of the dinucleotide at any position of the single-stranded or double-stranded siRNA. For example, a 4 '-alkylated nucleotide may be introduced at the 3' end of a dinucleotide at any position of a single-stranded or double-stranded siRNA. The alkyl group at the 4' position of ribose may be a racemic or chirally pure R or S isomer. An exemplary 4 '-alkylated nucleotide is a 4' -methyl nucleotide. The 4' -methyl group may be a racemic or chirally pure R or S isomer. Alternatively, the 4 '-O-alkylated nucleotide may be introduced at the 3' end of the dinucleotide at any position of the single-stranded or double-stranded siRNA. The 4' -O-alkyl group of ribose may be a racemic or chirally pure R or S isomer. An exemplary 4 '-O-alkylated nucleotide is a 4' -O-methyl nucleotide. The 4' -O-methyl group may be a racemic or chirally pure R or S isomer.

In some embodiments, a 5' -alkylated nucleotide is introduced at any position of the sense strand or antisense strand of the dsRNA, and such modification maintains or increases the efficacy of the dsRNA. The 5' -alkyl group may be a racemic or chirally pure R or S isomer. An exemplary 5 '-alkylated nucleotide is a 5' -methyl nucleotide. The 5' -methyl group may be the racemic or chirally pure R or S isomer.

In some embodiments, the 4' -alkylated nucleotide is introduced at any position of the sense strand or antisense strand of the dsRNA, and such modification maintains or increases the efficacy of the dsRNA. The 4' -alkyl group may be a racemic or chirally pure R or S isomer. An exemplary 4 '-alkylated nucleotide is a 4' -methyl nucleotide. The 4' -methyl group may be a racemic or chirally pure R or S isomer.

In some embodiments, a 4' -O-alkylated nucleotide is introduced at any position of the sense strand or antisense strand of the dsRNA, and such modification maintains or increases the efficacy of the dsRNA. The 5' -alkyl group may be a racemic or chirally pure R or S isomer. An exemplary 4 '-O-alkylated nucleotide is a 4' -O-methyl nucleotide. The 4' -O-methyl group may be a racemic or chirally pure R or S isomer.

In some embodiments, dsRNA molecules of the present disclosure may comprise 2' -5' linkages (with 2' -H, 2' -OH, and 2' -OMe, and with p=o or p=s). For example, 2' -5' bond modifications can be used to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to avoid activation of the sense strand by RISC.

In another embodiment, the dsRNA molecules of the present disclosure may comprise an L-sugar (e.g., L-ribose, L-arabinose with 2' -H, 2' -OH, and 2' -OMe). For example, these L sugar modifications may be used to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand, or may be used at the 5' end of the sense strand to avoid activation of the sense strand by RISC.

A number of publications describe multimeric siRNA that can be used with the dsRNA of the present disclosure. Such publications include WO2007/091269, US 7858769, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520, the entire contents of which are incorporated herein.

As described in more detail below, RNAi agents containing one or more saccharide moieties conjugated to themselves can optimize one or more properties of the RNAi agent. In many cases, the saccharide moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, such as a non-saccharide (e.g., cyclic) carrier having a saccharide ligand attached thereto. A ribonucleotide subunit in which the ribose of the subunit has been so replaced is referred to herein as a Ribose Replacement Modified Subunit (RRMS). The cyclic carrier may be a carbocyclic ring system, i.e. all ring atoms are carbon atoms, or a heterocyclic ring system, i.e. one or more ring atoms may be heteroatoms, such as nitrogen, oxygen, sulfur. The cyclic carrier may be a single ring system or may contain two or more rings, such as fused rings. The cyclic support may be a fully saturated ring system or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide by a carrier. The carrier comprises: (i) At least one "backbone attachment point", e.g., two "backbone attachment points", and (ii) at least one "tie attachment point (tethering attachment point)". As used herein, "backbone attachment point" refers to a functional group such as a hydroxyl group or a bond that is generally useful and suitable for incorporating the vector into the backbone of ribonucleic acid (e.g., a phosphate backbone or a modified phosphate backbone such as a sulfur-containing backbone). In some embodiments, "linkage attachment point" (TAP) refers to a constituent ring atom of a cyclic carrier that links selected moieties, e.g., a carbon atom or a heteroatom (other than the atom providing the backbone attachment point). The moiety may be, for example, a saccharide, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide. Optionally, the selected moiety is linked to the circular carrier by an intermediate linkage (intervening tether). Thus, a cyclic support will typically include a functional group such as an amino group or typically provide a bond suitable for incorporating or linking another chemical entity (e.g., a ligand) to the constituent ring.

The RNAi agent can be conjugated to the ligand via a carrier, wherein the carrier can be a cyclic group or an acyclic group; in some embodiments, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; in some embodiments, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

In certain embodiments, the RNAi agents used in the methods of the present disclosure are agents selected from the group of agents listed in any one of tables 2, 3, 5, and 6. These agents may also comprise ligands, such as one or more lipophilic moieties, one or more GalNAc derivatives, or both one or more lipophilic moieties and one or more GalNAc derivatives.

iRNA conjugated to ligand

Another modification of the RNAs of the iRNAs of the present invention involves chemical ligation of one or more ligands, moieties, or conjugates to the iRNA that enhance the activity of the iRNA into a cell, the cell distribution, or the cellular uptake. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al, proc.Natl.Acid.Sci.USA,1989, 86:6553-6556), cholic acids (Manoharan et al, biorg.Med.chem.Let.,1994, 4:1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al, ann.N.Y. Acad.Sci.,1992,660:306-309;Manoharan et al, biorg.Med.chem.Let.,1993, 3:2765-2770), mercapto cholesterol (Obohauser et al, nucl.acids Res.,1992, 20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmas et al, EMJ, 1991,10:1111-1118;Kabanov et al, BS, 1990: 330;Svinarchuk et al, biochi.35: 330;Svinarchuk et al), 1993, 75:49-54), phospholipids such as bis-hexadecyl-rac-glycerol or 1, 2-di-O-hexadecyl-rac-glycerol-3-phosphonic acid triethylammonium (Manoharan et al, tetrahedron Lett.,1995,36:3651-3654; shea et al, nucleic acids Res.,1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al, nucleic acids & Nucleotodes, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al, tetrahedron Lett.,1995, 36:3651-3654), palmityl moieties (Mishra et al, biochem. Acta,1995, 1264:229-237), or octadecylamine or hexylamino-carbonyl oxy sterol moieties (J.1996:277-93, 1997).

In certain embodiments, the ligand alters the distribution, targeting, or lifetime of the iRNA agent into which it is incorporated. In some embodiments, the ligand provides a higher affinity for a selected target, e.g., a molecule, cell or cell type, a compartment, e.g., a cell compartment or organ compartment, a tissue, organ or region of the body, e.g., as compared to a species without such ligand. Typical ligands do not participate in duplex pairing in duplex nucleic acids.

The ligand may include naturally occurring substances, such as proteins (e.g., human Serum Albumin (HSA), low Density Lipoprotein (LDL), or globulin); saccharides (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or a lipid. The ligand may also be a recombinant molecule or a synthetic molecule, such as a synthetic polymer, e.g. a synthetic polyamino acid. Examples of the polyamino acid include Polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymer, poly (L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N- (2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethyl acrylic acid), N-isopropyl acrylamide polymer, or polyphosphazine (polyphosphazene). Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salts of polyamines, or alpha helical peptides.

The ligand may also include a targeting group that binds to a specific cell type (e.g., kidney cells), such as a cell targeting agent or a tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein, such as an antibody. The targeting group may be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein a, mucin saccharide, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl glucosamine, multivalent mannose, multivalent trehalose, glycosylated polyamino acid, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, biotin, or RGD peptide mimetic. In certain embodiments, the ligand is a multivalent galactose, such as N-acetyl-galactosamine.

Other examples of ligands include dyes, intercalators (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC 4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranyloxyhexyl group, hexadecyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) cholanic acid, dimethoxytrityl, or phenoxazine), and peptide conjugates (e.g., antennapedia peptide, tat peptide), alkylating agents, phosphoric acid, amino groups, mercapto groups, PEG (e.g., PEG-40K), MPEG, [ MPEG ] ₂ Polyamino, alkyl, substituted alkyl, radiolabeled marker, enzyme, hapten (e.g. biotin), transport/absorption enhancer (e.g. aspirin, vitamin E, folic acid), synthetic ribonuclease (e.g. imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, eu3+ complex of tetraazamacrocycle), dinitrophenyl, HRP, or AP.

The ligand may be a protein such as a glycoprotein, or a peptide, e.g., a molecule having specific affinity for a co-ligand, or an antibody, e.g., an antibody that binds to a particular cell type, e.g., a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They may also include non-peptide substances such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent trehalose. The ligand may be, for example, lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF- κB.

The ligand may be a substance, such as a drug, that increases uptake of the iRNA agent by the cell, for example, by disrupting the cytoskeleton of the cell (e.g., by disrupting microtubules, microfilaments, or intermediate filaments of the cell). The drug may be, for example, taxol (taxon), vincristine (vincristine), vinblastine (vinblastine), cytochalasin, nocodazole (nocodazole), jasmonate (japlakinelide), halichondrin A (latrunculin A), phalloidin (phalloidin), spongosine A, indenone derivative (indacenine), or myoservin.

In some embodiments, the ligand linked to the iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen (naproxen), ibuprofen (ibuprofen), vitamin E, biotin, and the like. Oligonucleotides comprising some phosphorothioate linkages are also known to bind to serum proteins, and thus short oligonucleotides comprising multiple phosphorothioate linkages in the backbone, such as about 5 bases, 10 bases, 15 bases, or 20 bases, may also be ligands of the invention (e.g., as PK modulating ligands). Furthermore, in the embodiments described herein, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK modulating ligands.

The ligand-conjugated iRNA of the invention can be synthesized by using oligonucleotides with side chain reactive functionalities, such as derived from the attachment of a linker molecule to the oligonucleotide (described below). This reactive oligonucleotide may react directly with the following ligands: commercially available ligands, synthetic ligands having any of a variety of protecting groups, or ligands having a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the invention may be conveniently and routinely prepared by well known solid phase synthesis techniques. The equipment used for such synthesis may be sold by a number of vendors, including, for example, applied(Foster City, calif.). Any other method known in the art for such synthesis may additionally or alternatively be employed. The preparation of other oligonucleotides, such as phosphorothioates and alkylated derivatives, using similar techniques is also known.

In the ligand-conjugated oligonucleotides and sequence-specifically linked nucleosides with ligand molecules of the invention, the oligonucleotides and oligonucleotides can be assembled on a suitable DNA synthesizer using: standard nucleotides or nucleoside precursors, or nucleotide or nucleoside conjugated precursors already bearing a linking moiety, ligand-nucleotide or nucleoside conjugated precursors already bearing a ligand molecule or a non-nucleoside ligand building block.

When using nucleotide conjugation precursors that already carry a linking moiety, synthesis of sequence-specific linked nucleosides is typically completed, followed by reaction of the ligand molecule with the linking moiety to form an oligonucleotide conjugated to the ligand. In some embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to standard and non-standard phosphoramidites commercially available and conventionally used in oligonucleotide synthesis.

A. Lipid conjugates

In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such lipids or lipid-based molecules may typically bind to serum proteins such as Human Serum Albumin (HSA). Binding of HSA to the ligand allows the conjugate to be distributed to target tissues, such as non-renal target tissues of the body. For example, the target tissue may be the liver, including parenchymal cells of the liver. Other molecules that bind HSA may also be used as ligands. For example, naproxen or aspirin may be used. The lipid or lipid-based ligand may (a) increase resistance to degradation of the conjugate, (b) increase targeting or delivery to a target cell or cell membrane, or (c) be used to modulate binding to a serum protein such as HSA.

Lipid-based ligands can be used to modulate, e.g., control (e.g., inhibit) the binding of conjugates to target tissue. For example, lipids or lipid-based ligands that bind HSA more strongly will be less likely to target the kidneys and therefore less likely to be cleared from the body. Lipids or lipid-based ligands that bind poorly to HSA can be used to target the conjugate to the kidney.

In certain embodiments, the lipid-based ligand binds HSA. For example, the ligand may bind HSA with sufficient affinity to enhance the distribution of the conjugate in non-kidney tissue. However, this affinity is typically not so strong as to render the binding of the HSA-ligand irreversible.

In certain embodiments, the lipid-based ligand binds to HSA weakly or not at all, thereby enhancing the distribution of the conjugate in the kidney. Other moieties targeting kidney cells may also be administered in place of or in addition to the lipid-based ligand.

In another aspect, the ligand is a moiety, such as a vitamin, that is taken up by the target cell (e.g., a proliferating cell). These are particularly useful in the treatment of conditions characterized by unwanted cell proliferation (e.g., malignant or non-malignant types, such as cancer cells). Exemplary vitamins include vitamin a, vitamin E, and vitamin K. Other exemplary vitamins include B vitamins such as folic acid, vitamin B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are ingested by cancer cells. HSA and Low Density Lipoprotein (LDL) are also included.

B. Cell penetrating agent

In another aspect, the ligand is a cell penetrating agent, such as a helical cell penetrating agent. In certain embodiments, the agent is amphiphilic. Exemplary agents are peptides, such as tat or antennapedia (antennapedia). If the agent is a peptide, it may be modified, including peptidomimetics, retro-isomers, non-peptide or pseudo-peptide bonds, and the use of D-amino acids. The helicant is typically an alpha-helicant and may have a lipophilic phase and a lipophobic phase.

The ligand may be a peptide or a peptidomimetic. Peptide mimetics (also referred to herein as oligopeptide mimetics) are molecules that fold into a defined three-dimensional structure similar to a natural peptide. Attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic profile of iRNA, for example, by enhancing cell recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

The peptide or peptidomimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., consisting essentially of Tyr, trp, or Phe). The peptide moiety may be a dendrimer peptide, a restriction peptide or a cross-linked peptide. In another alternative, the peptide moiety may include a hydrophobic Membrane Translocation Sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence: AAVALLPAVLLALLAP (SEQ ID NO: 9). RFGF analogs containing a hydrophobic MTS, such as amino acid sequence AALLPVLLAAP (SEQ ID NO: 10), may also be targeting moieties. The peptide moiety may be a "delivery" peptide that can carry polar macromolecules including peptides, oligonucleotides, and proteins across a cell membrane. For example, it has been found that the sequence from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and the sequence from drosophila antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)) can function as delivery peptides. Peptides or peptidomimetics can be encoded by random DNA sequences, such as peptides identified from phage display libraries or a combinatorial library of one-bead-one compounds (OBOC) (Lam et al, nature,354:82-84,1991). Typically, the peptide or peptidomimetic linked to the dsRNA agent by the incorporated monomer units is a cell-targeted peptide, such as an arginine-glycine-aspartic acid (RGD) -peptide or RGD mimetic. The peptide portion may range in length from about 5 amino acids to about 40 amino acids. The peptide moiety may have structural modifications, for example to increase stability or to direct conformational properties. Any of the structural modifications described below may be used.

RGD peptides for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting of one or more specific tissues. RGD-containing peptides and peptide mimetics may include D-amino acids, as well as synthetic RGD mimetics. In addition to RGD, other moieties that target integrin ligands, such as PECAM-1 or VEGF, may also be used.

The RGD peptide moiety can be used to target specific cell types, for example tumor cells, such as endothelial tumor cells or breast Cancer tumor cells (Zitzmann et al, cancer Res.,62:5139-43,2002). RGD peptides can promote targeting of dsRNA agents to tumors of a variety of other tissues, including lung, kidney, spleen, or liver (Aoki et al Cancer Gene Therapy 8:783-787,2001). Typically, RGD peptides help the iRNA agent target the kidneys. RGD peptides may be linear or cyclic and may be modified, e.g., glycosylated or methylated, to facilitate their targeting to specific tissues. For example, glycosylated RGD peptides may deliver iRNA agents to express αvβ ₃ Is described (Haubner et al, journal. Nucl. Med.,42:326-336,2001).

"cell penetrating peptide" is capable of penetrating a cell, such as a microbial cell, e.g., a bacterial or fungal cell, or a mammalian cell, e.g., a human cell. The microbial cell penetrating peptide may be, for example, an alpha-helical linear peptide (e.g., LL-37 or cerpin P1), a disulfide-containing peptide (e.g., alpha-defensin, beta-defensin, or bovine antibacterial peptide (bacterin)), or a peptide containing only one or two major amino acids (e.g., PR-39 or indomethacin). Cell penetrating peptides may also include Nuclear Localization Signals (NLS). For example, the cell penetrating peptide may be a bi-directional amphiphilic peptide such as MPG, which is derived from the fusion peptide domain of HIV-1gp41 and NLS of the SV40 large T antigen (Simeoni et al, nucleic acids Res.31:2717-2724, 2003).

C. Saccharide conjugates

In some embodiments of the compositions and methods of the invention, the iRNA further comprises a saccharide. As described herein, saccharide-conjugated iRNA facilitates in vivo delivery of nucleic acids, and the compositions are suitable for therapeutic use in vivo. As used herein, "saccharide" refers to a compound that itself consists of one or more monosaccharide units (which may be linear, branched, or cyclic) having at least 6 carbon atoms, and an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound having as part thereof a saccharide moiety consisting of one or more monosaccharide units having at least six carbon atoms, which may be linear, branched or cyclic, and an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative sugars include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), as well as polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; disaccharides and trisaccharides, which include saccharides having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In certain embodiments, the saccharide conjugates comprise monosaccharides.

In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates comprising one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in US 8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, galNAc conjugates are used as ligands to target iRNA to a specific cell. In some embodiments, galNAc conjugates target iRNA to liver cells, for example, by acting as a ligand for an asialoglycoprotein receptor of a liver cell (e.g., a liver cell).

In some embodiments, the saccharide conjugates comprise one or more GalNAc derivatives. GalNAc derivatives may be attached by a linker, for example a divalent or trivalent branching linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3' end of the sense strand) via a linker, such as the linkers described herein. In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5' end of the sense strand) via a linker, such as the linkers described herein.

In certain embodiments of the invention, galNAc or GalNAc derivatives are attached to the iRNA agents of the invention by a monovalent linker. In some embodiments, galNAc or GalNAc derivatives are attached to the iRNA agents of the invention by a divalent linker. In still other embodiments of the invention, galNAc or GalNAc derivative is attached to the iRNA agent of the invention by a trivalent linker. In other embodiments of the invention, galNAc or GalNAc derivatives are attached to the iRNA agents of the invention by a tetravalent linker.

In certain embodiments, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to an iRNA agent. In certain embodiments, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) of galnacs or GalNAc derivatives, each GalNAc or GalNAc derivative being independently attached to multiple nucleotides of the double stranded RNAi agent by multiple monovalent linkers.

In some embodiments, for example, when two strands of an iRNA agent of the invention are part of one larger molecule and form a hairpin loop comprising a plurality of unpaired nucleotides by uninterrupted nucleotide strand ligation between the 3 'end of one strand and the 5' end of the other strand, each unpaired nucleotide within the hairpin loop can independently comprise GalNAc or a GalNAc derivative attached by a monovalent linker. The hairpin loop may also be formed by an extended overhang of one strand of the duplex.

In some embodiments, the GalNAc conjugate is

In some embodiments, as shown in the following schemes, the RNAi agent is attached to the saccharide conjugate via a linker, wherein X is O or S

In some embodiments, the RNAi agent is conjugated to L96 as defined in table 1 as follows:

in certain embodiments, saccharide conjugates used in the compositions and methods of the invention are selected from the group consisting of:

wherein Y is O or S and n is 3 to 6 (formula XXIV);

wherein Y is O or S and n is 3 to 6 (formula XXV);

wherein X is O or S (formula XXVII); />

In certain embodiments, saccharide conjugates used in the compositions and methods of the present invention are monosaccharides. In certain embodiments, the monosaccharide is N-acetylgalactosamine, e.g

Another representative saccharide conjugate for use in the embodiments described herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is hydrogen.

In some embodiments, suitable ligands are those disclosed in WO 2019/055633, the entire contents of which are incorporated herein by reference. In one embodiment, the ligand comprises the following structure:

in certain embodiments, RNAi agents of the present disclosure can include GalNAc ligands, even though such GalNAc ligands are currently expected to be of limited value for the intrathecal/CNS delivery routes of the present disclosure.

In certain embodiments of the invention, galNAc or GalNAc derivatives are linked to the iRNA agents of the invention by a monovalent linker. In some embodiments, galNAc or GalNAc derivatives are linked to the iRNA agents of the invention through a divalent linker. In other embodiments of the invention, galNAc or GalNAc derivative is linked to the iRNA agent of the invention via a trivalent linker. In other embodiments of the invention, galNAc or GalNAc derivatives are linked to the iRNA agents of the invention via tetravalent linkers.

In certain embodiments, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative linked to an iRNA agent, e.g., linked to the 5 'end of the sense strand of a dsRNA agent or the 5' end of one or both sense strands of a dual targeted RNAi agent, as described herein. In certain embodiments, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) galnacs or GalNAc derivatives, each derivative being independently linked to multiple nucleotides of the double stranded RNAi agent by a plurality of monovalent linkers.

In some embodiments, for example, when two strands of an iRNA agent of the invention are part of one larger molecule and are joined by an uninterrupted nucleotide chain between the 3 'end of one strand and the 5' end of the corresponding other strand to form a hairpin loop comprising a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop can independently comprise GalNAc or a GalNAc derivative joined by a monovalent linker.

In some embodiments, the saccharide conjugates also comprise one or more additional ligands as described above, such as, but not limited to, PK modulators or cell penetrating peptides.

Other saccharide conjugates and linkers suitable for use in the present invention include those described in WO 2014/179620 and WO 2014/179627, the respective disclosures of which are incorporated herein by reference in their entirety.

D. Joint

In some embodiments, the conjugates or ligands described herein can be attached to an iRNA oligonucleotide through a variety of linkers, which may be cleavable or non-cleavable.

The term "linker" or "linking group" means an organic moiety that links two parts of a compound, e.g., covalently links two parts of a compound. The linker usually comprises a direct bond or atom, e.g. oxygen or sulfur, units such as NR8, C (O) NH, SO ₂ 、SO ₂ NH or an atomic chain such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylaryl alkyl, alkylaryl alkenyl, alkylaryl alkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkynylalkynyl, alkynylalkyls, alkynylalkynyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylalkyl, alkynylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, heteroarylone or more of which may be interrupted by one or more of the following groups: o, S, S (O), SO ₂ N (R8), C (O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclyl; wherein R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In certain embodiments, the linker is about 1 to 24 atoms, 2 to 24 atoms,3 to 24 atoms, 4 to 24 atoms, 5 to 24 atoms, 6 to 18 atoms, 7 to 18 atoms, 8 to 18 atoms, 7 to 17 atoms, 8 to 17 atoms, 6 to 16 atoms, 7 to 16 atoms, or 8 to 16 atoms.

The cleavable linking group is sufficiently stable extracellular but is cleaved upon entry into the target cell to release the two moieties held together by the linker. In one embodiment, the cleavable linking group is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or more, or at least about 100-fold faster in a target cell or under a first reference condition (which may be, for example, a condition selected to mimic or present in a cell) than in a subject's blood or under a second reference condition (which may be, for example, a condition selected to mimic or present in blood or serum).

Cleavable linking groups are susceptible to cleavage agents such as pH, redox potential or the presence of a degradable molecule. Generally, the lysing agent is more prevalent or present at a higher level or activity within the cell than in serum or blood. Examples of such degradation agents include: redox agents selected for a particular substrate or which have no substrate specificity, including, for example, oxidases, reductases or reducing agents present in the cell, such as thiols, which can degrade redox cleavable linkers by reduction; an esterase; endosomes or agents that can create an acidic environment, such as those that result in a pH of 5 or less; enzymes that hydrolyze or degrade acid-cleavable linkers can be prepared by a method that is general-purpose for acids, peptidases (which may be substrate-specific), and phosphatases.

Cleavable linking groups such as disulfide bonds may be pH sensitive. The pH of human serum is 7.4, while the average pH in the cells is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH in the range of about 5.5 to 6.0; while lysosomes have even more acidic pH, about 5.0. Some linkers will have cleavable linking groups that cleave at a selected pH, thereby releasing the cationic lipid from the intracellular ligand or into a desired cellular compartment.

The linker may comprise a cleavable linking group cleavable by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the cell to be targeted. For example, the liver targeting ligand may be linked to the cationic lipid through a linker comprising an ester group. Liver cells are rich in esterases and therefore the linker is more efficiently cleaved in liver cells than in cell types that are not rich in esterases. Other esterase-enriched cell types include lung cells, kidney cortical cells and testis cells.

When targeting peptidase-rich cell types such as liver cells and synovial cells, linkers containing peptide bonds may be used.

In general, the suitability of a candidate cleavable linking group can be assessed by testing the ability of the degrading agent (or condition) to cleave the candidate linking group. It is also desirable to test candidate cleavable linking groups for their ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative sensitivity of lysis between a first condition selected to indicate lysis in target cells and a second condition selected to indicate lysis in other tissues or biological fluids such as blood or serum can be determined. The evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to perform an initial assessment in cell-free or culture conditions and to confirm by further assessment in whole animals. In certain embodiments, useful candidate compounds are at least about 2-fold, 4-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions). i. Redox cleavable linking groups

In certain embodiments, the cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of a linker that can be cleaved by reduction is a disulfide linker (-S-S-). Reference may be made to the methods described herein to determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or, for example, suitable for use with a particular iRNA moiety and a particular targeting agent. For example, the candidate may be evaluated by incubating Dithiothreitol (DTT) or other reducing agent with the candidate using reagents known in the art that mimic the rate of lysis that would be observed in a cell, such as a target cell. Candidates may also be evaluated under conditions selected to mimic blood or serum conditions. In one embodiment, the candidate compound is cleaved in the blood up to about 10%. In other embodiments, the degradation rate of a useful candidate compound is at least about 2-fold, 4-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold faster in a cell (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound may be determined using standard enzymatic kinetic analysis under conditions selected to mimic the intracellular medium and compared to the cleavage rate of the candidate compound determined under conditions selected to mimic the extracellular medium.

Phosphate-based cleavable linking groups

In certain embodiments, the cleavable linker comprises a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of agents that cleave phosphate groups within cells are enzymes such as intracellular phosphatases. Examples of phosphate-based linking groups are-O-P (O) (ORk) -O-, -O-P (S) (ORk) -O-, -O-P (S) (SRk) -O-, -S-P (O) (ORk) -O-, -O-P (O) (ORk) -S-, -S-P (O) (ORk) -S-, O-O (ORk) -O-, O-O (ORk) O-, O-O (O) S-O-S- -O-P (S) (ORk) -S-, -S-P (S) (ORk) -O-, -O-P (O) (Rk) -O-, -O-P (S) (Rk) -O-, -S-P (O) (Rk) -O-, -S-P (S) (Rk) -O-, -S-P (O) (Rk) -S-, -O-P (S) (Rk) -S, wherein Rk may be independently at each occurrence C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7-C12 aralkyl. -S-P (O) (OH) -O- -O-P (O) (OH) -S-, -S-P (O) (OH) -O-, -O-P (O) (OH) -S-, and-S-P (O) (OH) -S-, -O-P (S) (OH) -S-, -S-P (S) (OH) -O-, -O-P (O) (H) -O-, -O-P (S) (H) -O-, -S-P (O) (H) -O, -S-P (S) (H) -O-, -S-P (O) (H) -S-and-O-P (S) (H) -S-. In one embodiment, the phosphate-based linking group is-O-P (O) (OH) -O-. These candidates can be evaluated using methods similar to those described above.

Acid cleavable linking groups

In certain embodiments, the cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In some embodiments, the acid-cleavable linking group is cleaved in an acidic environment at a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or less), or by an agent such as an enzyme useful as a generalized acid. In cells, specific low pH organelles such as endosomes and lysosomes can provide a cleavage environment for acid cleavable linkers. Examples of acid cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. The acid cleavable linking group may have the general formula-c=nn-, C (O) O, or-OC (O). An exemplary embodiment is that the carbon attached to the oxygen of the ester (alkoxy) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or tertiary butyl. These candidates can be evaluated using methods similar to those described above.

Ester-based cleavable linking groups

In certain embodiments, the cleavable linker comprises an ester-based cleavable linking group. The cleavable ester-based linking group is cleaved by an enzyme in the cell, such as an esterase or amidase. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene groups, alkenylene groups, and alkynylene groups. The ester cleavable linking group has the general formula-C (O) O-or-OC (O) -. These candidates can be evaluated using methods similar to those described above.

Peptide-based cleavable linking groups

In another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in the cell. Peptide-based cleavable groups are peptide bonds formed between amino acids to obtain oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not include an amide group (-C (O) NH-). The amide groups may be formed between any alkylene, alkenylene or alkynylene groups. Peptide bonds are a special type of amide bond formed between amino acids to obtain peptides and proteins. Peptide-based cleavage groups are typically limited to creating peptide bonds (i.e., amide bonds) between amino acids of peptides and proteins, and do not include intact amide functionalities. The peptide-based cleavable linking group has the general formula-NHCHRAC (O) NHCHRBC (O) -, wherein RA and RB are R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

In some embodiments, the iRNA of the invention is conjugated to a saccharide through a linker. Non-limiting examples of iRNA saccharide conjugates having a linker in the compositions and methods of the invention include, but are not limited to,

When one of X or Y is an oligonucleotide, the other is hydrogen.

In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a divalent or trivalent branching linker.

In certain embodiments, the dsRNA of the invention is conjugated to a bivalent or trivalent branch linker selected from the group of structures shown in any one of formulas (XLV) to (XLVIII):

wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C independently represent from 0 to 20 at each occurrence, and the repeat units therein 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 each occurrence independently represents: absence, CO, NH, O, S, OC (O), NHC (O), CH ₂ 、CH ₂ NH or CH ₂ O；

Q ^2A 、Q ^2B 、Q ^3A 、Q ^3B 、Q ^4A 、Q ^4B 、Q ^5A 、Q ^5B 、Q ^5C Each occurrence independently represents: non-existent, alkylene, substituted alkylene, wherein one or more methylene groups may be interrupted or terminated by one or more of the following groups: o, S, S (O), SO ₂ 、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 each occurrence independently represents: absence of NH, O, S, CH ₂ 、C(O)O、C(O)NH、NHCH(R ^a )C(O)、-C(O)-CH(R ^a )-NH-、CO、CH＝N-O、 Or a heterocyclic group;

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 is ^a Is H or an amino acid side chain. The use of trivalent conjugated GalNAc derivatives with RNAi agents is particularly useful for inhibiting expression of a target gene, such as those of formula (XLIX):

XLIX

Wherein L is ^5A 、L ^5B And L ^5C Represents a monosaccharide such as GalNAc derivatives.

Examples of suitable divalent and trivalent branching linker groups for conjugation to GalNAc derivatives include, but are not limited to, the structures listed above as formula II, formula VII, formula XI, formula X, and formula XIII.

Representative U.S. patents teaching the preparation of RNA conjugates include but are not limited to, U.S. Pat. nos. 5,218,105, no. 5, no. 2, no. 5, no. 138,045, no. 5, no. 2, no. 5, no. 4, no. 5, no. 2, no. 830, no. 5, no. 082,830, no. 5, no. 2, no. 830, no. 5, no. 2 No. 5, no. 481, no. 5, no. 587, no. 371, no. 5, no. 597, no. 696, no. 6, no. 320, no. 017, no. 5, no. 565, no. 552, no. 5, no. 567, no. 810, no. 5,574, no. 142, no. 5,585, no. 481, no. 5,587, no. 371, the entire contents of each patent are incorporated herein by reference.

All positions of a given compound need not be uniformly modified, and in fact, more than one of the aforementioned modifications can be incorporated into a single compound or even at a single nucleoside of an iRNA. The invention also includes iRNA compounds as chimeric compounds.

In the context of the present invention, a "chimeric" iRNA compound or "chimera" is an iRNA compound, e.g. a dsRNA agent, comprising two or more chemically distinct regions, each region being composed of at least one monomer unit, i.e. in the case of a dsRNA compound, the monomer unit is a nucleotide. These irnas typically contain at least one region, wherein the RNA is modified to confer to the iRNA: increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity to the target nucleic acid. Another region of iRNA can be used to cleave RNA: DNA hybrids or RNA: substrates for enzymes of RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves RNA: RNA strand of DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly improving the efficiency of iRNA inhibition of gene expression. Thus, when chimeric dsrnas are used, the use of shorter irnas can achieve comparable results to the use of phosphorothioate deoxydsrnas that hybridize to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis, and if desired, gel electrophoresis can be used in conjunction with related nucleic acid hybridization techniques known in the art.

In some cases, the RNA of the iRNA can be modified by a non-ligand group. A large number of non-ligand molecules have been conjugated to iRNA to increase the activity, cellular distribution or cellular uptake of iRNA, and the process of performing such conjugation is available in the scientific literature. Such non-ligand moieties have included lipid moieties such as cholesterol (Kubo, t.et al., biochem. Biophys. Res. Comm.,2007,365 (1): 54-61;Letsinger et al, proc.Natl. Acad.Sci.USA,1989, 86:6553), cholic acid (Manoharan et al, biorg.Med.chem.Lett., 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al, ann.N.Y. Acad.Sci.,1992,660:306;Manoharan et al, biorg.Med.chem.Let., 1993, 3:2765), mercapto cholesterol (Obohauser et al, nucl.acids Res.,1992, 20:533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al, EMBO J.,1991,10:111;Kabanov et al, FEBS Lett.,1990,259:327;Svinarchuk etal, biomie, 1993, 75:49), phospholipids such as bis-hexadecyl-rac-glycerol or 1, 2-di-O-hexadecyl-rac-glycerol-3-phosphonic acid triethylammonium (Manoharan et al, tetrahedron lett.,1995,36:3651;Shea et al, nucleic acids res.,1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al, nucleic acids & Nucleotides,1995, 14:969), or adamantane acetic acid (Manoharan et al, tetrahedron lett.,1995, 36:3651), palmityl moieties (Mishra et al, biochem. Acta,1995, 1264:229), or octadecylamine or hexylamino-carbonyloxy cholesterol moieties (croooet al, j. Exp. Thoer, 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of RNA with an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule to be conjugated using a suitable coupling agent or activator. The conjugation reaction can be carried out using RNA that is still bound to the solid support, or in the solution phase after cleavage of the RNA. Purification of the RNA conjugate by HPLC will typically yield a pure conjugate.

V. delivery of RNAi agents of the present disclosure

Delivering RNAi agents of the present disclosure to cells, e.g., cells in a subject, e.g., cells in a human subject (e.g., a subject in need thereof, e.g., a subject having a GPR 75-related disorder (e.g., a body weight disorder, e.g., obesity), e.g., a subject at risk of developing a body weight disorder (e.g., obesity), or a subject at risk of developing a body weight disorder (e.g., obesity), can be accomplished in a number of different ways. For example, delivery may be performed by contacting the cells with RNAi agents of the present disclosure in vitro or in vivo. In vivo delivery may also be directly performed by administering a composition comprising an RNAi agent (e.g., dsRNA) to a subject. Alternatively, in vivo delivery may be indirectly effected by administration of one or more vectors encoding and directing expression of the RNAi agent. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) may be suitable for use with the RNAi agents of the present disclosure (see, e.g., akhtar S.and Julian RL. (1992) Trends cell. Biol.2 (5): 139-144 and WO94/02595, the entire contents of which are incorporated herein by reference). For in vivo delivery, factors that need to be considered in order to deliver the RNAi agent include, for example, biostability of the delivered agent, prevention of non-specific effects, and accumulation of the delivered agent in the target tissue. Nonspecific effects of RNAi agents can be minimized by topical administration (e.g., by direct injection or implantation into tissue or topical administration of the formulation). Local administration to the treatment site maximizes the local concentration of the agent, limits contact of the agent with systemic tissue that would otherwise be damaged or degraded by the agent, and allows for administration of lower total doses of RNAi agent. Several studies have shown that gene products were successfully knocked down when RNAi agents were administered locally. For example, pulmonary systemic delivery (e.g., inhalation) of dsRNA (e.g., SOD 1) has been shown to be effective in knocking down gene and protein expression in lung tissue, and the bronchioles and alveoli of the lung have excellent uptake of dsRNA. 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 also been shown to prevent neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice reduced tumor volume (Pille, J.et al. (2005) mol. Ther.11:267-274) and could extend survival time 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 delivered locally to the CNS by direct injection (Dorn, G. (2004) Nucleic Acids 32:e49; tan, pH., et al (2005) Gene Ther.12:59-66; makimura, H., et al (2002) BMC neurosci.3:18; shishkina, GT., et al (2004) neurosci 129:521-528; thakker, ER., et al (2004) Proc.Natl. Acad. Sci.A.101:17270-17275; akaneya, Y.; et al (2005) J.Neurohyeol.93:594-602) and RNA interference delivered to the lungs by intranasal administration (Howard, KA.et al., (2006) mol. Th. 14:476-484;Zhang,X.et al. (2004) J.Natl.Acad. Sci.U.101:17270-17275; akanya, Y. (2005) J.Neurohyol.93:594-602) and (2006: J.Biol.5777. 10684;Bitko,V.et al). For systemic administration of RNAi agents to treat diseases, RNA can be modified or delivered using a drug delivery system; both methods prevent rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of RNA or drug carriers may also allow for targeting of RNAi agents to target tissues and avoid unwanted off-target effects (e.g., without wishing to be bound by theory, use of GNAs as described herein has been confirmed to destabilize seed regions of dsRNA, resulting in increased preference for the effectiveness of such dsRNA against hit targets over off-target effects, as such seed region destabilization would significantly attenuate such off-target effects). RNAi agents can be modified by chemical binding to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, RNAi agents against ApoB conjugated to a lipophilic cholesterol moiety are injected systemically into mice resulting in knockdown of apoB mRNA in the liver and jejunum (Sonschek, J.et al, (2004) Nature 432:173-178). In a mouse model of prostate cancer, binding of RNAi agents to aptamers has been shown to inhibit tumor growth and mediate tumor regression (McNamara, JO.et al., (2006) Nat. Biotechnol.24:1005-1015). In another embodiment, the RNAi agent can be delivered using a drug delivery system, such as a nanoparticle, dendrimer, polymer, liposome, or cationic delivery system. Positively charged cation delivery systems facilitate binding of molecular RNAi agents (negatively charged) and also enhance interactions on negatively charged cell membranes to allow efficient uptake of RNAi agents by cells. The cationic lipid, dendrimer or polymer may be conjugated to or induced to form vesicles or micelles encapsulating the RNAi agent (see, e.g., kimsh.et al, (2008) Journal of Controlled Release 129 (2): 107-116). When administered systemically, the formation of vesicles or micelles further prevents degradation of the RNAi agent. Methods of preparing and administering cation-RNAi agent complexes are well within the ability of those skilled in the art (see, e.g., sorensen, DR., et al (2003) J.mol. Biol 327:761-766;Verma,UN.et al, (2003) Clin. Cancer Res.9:1291-1300;Arnold,AS et al (2007) J.hypertens.25:197-205, the entire contents of which are incorporated herein by reference). Some non-limiting examples of drug delivery systems that can be used for systemic delivery of RNAi agents include DOTAP (Sorensen, DR., et al (2003), supra; verma, un. Et al, (2003), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, ts.et al., (2006) Nature 441:111-114), cardiolipin (Chien, py.et al., (2005) Cancer Gene Ther.12:321-328; pal, a.et al., (2005) Int j.Oncol.26:1087-1091), polyethylenimine (Bonnet et al., (2008) pharm.Res.aug.16 electronics priority release; aigner, a. (2006) j.biomed.biotechn.659), poly-Gly-Asp (RGD) peptide (Liu, S. (2006) Phami.487:35-35; and poly-35:35; shock, and (35:35.35). In some embodiments, the RNAi agent forms a complex with cyclodextrin for systemic administration. Methods of administering RNAi agents and cyclodextrins and pharmaceutical compositions of RNAi agents and cyclodextrins can be found in U.S. patent No. 7,427,605, the entire contents of which are incorporated herein by reference.

Certain aspects of the present disclosure relate to methods of reducing expression of a GPR75 gene in a cell comprising contacting the cell with a double stranded RNAi agent of the present disclosure. In one embodiment, the cell is a liver cell (hepatocyte), optionally a hepatocyte (hepatocyte). In one embodiment, the cell is a neuronal cell.

In certain embodiments, the RNAi agent is taken up by one or more tissues or cell types present in an organ, such as the liver, kidney.

Another aspect of the disclosure relates to a method of reducing expression and/or activity of a GPR75 gene in a subject, the method comprising administering to the subject a double stranded RNAi agent of the disclosure.

Another aspect of the present disclosure relates to a method of treating a subject having or at risk of developing a GPR 75-related disorder, the method comprising administering to the subject a therapeutically effective amount of a double stranded RNAi agent of the disclosure, thereby treating the subject. In some embodiments, the GPR 75-related disorder comprises a weight disorder, such as obesity.

In one embodiment, the double stranded RNAi agent is administered subcutaneously.

In one embodiment, the double stranded RNAi agent is administered intrathecally. By intrathecal administration of the double stranded RNAi agent, the method can reduce expression of the GPR75 target gene in brain (e.g., striatum) or spinal tissue (e.g., cortex, cerebellum, cervical, lumbar, and thoracic).

In one embodiment, the double stranded RNAi agent is administered intravenously.

For ease of illustration, the formulations, compositions and methods in this section are discussed primarily with respect to modified siRNA compounds. However, it is understood that these formulations, compositions and methods may be practiced with other siRNA compounds, such as unmodified siRNA compounds, and such implementations are within the scope of the present disclosure. Compositions comprising RNAi agents can be delivered to a subject by a variety of routes. Exemplary routes include intrathecal administration, pulmonary system, intravenous, subcutaneous, intraventricular, buccal, topical, rectal, anal, vaginal, nasal, and ocular.

The RNAi agents of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more RNAi agents and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional medium or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds may also be incorporated into the compositions.

The pharmaceutical compositions of the present invention may be administered in a variety of ways depending on whether local or systemic treatment is desired and the area to be treated. Administration may be intratracheal, intranasal, topical (including ocular, vaginal, rectal, intranasal, transdermal), buccal, parenteral or pulmonary, for example by inhalation or insufflation of powders or aerosols, including by nebulizer. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

The route of administration and the site of administration may be selected to enhance targeting. For example, for targeting muscle cells, intramuscular injection of the muscle of interest would be a logical option. The lung cells may be targeted by administration of RNAi agents in powder or aerosol form. Vascular endothelial cells can be targeted by coating the balloon catheter with RNAi agents and mechanically introducing RNA.

Compositions for pulmonary system delivery may include aqueous solutions (e.g., aqueous solutions for intranasal or oral inhalation administration), suitable carriers comprising, for example, lipids (liposomes, vesicles, microemulsions, lipid micelles, solid lipid nanoparticles) or polymers (polymer micelles, dendrimers, polymer nanoparticles, nanogels, nanocapsules), adjuvants (e.g., adjuvants for oral inhalation administration). The aqueous composition may be sterile and may optionally contain buffers, diluents, absorption enhancers, and other suitable additives. Such administration allows for systemic and local delivery of the double stranded RNAi agents of the invention.

Intranasal administration may include instilling or insufflating the double stranded RNAi agent into the nasal cavity by administration of a few drops at a time with a syringe or dropper, or by nebulization. Suitable dosage forms for intranasal administration include drops, powders, nebulizers and sprays. Intranasal delivery devices include, but are not limited to, vapor inhalers, nasal drops, spray bottles, metered dose spray pumps, gas-driven spray atomizers (gas driven spray atomizer), atomizers (nebulizers), mechanical powder atomizers (mechanical powder sprayer), breath-driven inhalers, and insufflators. Devices for delivery deeper into the respiratory system, such as in the lungs, include nebulizers, pressurized metered dose inhalers, dry powder inhalers, and thermal vaporization aerosol devices. Devices for delivery by inhalation are available from commercial suppliers. The device may be fixed or variable dose, single or multi-dose, disposable or reusable, depending on, for example, the disease or condition to be prevented or treated, the volume of agent to be delivered, the frequency of delivery of the agent, and other considerations in the art.

Oral inhalation administration may include the use of a device, such as a passive breath-actuated or active power-actuated single/multi-dose Dry Powder Inhaler (DPI), to deliver double stranded RNAi agents to the pulmonary system. Suitable dosage forms for oral inhalation administration include powders and solutions. Suitable devices for oral inhalation administration include nebulizers, metered dose inhalers, and dry powder inhalers. Dry powder inhalers are the most commonly used devices for delivering drugs, especially proteins, to the lungs. Exemplary commercially available dry powder inhalers include Spinhaler (Fisons pharma ceuticals, rochester, NY) and Rotahaler (GSK, RTP, NC). Several types of atomizers are available, namely jet atomizers, ultrasonic atomizers, vibrating mesh atomizers. The jet atomizer is driven by compressed air. Ultrasonic atomizers use piezoelectric transducers to produce droplets from an open liquid reservoir. Vibrating mesh atomizers use perforated membranes driven by annular piezoelectric elements to vibrate in a resonant bending mode. The pores in the membrane have a large cross-sectional dimension on the liquid supply side and a narrow cross-sectional dimension on the side where the droplets appear. The size and number of holes may be adjusted depending on the therapeutic application. The choice of suitable means depends on parameters such as the nature of the drug and its formulation, the site of action and the pathophysiology of the lung. The aqueous suspensions and solutions are effectively atomized. Aerosols based on mechanically generated vibrating mesh technology have also been successfully used to deliver proteins to the lungs.

The amount of RNAi agent used for pulmonary system administration can vary from target gene to target gene, and the appropriate dose to be administered needs to be determined separately for each target gene. Typically, the amount ranges from 10 μg to 2mg, 50 μg to 1500 μg, or 100 μg to 1000 μg.

Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules, suspensions or aqueous solutions, syrups, elixirs or non-aqueous media, tablets, capsules, troches or lozenges. In the case of tablets, carriers that can be used include lactose, sodium citrate, and phosphate. Various disintegrants such as starch and lubricants such as magnesium stearate, sodium lauryl sulfate and talc are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are desired for oral use, the nucleic acid composition may be combined with emulsifying and suspending agents. If desired, certain sweeteners or flavoring agents may be added. Compositions suitable for oral administration of the agents of the present invention are further described in PCT application No. PCT/US20/33156, the entire contents of which are incorporated herein by reference.

Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection can be accomplished, for example, by a ventricular catheter connected to a reservoir. For intravenous use, the total concentration of solutes can be controlled to render the formulation isotonic.

In one embodiment, the administration of the siRNA compound (e.g., a double stranded siRNA compound) is parenteral administration, such as intravenous (e.g., as bolus infusion or as diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary system, intranasal, urethral, or ocular administration. Administration may be provided by the subject or another person, such as a health care provider. The medicament may be provided in metered doses or in a dispenser which provides metered doses. The selected delivery mode will be discussed in more detail below.

Intrathecal administration.

In one embodiment, the double stranded RNAi agent is delivered by intrathecal injection (i.e., injection into spinal fluid in which brain and spinal tissue is bathed). Intrathecal injection of the RNAi agent into spinal fluid can be performed by bolus infusion or by implantable subcutaneous micropump to deliver the siRNA periodically and continuously into spinal fluid. Circulation of spinal fluid begins with the choroid plexus producing spinal fluid down the spinal cord and dorsal root ganglion, then up through the cerebellum, across the cortex, to the arachnoid particles, where the spinal fluid may leave the CNS, i.e., intrathecally delivered molecules may hit targets throughout the CNS, depending on the size, stability, and solubility of the injected compound.

In some embodiments, intrathecal administration is by pump. The pump may be a surgically implanted osmotic pump. In one embodiment, the osmotic pump is implanted in the subarachnoid space of a spinal canal to facilitate intrathecal administration.

In some embodiments, intrathecal administration is performed by an intrathecal delivery system for a drug comprising a reservoir containing a volume of a medicament and a pump configured to deliver a portion of the medicament contained in the reservoir. Further details regarding this intrathecal delivery system can be found in WO 2015/116658, the entire contents of which are incorporated herein by reference.

The amount of RNAi agent injected intrathecally may vary from target gene to target gene and it is desirable to determine the appropriate dose to administer separately for each target gene. . Typically, the amount ranges from 10 μg to 2mg, 50 μg to 1500 μg or 100 μg to 1000 μg.

Vectors encoding RNAi agents of the present disclosure

RNAi agents targeting the GPR75 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, WO 00/22113, WO 00/22114, and US 6,054,299). Depending on the specific construct used and the target tissue or cell type, expression may be sustained (months or longer). These transgenes may be introduced as linear constructs, circular plasmids, or viral vectors, which may be integrating or non-integrating vectors. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (Gassmann, et al, (1995) Proc. Natl. Acad. Sci. USA 92:1292).

The single strand or multiple strands of the RNAi agent can be transcribed from the promoter on the expression vector. When two separate strands are to be expressed to produce, for example, dsRNA, the two separate expression vectors can be co-introduced (e.g., by transfection or infection) into the target cell. Alternatively, each individual strand of dsRNA may 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 such that the dsRNA has a stem and loop structure.

RNAi agent expression vectors are typically DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, such as vertebrate cells, can be used to generate recombinant constructs for expression of RNAi agents as described herein. Delivery of the RNAi agent expression vector can be systemic, such as by intravenous or intramuscular administration, by administration to target cells removed from the patient, which are then introduced into the patient, or by any other means that allows for the introduction of the desired target cells.

Viral vector systems useful in 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) an adeno-associated viral vector; (d) a herpes simplex virus vector; (e) SV 40 vector; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) a picornaviral vector; (i) Poxvirus vectors, for example smallpox, for example vaccinia virus vectors or avian poxviruses such as canary pox or chicken poxvirus vectors; and (j) helper-dependent adenovirus or naked adenovirus. Replication-defective viruses may also be advantageous. The different vectors will or will not be incorporated into the genome of the cell. If necessary, the construct may include viral sequences for transfection. Alternatively, the construct may be incorporated into vectors capable of episomal replication (episomal replication), such as EPV vectors and EBV vectors. Constructs for recombinant expression of RNAi agents will typically require regulatory elements, such as promoters, enhancers, and the like, to ensure expression of the RNAi agent in the target cell. Other aspects to be considered for vectors and constructs are known in the art.

VI pharmaceutical compositions of the invention

The present disclosure also includes pharmaceutical compositions and formulations comprising the RNAi agents of the present disclosure. In one embodiment, provided herein are pharmaceutical compositions comprising an RNAi agent as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing RNAi agents are useful for treating subjects that would benefit from inhibiting or reducing GPR75 gene expression, e.g., subjects having a GPR 75-related disorder, e.g., subjects having a weight disorder (e.g., obesity) or at risk of developing a weight disorder (e.g., obesity).

Such pharmaceutical compositions are formulated according to the mode of delivery. One example is a composition formulated for systemic administration by parenteral delivery, such as by Intravenous (IV), intramuscular (IM), or subcutaneous (subQ) delivery. Another example is to formulate the composition for direct delivery to the CNS, e.g., by intrathecal or intravitreal injection routes, optionally by infusion to the brain (e.g., striatum), e.g., by continuous pump infusion to the brain.

In some embodiments, the pharmaceutical compositions of the invention are pyrogen-free or pyrogen-free.

The pharmaceutical compositions of the invention may be administered in a dose sufficient to inhibit GPR75 gene expression. Typically, suitable dosages of RNAi agents of the present disclosure will be fixed dosages ranging from about 0.001mg to about 200.0mg, from about once a month to about once a year, typically from about once a quarter (i.e., about once every three months) to about once a year, typically fixed dosages ranging from about 1mg to 50mg, from about once a month to about once a year, typically from about once a quarter to about once a year. In certain embodiments, the dose will be a fixed dose, for example a fixed dose of about 25 μg to about 5 mg.

Repeated doses of the dosage regimen may include the periodic administration of a therapeutic amount of the RNAi agent, e.g., once a month to once every six months. In certain embodiments, the RNAi agent is administered about once a quarter (i.e., about once every three months) to about twice a year, particularly for the treatment of chronic diseases.

Treatment may be performed less frequently following an initial treatment regimen (e.g., a rapid-acting dose) once a day, twice a week, once a week.

In other embodiments, a single dose of the pharmaceutical composition may be long-acting such that subsequent doses are administered at intervals of no more than 1, 2, 3, or 4 months or more. In some embodiments of the present disclosure, a single dose of the pharmaceutical composition of the present disclosure is administered once a month. In other embodiments of the present disclosure, the pharmaceutical composition of the present disclosure is administered once a quarter to twice a year in a single dose.

Those of skill in the art will appreciate that certain factors will affect the dosage and time required to effectively treat a subject, including but not limited to the severity of the disease or condition, past treatment, the general health or age of the subject, and other diseases presently suffering from. Furthermore, the treatment of a subject with a therapeutically effective amount of the composition may include a single treatment or a series of treatments.

Advances in mouse genetics have produced a number of mouse models for studying various GPR 75-related diseases that would benefit from reduced GPR75 expression. Such models can be used for in vivo testing of RNAi agents and for determining therapeutically effective dosages. Suitable mouse models are known in the art and include, for example, the mouse models described elsewhere herein.

The pharmaceutical compositions of the present disclosure may be administered in a variety of ways depending on whether local or systemic treatment is desired and the area to be treated. Administration may be topical (e.g., by transdermal patch), by intranasal administration or oral inhalation (e.g., by inhalation or insufflation of a powder or aerosol, including by nebulizer) pulmonary system administration; intratracheal, epidermal and transdermal, oral or parenteral administration. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous administration, for example by implantation of a device; or intracranial administration, for example, by intraparenchymal, intrathecal, or intraventricular administration.

RNAi agents can be delivered in a manner that targets specific tissues (e.g., liver, CNS (e.g., neuronal, glial, or vascular tissue of the brain), or both liver and CNS).

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like are also useful. Suitable topical formulations include those wherein an RNAi agent described in the present disclosure is admixed with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelators, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoyl phosphatidyl DOPE ethanolamine, dimyristoyl phosphatidylcholine DMPC, distearoyl phosphatidylcholine), negative (e.g., dimyristoyl phosphatidyl glycerol DMPG), and cationic (e.g., dioleoyl tetraminopropyl DOTAP and dioleoyl phosphatidylethanolamine DOTMA). The RNAi agents described in the present disclosure can be encapsulated within liposomes, or can form complexes with liposomes, particularly with cationic liposomes. Alternatively, the RNAi agent can be complexed with lipids, particularly with 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, monoglyceryl oleate, diglycerol laurate, 1-monocaprylate, 1-dodecylazepan-2-one, acyl carnitine, acyl choline, 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 US 6,747,014, which is incorporated herein by reference.

A. RNAi agent formulations comprising membrane molecule modules

RNAi agents for use in the compositions and methods of the present disclosure can be formulated for delivery in membrane molecule assemblies, such as liposomes or micelles (micelles). As used herein, the term "liposome" refers to a vesicle comprising amphiphilic lipids arranged in at least one bilayer (e.g., one bilayer or multiple bilayers). Liposomes include unilamellar vesicles and multilamellar vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion comprises an RNAi agent composition. The lipophilic material separates the aqueous interior from the aqueous exterior, which generally does not contain the RNAi agent composition, although in some embodiments it may contain the RNAi agent composition. Liposomes can be used to transfer and deliver active ingredients to the site of action. Because the liposome membrane is structurally similar to a biological membrane, when the liposome is applied to a tissue, the liposome bilayer fuses with the bilayer of the cell membrane. As liposome and cell fusion proceeds, the internal aqueous content comprising the RNAi agent is delivered into the cell, where the RNAi agent can specifically bind to the target RNA and can mediate RNAi. In some cases, the liposomes are also specifically targeted, e.g., to direct RNAi agents to specific cell types.

Liposomes containing RNAi agents can be prepared by a variety of methods. In one example, the lipid component of the liposome is solubilized in the detergent such that the lipid component forms micelles. For example, the lipid component may be an amphiphilic cationic lipid or a lipid conjugate. The detergent may have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles comprising the lipid component. Cationic groups on the lipid 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 liposomal formulation of the RNAi agent.

If desired, carrier compounds which aid in the condensation can be added during the condensation reaction, for example by controlled addition. For example, the carrier compound may be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to facilitate condensation.

Methods for producing a stable polynucleotide delivery vehicle incorporating a polynucleotide/cationic lipid complex as a structural component of the delivery vehicle are further described, for example, in WO 96/37194, the entire contents of which are incorporated herein by reference. The formation of liposomes may also include one or more aspects of the exemplary methods described in the following documents: felgner, P.L.et al, (1987) Proc.Natl.Acad.Sci.USA 8:7413-7417; united States Patent No.4,897,355; united States Patent No.5,171,678; bangham et al, (1965) M.mol.biol.23:238; olson et al, (1979) biochem. Biophys. Acta557:9; szoka et al, (1978) proc.Natl. Acad.Sci.75:4194; mayhew et al, (1984) biochem. Biophys. Acta 775:169; kim et al, (1983) biochem. Biophys. Acta 728:339; fukunaga et al, (1984) Endocrinol.115:757. Common techniques for preparing lipid aggregates of suitable size for use as delivery vehicles include sonication and freeze-thawing extrusion (Mayer et al, (1986) biochem. Biophys. Acta858:161. Microfluidization may be used when consistently small (50 nm to 200 nm) and relatively uniform aggregates are desired (Mayhew et al, (1984) biochem. Biophys. Acta 775:169. These methods are readily applicable to packaging RNAi agent formulations into liposomes.

Liposomes fall into two broad categories. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized into endosomes. Due to the acidic pH in the endosome, the liposomes burst, releasing their contents into the cytoplasm (Wang et al (1987) biochem. Biophys. Res. Commun., 147:980-985).

pH sensitive liposomes or negatively charged liposomes capture nucleic acids rather than complex with nucleic acids. Because both nucleic acids and lipids carry similar charges, rejection occurs without complex formation. However, some nucleic acids are trapped inside the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding thymidine kinase genes to cell monolayers in culture. The expression of the foreign gene was detected in the target cells (Zhou et al (1992) Journal of Controlled Release, 19:269-274).

One major type of liposome composition includes phospholipids other than phosphatidylcholine of natural origin. For example, the neutral liposome composition can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are typically formed from dimyristoyl phosphatidylglycerol, whereas anionic fusogenic liposomes are formed predominantly from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from Phosphatidylcholine (PC), such as soybean PC and egg PC. The other is formed from a mixture of phospholipids or phosphatidylcholine or cholesterol.

Examples of other methods of introducing liposomes into cells in vitro and in vivo include U.S. patent No. 5,283,185; U.S. patent No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; felgner, (1994) J.biol. Chem.269:2550; nabel, (1993) Proc.Natl. Acad.Sci.90:11307; nabel, (1992) Human Gene Ther.3:649; gershon, (1993) biochem.32:7143; and Strauss, (1992) EMBO J.11:417.

Nonionic liposome systems have also been tested to determine their utility in delivering drugs to the skin, particularly systems comprising nonionic surfactants and cholesterol. Using a Novasome containing ^TM I (glycerol dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome ^TM Nonionic liposome formulations of II (glycerol distearate/cholesterol/polyoxyethylene-10-stearyl ether) deliver cyclosporin-A into the dermis of the mouse skin. The results indicate that this nonionic liposome system is effective in promoting the deposition of cyclosporin A on the various layers of the skin (Hu et al, (1994) S.T.P.Pharma.Sci.,4 (6): 466).

Liposomes also include "sterically stabilized" liposomes, which term, as used herein, refers to liposomes comprising one or more specific lipids which, when incorporated into the liposome, result in an increased circulation lifetime relative to liposomes lacking such specific lipids. Examples of sterically stabilized liposomes are those in which part (A) of the vesicle-forming lipid fraction of the liposome comprises one or more glycolipids such as monosialoganglioside G _M1 Or (B) one or more hydrophilic polymers such as polyethylene glycolDerivatization of (PEG) moieties. While not wishing to be bound by any particular theory, it is believed that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelins, or PEG-derivatized lipids, the increase in circulation half-life of these sterically stabilized liposomes is due to reduced cellular uptake by the reticuloendothelial system (RES) (Allen et al, (1987) FEBS Letters,223:42; wu et al, (1993) Cancer Research, 53:3765).

A variety of liposomes comprising one or more glycolipids are known in the art. Papahadjoulous et al (Ann.N.Y. Acad.Sci., (1987), 507:64) reported monosialoganglioside G _M1 The ability of galactocerebroside sulfate and phosphatidylinositol to improve the blood half-life of liposomes. Gabizon et al set forth these findings (Proc. Natl. Acad. Sci. U.S. A., (1988), 85:6949). U.S. Pat. No. 4,837,028 to Allen et al and WO 88/04924 disclose a pharmaceutical composition comprising (1) a sphingomyelin and (2) a ganglioside G _M1 Or a liposome of galactocerebroside sulfate. U.S. patent No. 5,543,152 (Webb et al) discloses liposomes comprising sphingomyelin. WO 97/13499 (Lim et al) discloses liposomes comprising 1, 2-sn-dimyristoyl phosphatidylcholine.

In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, while not able to fuse effectively with the plasma membrane, are absorbed by macrophages in vivo and can be used to deliver RNAi agents to macrophages.

Other 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; liposomes can protect the RNAi agents encapsulated in their internal compartments 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 liposome.

The positively charged synthetic cationic lipid N- [1- (2, 3-dioleyloxy) propyl ] -N, N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with negatively charged lipids of cell membranes of tissue culture cells to deliver RNAi agents (see, e.g., felgner, p.l.et al, (1987) proc.Natl. Acad. Sci. Usa 8:7413-7417, and description of DOTMA and its use with DNA in U.S. patent No. 4,897,355).

The DOTMA analogue 1, 2-bis (oleoyloxy) -3- (trimethylammonio) propane (DOTAP) can be used in combination with phospholipids to form DNA complex vesicles. Lipofectin ^TM (Bethesda Research Laboratories, gaithersburg, md) is an agent effective for delivering highly anionic nucleic acids to living tissue culture cells comprising positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. When sufficiently positively charged liposomes are used, the net charge on the resulting complex is also positive. Positively charged complexes prepared in this way spontaneously adhere to negatively charged cell surfaces, fuse with plasma membranes, and effectively deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1, 2-bis (oleoyloxy) -3,3- (trimethylammonio) propane ("DOTAP") (Boehringer Mannheim, indianapolis, indiana), differs from DOTMA in that the oleoyl moiety is linked by an ester rather than an ether linkage.

Other reported cationic lipid compounds include those that have been conjugated to multiple moieties, including, for example, carboxy spermine that has been conjugated to one of two types of lipids, and include, for example, 5-carboxy cetyl glycine dioctyl amide (5-carboxyspermylglycine dioctaoleoylamide, "DOGS") (Transfectam ^TM Promega, madison, wisconsin) and dipalmitoyl phosphatidylethanolamine 5-carboxycetyl amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes a lipid derivatized with cholesterol ("DC-Chol"), which has been formulated into liposomes in combination with DOPE (see, gao, X and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine prepared by conjugation of polylysine to DOPE was reported to be effective for transfection in the presence of serum (Zhou, X.et al, (1991) Biochim. Biophys. Acta 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than compositions containing DOTMA. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (visual, 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.

Liposome formulations are particularly suitable for topical administration, and liposomes have several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer the RNAi agent to the skin. In some embodiments, the liposomes are used to deliver the RNAi agent to epidermal cells, and are also used to enhance penetration of the RNAi agent into dermal tissue, e.g., into the skin. For example, liposomes may be applied topically. The topical delivery of drugs formulated as liposomes to the skin has been described (see, e.g., weiner et al, (1992) Journal of Drug Targeting, vol.2,405-410 and du plasis et al, (1992) Antiviral Research,18:259-265;Mannino,R.J.and Fould-Fogerite, S., (1998) Biotechniques 6:682-690;Itani,T.et al., (1987) Gene 56:267-276;Nicolau,C.et al. (1987) meth.enzymol.149:157-176;Straubinger,R.M.and Papahadjopoulos,D. (1983) meth.enzymol.101:512-527;Wang,C.Y.and Huang,L., (1987) Proc.Natl. Acad. Sci. USA 84:7851-7855).

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

Liposomes containing RNAi agents can be made highly deformable. This deformability allows the liposomes to penetrate pores smaller than the average radius of the liposomes. For example, the delivery body is a deformable liposome. The transfer body may be prepared by adding a surface edge activator (typically a surfactant) to a standard liposome composition. The delivery body comprising the RNAi agent can be delivered, for example, by subcutaneous delivery of the infection, to deliver the RNAi agent to keratinocytes in the skin. In order to pass through intact mammalian skin, lipid vesicles must pass through a series of pores each less than 50nm in diameter under the influence of a suitable transdermal gradient. Furthermore, due to lipid properties, these transmitters can self-optimize (adapt to the shape of the pores, e.g. in the skin), self-repair, and can often reach their targets without breaking, and often self-load.

Other formulations suitable for use in the present disclosure are described in U.S. provisional application Ser. No. 61/018,616, filed on 1/2/2008; U.S. provisional application No. 61/018,611 filed on 1/2/2008; U.S. provisional application No. 61/039,748 filed on 26/3/2008; U.S. provisional application No. 61/047,087 filed on month 4 and 22 of 2008, and U.S. provisional application No. 61/051,528 filed on month 5 and 8 of 2008. PCT application No. PCT/US2007/080331, filed on month 10 and 3 of 2007, also describes formulations suitable for use in the present disclosure.

The carrier is another type of liposome, a highly deformable lipid aggregate, an attractive candidate for drug delivery vehicles. The transfer bodies can be described as highly deformable lipid droplets that easily pass through smaller pores than the droplets. The transfer bodies may be adapted to the environment in which they are used, e.g., they are self-optimizing (adapt to the shape of the skin pores), self-repairing, often reach their targets without rupture, and often self-loading. To prepare the transfer body, a surfactant (typically a surfactant) may be added to the standard liposome composition. Transfer bodies have been used to deliver serum albumin to the skin. The carrier-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as those described herein, particularly in emulsions (including microemulsions) and liposomes. The most common method of classifying and ordering the nature of many different types of surfactants (natural and synthetic) is to use a hydrophilic/lipophilic balance (HLB). The nature of the hydrophilic group (also referred to as "head") provides the most useful method for classifying the different surfactants used in the formulation (Rieger, in Pharmaceutical Dosage Forms, marcel Dekker, inc., new York, n.y.,1988, p.285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceuticals and cosmetics and can be used in a wide range of pH values. Typically, their HLB value ranges from 2 to about 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glycerol esters, polyglycerol 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 popular members of the nonionic surfactant class.

Surfactants are classified as anionic if they have a negative charge when dissolved or dispersed in water. Anionic surfactants include carboxylic acid esters such as soaps, acyl lactylates, acyl amides of amino acids, sulfuric acid esters such as alkyl sulfuric acid esters and ethoxylated alkyl sulfuric acid esters, sulfonic acid esters such as alkylbenzenesulfonic acid esters, acyl isethionates, acyl taurates and sulfosuccinates, and phosphoric acid esters. The most important members of the class of anionic surfactants are alkyl sulfates and soaps.

Surfactants are classified as cationic surfactants if the surfactant molecules are positively charged when dissolved or dispersed in water. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

Surfactants are classified as amphoteric surfactants if they have the ability to carry a positive or negative charge. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines and phospholipids.

The use of surfactants in pharmaceutical products, formulations and emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, marcel Dekker, inc., new York, n.y.,1988, p.285).

RNAi agents for use in the methods of the present disclosure can also be provided in the form of micelle formulations. "micelle" is defined herein as a special type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all hydrophobic portions of the molecule are directed inward, leaving hydrophilic portions in contact with the surrounding water. If the environment is hydrophobic, the opposite arrangement exists.

By mixing an aqueous solution of an siRNA composition with an alkali metal C ₈ -C ₂₂ The alkyl sulfate and micelle forming compound are mixed to produce a mixed micelle formulation suitable for delivery through a transdermal membrane. 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, monooleate, monolaurate, borage oil, evening primrose oil (evening of primrose oil), menthol, trihydroxy oxo-cholate and pharmaceutically acceptable salts thereof, glycerol, polyglycerol, lysine, polylysine, triolein, polyoxyethylene ethers and analogs thereof, polidocanol alkyl ethers and analogs thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compound may be added simultaneously with or after the addition of the alkali metal alkyl sulfate. Mixing of essentially any kind of ingredients will form mixed micelles, but vigorous mixing is to provide smaller size micelles.

In one method, a first micelle composition comprising 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 compound with vigorous mixing.

Phenol or m-cresol may be added to the mixed micelle composition to stabilize the formulation and prevent bacterial growth. Alternatively, phenol or m-cresol may be added with the micelle-forming ingredients. Isotonic agents such as glycerol may also be added after formation of the mixed micelle composition.

To deliver the micelle formulation as a spray, the formulation may be placed into an aerosol dispenser and the dispenser filled with a propellant. The propellant under pressure is in liquid form in the dispenser. The proportions of the components are adjusted so that the aqueous phase and the propellant phase are in one phase, i.e. only one phase. If there are two phases, the dispenser must be shaken, for example by a metering valve, before a portion of the contents is dispensed. The dispensed dose of medicament is ejected from the metering valve in the form of a fine spray.

The propellant may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1, 2 tetrafluoroethane) may be used.

The specific concentrations of the essential components can be determined by relatively simple experimentation. For absorption through the oral cavity it is often desirable to increase the dose administered by injection or through the gastrointestinal tract, for example by at least two or three times.

Lipid particles

RNAi agents, such as dsRNA in the present disclosure, can be fully encapsulated in lipid formulations, such as LNP or other nucleic acid-lipid particles.

As used herein, the term "LNP" refers to stable nucleic acid-lipid particles. LNP typically comprises cationic lipids, non-cationic lipids, and lipids that prevent aggregation of particles (e.g., PEG-lipid conjugates). LNPs are extremely useful for systemic applications because they exhibit extended cycle life following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the site of administration). LNPs include "pSPLP" which includes encapsulated condensing agent-nucleic acid complexes as described in WO 00/03683. The particles of the present disclosure typically have an average diameter of about 50nm to about 150nm, more typically about 60nm to about 130nm, more typically about 70nm to about 110nm, most typically about 70nm to about 90nm, and are substantially non-toxic. Furthermore, when present in the nucleic acid-lipid particles of the present disclosure, the nucleic acids are resistant to degradation by nucleases in aqueous solutions. Nucleic acid-lipid particles and methods of making the same are disclosed, for example, in U.S. patent No. 5,976,567;5,981,501;6,534,484;6,586,410;6,815,432; U.S. patent publication No. 2010/0325420 and WO 96/40964.

In one embodiment, the ratio of lipid to drug (mass/mass ratio) (e.g., ratio of lipid to dsRNA) will range from about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Intermediate ranges of the above ranges are also considered part of the present disclosure.

Certain specific LNP formulations for delivering RNAi agents have been described in the art, including, for example, "LNP01" formulations described in WO 2008/042973, which is incorporated herein by reference.

The following table lists other exemplary lipid-dsRNA formulations.

DSPC: distearoyl phosphatidylcholine

DPPC: dipalmitoyl phosphatidylcholine

PEG-DMG: PEG-dimyristoylglycerol (C14-PEG, or PEG-C14) (PEG having an average molar mass of 2000)

PEG-DSG: PEG-Biphenylvinyl Glycerol (C18-PEG or PEG-C18) (PEG with average molar mass of 2000)

PEG-cDMA: PEG-carbamoyl-1, 2-dimyristoyloxypropylamine (PEG with average molar mass of 2000)

Formulations comprising SNALP (1, 2-dioleenyloxy-N, N-dimethylaminopropane (DLinDMA)) are described in WO 2009/127060, which is incorporated herein by reference.

Formulations comprising XTC are described in WO 2010/088537, the entire contents of which are incorporated herein by reference.

Formulations comprising MC3 are described, for example, in U.S. patent publication No. 2010/0325420, which is incorporated herein by reference in its entirety.

Formulations comprising ALNY-100 are described in WO 2010/054406, the entire contents of which are incorporated herein by reference.

Formulations comprising C12-200 are described in WO 2010/129709, the entire contents of which are incorporated herein by reference.

Compositions and formulations for oral administration include powders or granules (granule), microparticles, nanoparticles, suspensions or solutions in aqueous or nonaqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which the dsRNA described in the present disclosure is administered in combination with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids or esters or salts thereof, bile acids or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycocholic acid (glycocholic acid), glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24, 25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoglyceryl oleate, diglycerol laurate, 1-monocaprylate, 1-dodecylazepan-2-one, acyl carnitine, acyl choline or monoglycerides, diglycerides or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, a combination of permeation enhancers is used, such as a combination of fatty acids/salts and bile acids/salts. An exemplary combination is the sodium salts of capric acid, UDCA and lauric acid. Other permeation enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The dsRNA described in the present disclosure may be delivered orally, in particulate form, including spray-dried particles, or complexed to form microparticles or nanoparticles. The dsRNA complexing agent comprises polyamino acid; a polyimine; a polyacrylate; polyalkylacrylates, polyoxetanes, polyalkylcyanoacrylates; cationized gelatin, albumin, starch, acrylate, polyethylene glycol (PEG) and starch; polyalkylene cyanoacrylates; DEAE-derived polyimines, pullulan (pollulan), cellulose and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, spermine, protamine, polyvinylpyridine, polythiodiethylaminomethyl ethylene 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, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethacrylate, polyhexyl acrylate, poly (D, L-lactic acid), poly (DL-lactic acid-co-glycolic acid) (PLGA), alginate and polyethylene glycol (PEG). Oral formulations of dsrnas and their preparation are described in detail in U.S. patent 6,887,906, U.S.2003/0027780 and U.S. patent No. 6,747,014, each of which is incorporated herein by reference.

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

Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be produced from a variety of components including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Particularly useful formulations include formulations that target the brain in the treatment of GPR 75-related diseases or disorders.

The pharmaceutical formulations of the present disclosure may conveniently be presented in unit dosage form and may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of combining the active ingredient with a pharmaceutical carrier or excipient. Formulations are typically prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present disclosure may be formulated in any of a number of possible dosage forms, such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft capsules, suppositories, and enemas. The compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous or mixed media. The aqueous suspension may also contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension may also contain stabilizers.

Other formulations

i. Emulsion

The compositions of the present disclosure may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems in which one liquid is dispersed in another liquid in the form of droplets of a diameter typically exceeding 0.1 μm (see, e.g., ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, allen, LV., popovich ng, and Ansel HC.,2004,Lippincott Williams&Wilkins (8 th ed.), new York, NY, idson, in Pharmaceutical Dosage Forms, lieberman, rieger and Banker (eds.), 1988,Marcel Dekker,Inc, new York, n.y., volume 1,p.199;Rosoff,in Pharmaceutical Dosage Forms,Lieberman,Rieger and Banker (eds.), 1988,Marcel Dekker,Inc, new York, n.y., volume 1,p.245;Block in Pharmaceutical Dosage Forms,Lieberman,Rieger and Banker (eds.), 1988,Marcel Dekker,Inc, new York, n.y., volume 2,p.335;Higuchi et al, in Remington's Pharmaceutical Sciences, mack Publishing co., ston, pa.,1985, p.301). Emulsions are generally two-phase systems comprising two immiscible solution phases intimately mixed and dispersed with each other. Typically, the emulsion may be of the water-in-oil (w/o) type or of the oil-in-water (o/w) type. When the aqueous phase is subdivided into small droplets and dispersed in a substantially oil phase, the resulting composition is referred to as a water-in-oil (w/o) emulsion. Alternatively, when the oil phase is subdivided into small droplets and dispersed in a substantially aqueous phase, the resulting composition is referred to as an oil-in-water (o/w) emulsion. In addition to containing a dispersed phase and an active agent that may be present in solution as an aqueous phase, an oil phase, or as a separate phase itself, the emulsion may contain other components. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes and antioxidants may also be present in the emulsion, as desired. The pharmaceutical emulsion may also be a composite emulsion comprising more than two phases, for example, an oil-in-water-in-oil (o/w/o) emulsion and a water-in-oil-in-water (w/o/w) emulsion. Such formulations tend to provide certain advantages not possessed by simple biphasic emulsions. The composite emulsion wherein individual oil droplets of the o/w emulsion encapsulate water droplets to form the w/o/w emulsion. Also, a system in which oil droplets are encapsulated in water spheres that are stably present in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Typically, the dispersed or discontinuous phase of the emulsion is well dispersed in the external or continuous phase and this form is maintained by way of an emulsifier or by way of the viscosity of the formulation. Either phase of the emulsion may be semi-solid or solid, such as emulsion ointment bases and creams. Other methods of stabilizing emulsions require the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can be broadly divided into four categories: synthetic surfactants, natural emulsifiers, absorbent matrices, and finely divided solids (see, e.g., ansel' sPharmaceutical Dosage Forms and Drug Delivery Systems, allen, LV., popovich ng, and Ansel HC.,2004,Lippincott Williams&Wilkins (8 the.), new York, NY; idson, in Pharmaceutical Dosage Forms, lieberman, rieger and Banker (eds.), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, p.199).

Synthetic surfactants, also known as surfactants, have been widely used in emulsion formulations and have been reviewed in the literature (see, e.g., ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, allen, LV., popovich ng, and Ansel HC.,2004,Lippincott Williams&Wilkins (8 th ed.), new York, NY; rieger, in Pharmaceutical Dosage Forms, lieberman, rieger and Banker (eds.), 1988,Marcel Dekker,Inc, new York, n.y., volume 1,p.285;Idson,in Pharmaceutical Dosage Forms,Lieberman,Rieger and Banker (eds.), marcel Dekker, inc., new York, n.y.,1988,volume 1,p.199). Surfactants are generally amphiphilic and comprise a hydrophilic portion and a hydrophobic portion. The ratio of hydrophilicity to hydrophobicity of a surfactant has been defined as the hydrophilic/lipophilic balance (HLB) and is a valuable tool for selecting surfactants in classification and preparation of formulations. Surfactants can be classified into different categories based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphiphilic (see, e.g., anse's Pharmaceutical Dosage Forms and Drug Delivery Systems, allen, LV., popovich ng, and anse HC.,2004,Lippincott Williams&Wilkins (8 th ed.), new York, NY Rieger, in Pharmaceutical Dosage Forms, lieberman, rieger and Banker (eds.), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, p.285).

Natural emulsifiers used in emulsion formulations include lanolin, beeswax, phospholipids, lecithins and acacia. The absorbent matrices are hydrophilic so that they can absorb water to form w/o emulsions while maintaining their semi-solid consistency, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids are also used as good emulsifiers, especially in combination with surfactants and in viscous formulations. These include polar inorganic solids (e.g., heavy metal hydroxides), non-swelling clays (e.g., bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate), pigments and non-polar solids (e.g., carbon or glycerol tristearate).

A wide variety of non-emulsifying materials are also included in the emulsion formulation and contribute to the characteristics of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrocolloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, lieberman, rieger and Banker (eds.), 1988,Marcel Dekker,Inc, new York, n.y., volume 1,p.335;Idson,in Pharmaceutical Dosage Forms,Lieberman,Rieger and Banker (eds.), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, p.199).

Hydrocolloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (e.g. acacia, agar, alginic acid, carrageenan, guar gum, karaya gum and tragacanth), cellulose derivatives (e.g. carboxymethylcellulose and carboxypropylcellulose) and synthetic polymers (e.g. carbomers, cellulose ethers and carboxyvinyl polymers). They disperse or swell in water to form a colloidal solution, stabilizing the emulsion by forming a strong interfacial film around the dispersed phase droplets and by increasing the viscosity of the external phase.

Since emulsions generally contain many ingredients that readily support the growth of microorganisms, such as carbohydrates, proteins, sterols, and phospholipids, these formulations generally contain preservatives. Preservatives commonly used in emulsion formulations include methyl parahydroxybenzoate, propyl parahydroxybenzoate, quaternary ammonium salts, benzalkonium chloride, parabens and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. The antioxidants used may be free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant potentiators such as citric acid, tartaric acid and lecithin.

The use of emulsion formulations via the transdermal, oral and parenteral routes and methods for their preparation have been reviewed in the literature (see, for example, ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, allen, LV., popovich ng, and Ansel HC.,2004,Lippincott Williams&Wilkins (8 th ed.)), new York, NY; idson, in Pharmaceutical Dosage Forms, lieberman, rieger and Banker (eds.)), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, p.199). Emulsion formulations for oral delivery have been widely used for ease of formulation and efficacy in 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 (8 th ed.), new York, NY; rosoff, in Pharmaceutical Dosage Forms, lieberman, rieger and Banker (eds.), 1988,Marcel Dekker,Inc, new York, n.y., volume 1,p.245;Idson,in Pharmaceutical Dosage Forms,Lieberman,Rieger and Banker (eds.), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, p.199). Mineral oil-based laxatives, oil-soluble vitamins and high fat nutritional formulations are substances that are typically administered orally as o/w emulsions.

Microemulsion(s)

In one embodiment of the present disclosure, the composition of RNAi agent and nucleic acid is formulated as a microemulsion. Microemulsions can be defined as systems of water, oil and amphiphiles which are single optically isotropic and thermodynamically stable liquid solutions (see, e.g., ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, allen, LV., popovich ng, and AnselHC.,2004,Lippincott Williams&Wilkins (8 th ed.), new York, NY, rosoff, in Pharmaceutical Dosage Forms, lieberman, rieger and Banker (eds.), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, p.245). Generally, microemulsions are systems prepared by: first, the oil is dispersed in an aqueous surfactant solution, followed by the addition of a sufficient amount of a fourth component (typically a medium chain alcohol) to form a transparent system. Thus, microemulsions have also been described as thermodynamically stable, isotropic clear 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 typically prepared from a combination of three to five ingredients including oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or oil-in-water (o/w) type depends on the nature of the oil and surfactant used, as well as 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).

The phenomenological methods using phase diagrams have been widely studied and provide the person skilled in the art with a full knowledge of how to formulate microemulsions (see e.g. Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, allen, LV., popovich ng, and Ansel HC.,2004,Lippincott Williams&Wilkins (8 th ed.), new York, NY; rosoff, in Pharmaceutical Dosage Forms, lieberman, rieger and Banker (eds.), 1988,Marcel Dekker,Inc, new York, n.y., volume 1,p.245;Block,in Pharmaceutical Dosage Forms,Lieberman,Rieger and Banker (eds.), 1988,Marcel Dekker,Inc, new York, n.y., volume 1, p.335). The advantage of microemulsions over conventional emulsions is that the water-insoluble drug is dissolved in the spontaneously formed thermodynamically stable droplet formulation.

Surfactants for preparing the microemulsion include, but are not limited to, ionic surfactants, nonionic surfactants, brij96, polyoxyethylene oleyl ethers, polyglyceryl fatty acid esters, tetraglycerol monolaurate (ML 310), tetraglycerol monooleate (MO 310), hexaglycerol monooleate (PO 310), hexaglycerol pentaoleate (PO 500), decaglycerol monocaprylate (MCA 750), decaglycerol monooleate (MO 750), decaglycerol sesquioleate (SO 750), decaglycerol decaoleate (DAO 750), alone or in combination with co-surfactants. Cosurfactants are generally short-chain alcohols, such as ethanol, 1-propanol and 1-butanol, which increase interfacial flowability by penetrating into the surfactant film and thus creating disordered films due to the void spaces created between the surfactant molecules. However, microemulsions can be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, aqueous solutions of drugs, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, captex300, captex 355, capmul MCM, fatty acid esters, medium chain (C8-C12) monoglycerides, medium chain (C8-C12) diglycerides and medium chain (C8-C12) triglycerides, polyoxyethylated glycerol fatty acid esters, fatty alcohols, pegylated glycerides, saturated pegylated C8-C10 glycerides, vegetable oils, and silicone oils.

Microemulsions are of particular interest from the standpoint of drug solubility and enhanced drug absorption. Lipid-based microemulsions (o/w and w/o) have been proposed to improve the oral bioavailability of drugs including peptides (see, e.g., U.S. Pat. No. 6,191,105;7,063,860;7,070,802;7,157,099;Constantinides et al, pharmaceutical Research,1994,11,1385-1390; ritschel, meth.find.Exp.Clin.Pharmacol.,1993,13,205). Microemulsions offer the following advantages: improved drug solubility, protection of the drug from enzymatic hydrolysis, enhanced drug absorption possible due to surfactant-induced changes in membrane fluidity and permeability, ease of manufacture, easier oral administration than solid dosage forms, improved clinical efficacy and reduced toxicity (see, e.g., U.S. patent No. 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 general, the components of a microemulsion may spontaneously form a microemulsion when they are mixed together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or RNAi agents. Microemulsions are also effective in transdermal delivery of active ingredients in cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present disclosure are expected to promote increased systemic uptake of RNAi agents and nucleic acids from the gastrointestinal tract, and improve local cellular uptake of RNAi agents and nucleic acids.

The microemulsions of the present disclosure may also contain other components and additives, such as sorbitan monostearate (Grill 3), glycerol acid esters (Labrasol), and penetration enhancers, to improve the properties of the formulation and enhance the absorption of the RNAi agents and nucleic acids of the present invention. Permeation enhancers used in the microemulsions of the present disclosure can be classified as belonging to five broad classes-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 class has been discussed above.

Microparticle

RNAi agents of the present disclosure can be incorporated into particles, such as microparticles. The microparticles may be produced by spray drying, but may also be produced by other methods including freeze drying, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

Penetration enhancer

In one embodiment, the present disclosure uses a variety of permeation enhancers to achieve efficient delivery of nucleic acids, particularly RNAi agents, to the skin of an animal. Most drugs exist in solution in ionized and non-ionized forms. However, only lipid-soluble or lipophilic drugs can generally readily cross cell membranes. It has been found that if the cell membrane to be spanned is treated with a permeation enhancer, even a non-lipophilic drug is able to span the cell membrane. In addition to facilitating diffusion of the non-lipophilic drug across the cell membrane, the permeation enhancer also increases the permeability of the lipophilic drug.

Permeation enhancers can be classified as belonging to one of five classes: i.e., 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). The above-mentioned types of penetration enhancers will be described in more detail below.

Surfactants (or "surfactants") are chemical entities that, when dissolved in an aqueous solution, reduce the surface tension of the solution or interfacial tension between the aqueous solution and another liquid, with the result that the absorption of RNAi agents through the mucosa is enhanced. In addition to bile salts and fatty acids, such permeation enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (see, for example, malmsten, m.surfactants and polymers in drug delivery, informa Health Care, new York, NY,2002; lee et al Critical Reviews in Therapeutic Drug Carrier Systems,1991, p.92); and perfluorochemical emulsions such as FC-43.Takahashi et al, j.pharm.pharmacol.,1988,40,252).

Various fatty acids as permeation enhancersDerivatives thereof include, for example, oleic acid, lauric acid, capric acid (n-capric acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, glyceryl monooleate (1-monooleoyl-rac-glycerol), glyceryl dilaurate, caprylic acid, arachidonic acid, glyceryl 1-monodecanoate, 1-dodecylazepan-2-one, acyl carnitine, acyl choline, C _1-20 Alkyl esters (such as methyl, isopropyl, and tert-butyl), and their mono-and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see, e.g., 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).

Physiological effects of bile include promoting 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 38in:Goodman&Gilman's The Pharmacological Basis of Therapeutics,9th Ed, hardman et al eds., mcGraw-Hill, new York,1996, pp. 934-935). A variety of natural bile salts and synthetic derivatives thereof can act as permeation enhancers. Thus, the term "bile salt" includes any naturally occurring component of bile and any synthetic derivative thereof. Suitable bile salts include, for example, cholic acid (or a pharmaceutically acceptable sodium salt thereof), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glycocholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholate (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), tauro-24, 25-dihydro-sodium fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see, for example, malmsten, and polymers in drug delivery, informa Health Care, new York, NY,2002; lee et al Critical Reviews in Therapeutic Drug Carrier Systems,1991,page 92;Swinyard,Chapter 39In:Remington'sPharmaceutical Sciences,18th Ed, gennaro, ed., mack Publishing co, pa., chenodeoxycholic acid, 0, pages 782-783;Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems,1990,7,1-95, j.Pharm.976, pharm. 1993, pharm. Pharm, J.973, sc.5843.

Chelating agents used in connection with the present disclosure may be defined as compounds that remove metal ions from solution by forming complexes therewith, with the result that the uptake of RNAi agents through the mucosa is enhanced. With respect to their use as permeation enhancers in the present disclosure, chelators also have the additional advantage of acting as dnase inhibitors, as most characterized dnase enzymes require divalent metal ions for catalysis and thus would be inhibited by chelators (Jarrett, j.chromatogr.,1993,618,315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediamine tetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate, and homovanillic acid salts), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of beta-diketones (enamines) (see, e.g., katdare, A.et al, excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, danvers, MA,2006; lee et al, critical Reviews in Therapeutic Drug Carrier Systems,1991,page 92;Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems,1990,7,1-33; buur et al, J.control Rel.,1990,14,43-51).

As used herein, a non-chelating, non-surfactant penetration enhancing compound may be defined as a compound that exhibits insignificant chelating or surfactant activity, but still enhances the absorption of RNAi agents through the mucosa of the gut (see, e.g., muranishi, critical Reviews in Therapeutic Drug Carrier Systems,1990,7,1-33). Such permeation enhancers include, for example, unsaturated cyclic ureas, 1-alkyl-alkanone derivatives and 1-alkenyl-aza-alkanone derivatives (Lee et al Critical Reviews in Therapeutic Drug Carrier Systems,1991, page 92); and non-steroidal anti-inflammatory agents such as sodium diclofenac, indomethacin, and phenylbutazone (Yamashita et al, j.pharm.pharmacol.,1987,39,621-626).

Agents that enhance the uptake of RNAi agents at the cellular level can also be added to the pharmaceutical and other compositions of the present disclosure. For example, cationic lipids such as lipofectin (Junichi et al, U.S. Pat.No.705,188), cationic glycerol derivatives, and polycationic molecules such as polylysine (WO 97/30731) are also known to enhance cellular uptake of dsRNA.

Other agents may be used to enhance penetration of the applied nucleic acid, including glycols such as ethylene glycol and propylene glycol, pyrroles such as 2-pyrrole, azones and terpenes such as limonene and menthone.

Excipient(s)

In contrast to carrier compounds, "pharmaceutical carriers" or "excipients" are pharmaceutically acceptable solvents, suspending agents, or other pharmacologically inert vehicles for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid, and may be selected according to the intended mode of administration under consideration, when combined with the nucleic acid and other components of a given pharmaceutical composition, to provide a desired volume, viscosity, etc. Typical drug carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica colloid, stearic acid, metal stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, and the like); disintegrants (e.g., starch, sodium starch glycolate, etc.); and a wetting agent (e.g., sodium lauryl sulfate, etc.).

The compositions of the present disclosure may also be formulated using pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not adversely react with the nucleic acids. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone and the like.

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

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone and the like.

Other components

The compositions of the present disclosure may additionally contain other auxiliary components commonly found in pharmaceutical compositions in amounts of their levels as determined in the art. Thus, for example, the compositions may contain additional, compatible, pharmaceutically active materials, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain other materials such as dyes, flavors, preservatives, antioxidants, opacifying agents, thickening agents, and stabilizers useful in physically formulating the various dosage forms of the compositions of the present invention. However, when such materials are added, the biological activity of the components of the compositions of the present invention should not be unduly disturbed. The formulation may be sterilized and, if desired, mixed with adjuvants which do not adversely react with the nucleic acids of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavoring or aromatic substances, and the like.

The aqueous suspension may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension may also contain stabilizers.

In some embodiments, the pharmaceutical compositions described in the present disclosure comprise (a) one or more RNAi agents and (b) one or more agents that function by a non-iRNA mechanism and are useful for treating GPR 75-related disorders. Examples of such agents include, but are not limited to, antiviral agents, immunostimulants, therapeutic vaccines, inhibitors of viral entry, and combinations of any of the foregoing.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for LD ₅₀ (lethal dose to 50% of population) and ED ₅₀ (a therapeutically effective dose to 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index and can be expressed as LD ₅₀ /ED ₅₀ Ratio of the two components. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of the compositions disclosed herein is generally defined to include ED with low or no toxicity ₅₀ Is a cyclic concentration range of (c). The dosage may vary within this range depending upon the dosage form employed and the route of administration employed. For any compound used in the methods described in the present disclosure, a therapeutically effective dose can first be estimated from a cell culture assay. The dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, where appropriate, of the polypeptide product of the target sequence (e.g., to achieve a reduced polypeptide concentration), including IC as determined in cell culture ₅₀ (i.e., the concentration of test compound at which half-maximal inhibition of symptoms is achieved). Such information can be used to more accurately determine the dosage available to the human body. For example, the level in plasma can be measured by high performance liquid chromatography.

In addition to the administration of RNAi agents as described above, the RNAi agents described in this disclosure can also be administered in combination with other agents known to be effective in treating pathological processes mediated by repeated expression of nucleotides. In any event, the administering physician can adjust the amount and timing of RNAi agent administration based on the results observed by standard efficacy measurement methods known in the art or described herein.

VII kit

In certain aspects, the disclosure provides a kit comprising a suitable container containing a pharmaceutical formulation of an RNAi agent (e.g., a double stranded RNAi agent). In certain embodiments, the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation in two or more containers, respectively, such as one container for the RNAi agent and at least one other container for the carrier compound. The kits may be packaged in a variety of different configurations, such as one or more containers in a single box. The different components may be combined, for example, according to instructions provided by the kit. The components may be combined according to the methods described herein, for example, to prepare and administer a pharmaceutical composition. The kit may also include a delivery device, e.g., the kit may include a delivery device suitable for pulmonary administration, e.g., a device suitable for oral inhalation administration, including nebulizers, metered dose inhalers, and dry powder inhalers.

Methods of inhibiting GPR75 expression

The disclosure also provides methods of inhibiting the expression of the GPR75 gene in a cell. The method comprises contacting the cell with an RNAi agent (e.g., a double-stranded RNAi agent) in an amount effective to inhibit expression of the GPR75 gene in the cell, thereby inhibiting expression of GPR75 in the cell. In certain embodiments of the disclosure, the expression of the GPR75 gene in liver cells (e.g., hepatocytes) is inhibited.

The contacting of the cells with an RNAi agent, such as a double stranded RNAi agent, can be accomplished in vitro or in vivo. Contacting the cells with the RNAi agent in vivo includes contacting the cells or cell populations in a subject, e.g., a human subject, with the RNAi agent. Combinations of methods of contacting cells in vitro and methods of contacting cells in vivo are also possible.

As described above, the contact with the cells may be direct or indirect. Furthermore, contact with the cells may be achieved by targeting ligands, including any of the ligands described herein or known in the art. In some embodiments, the targeting ligand is a lipophilic moiety (e.g., C16) and/or a carbohydrate moiety (e.g., galNAc ligand), or any other ligand that directs the RNAi agent to the site of interest. In certain embodiments, the ligand is not a cholesterol moiety. In certain embodiments, the RNAi agent does not include a targeting ligand.

As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "down-regulate," "repression," and other similar terms, and includes any level of inhibition. In certain embodiments, the level of inhibition, e.g., for an RNAi agent of the present disclosure, can be assessed under cell culture conditions, e.g., wherein cells in the cell culture pass through Lipofectamine at a cell concentration of approximately 10nM or less, 1nM or less, etc ^TM Mediated transfection was performed. Knock-down of a given RNAi agent can be determined by comparing the pre-treatment level of the cell culture to the post-treatment level of the cell culture, optionally also compared to cells treated in parallel with a confounding or other form of control RNAi agent. Knock-down (e.g., 50% or more) in a cell culture may thus be identified as an indicator that "inhibition" or "alleviation", "downregulation" or "repression" or the like has occurred. It is expressly contemplated that the evaluation of the levels of target mRNA or encoded protein of the RNAi agents of the present disclosure (and the extent of "inhibition" caused by the RNAi agents of the present invention, etc.) can also be performed in an in vivo system under appropriately controlled conditions as described in the art.

As used herein, the phrase "inhibiting expression of the GPR75 gene" or "inhibiting expression of GPR 75" includes inhibiting expression of any GPR75 gene (e.g., a mouse GPR75 gene, a rat GPR75 gene, a monkey GPR75 gene, or a human GPR75 gene) or a variant or mutant of a GPR75 gene encoding a GPR75 protein. Thus, in the context of a genetically manipulated cell, cell population or organism, the GPR75 gene may be a wild-type GPR75 gene, a mutated GPR75 gene or a transgenic GPR75 gene.

"inhibiting expression of the GPR75 gene" includes any inhibition of the level of the GPR75 gene, such as at least partial repression of the expression of the GPR75 gene, such as at least 20% inhibition. In certain embodiments, inhibition is at least 30%, at least 40%, at least 50%, at least about 60%, at least 70%, at least about 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; or to a level below that detected by the assay. In one method, inhibition was measured at a concentration of 10nM siRNA using the luciferase assay provided in example 1.

The expression of the GPR75 gene can be assessed based on the level of any variable associated with GPR75 gene expression, such as GPR75 mRNA level or GPR75 protein level.

Inhibition may 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 may be any type of control level used in the art, e.g., a pre-dosing baseline level, or a level measured in a similar subject, cell or sample that is untreated or treated with a control (e.g., a buffer-only control or a non-active agent control).

In some embodiments of the methods of the present disclosure, the expression of the GPR75 gene is inhibited by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95%, or is inhibited to a level below the detection level of the assay. In certain embodiments, the methods comprise clinically relevant inhibition of GPR75 expression, e.g., as demonstrated by clinically relevant results after treatment of a subject with an agent that reduces GPR75 gene expression.

Inhibition of GPR75 gene expression may be manifested by a decrease in the amount of mRNA expressed by a first cell or first cell population (such cells may be present in a sample from a subject, for example) in which the GPR75 gene is transcribed and 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 or were located) such that expression of the GPR75 gene is inhibited compared to a second cell or cell population substantially identical to the first cell or cell population but not so treated (control cells not treated with iRNA or not treated with iRNA targeting a gene of interest). The extent of inhibition can be expressed as:

In other embodiments, inhibition of GPR75 gene expression can be assessed according to a decrease in a parameter functionally related to GPR75 gene expression, such as GPR75 protein expression, S protein priming, viral entry efficiency, viral load. GPR75 gene silencing can be determined in any cell expressing GPR75 (whether endogenous GPR75 or GPR75 from an expression construct) by any assay known in the art.

Inhibition of GPR75 protein expression may be manifested by a decrease in the level of GPR75 protein expressed by a cell or cell population (e.g., the level of protein expressed in a sample from a subject). As described above, to assess genomic inhibition, inhibition of protein expression levels in a treated cell or cell population can be similarly expressed as a percentage of protein levels in a control cell or cell population.

Control cells or control cell populations useful for assessing inhibition of GPR75 gene expression include cells or cell populations that have not been contacted with an RNAi agent of the invention. For example, the control cell or control cell population can be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with the RNAi agent.

The level of GPR75 mRNA expressed by a cell or cell population can be determined using any method known in the art for assessing RNA expression. In one embodiment, the level of expression of GPR75 in a sample is determined by detecting the transcribed polynucleotide or portion thereof, e.g., the mRNA of the GPR75 gene. RNA extraction techniques may be used, including, for example, the use of acidic phenol/guanidine isothiocyanate extraction (RNAzol B; biogenesis), RNeasy ^TM RNA preparation kitOr PAXgene (PreAnalytix, switzerland), RNA is extracted from cells. Common assay formats for ribonucleic acid hybridization include nuclear ligation assay, RT-PCR, RNase protection assay, northern blot, in situ hybridization and microarray assay. The circulating APOE mRNA can be detected using the method described in WO2012/177906, the entire contents of which are incorporated herein by reference.

In some embodiments, the expression level of GPR75 is determined using a nucleic acid probe. As used herein, the term "probe" refers to any molecule capable of selectively binding to a particular GPR75 nucleic acid or protein or fragment thereof. Probes may be synthesized by those skilled in the art or derived from suitable biological products. Probes may 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.

The isolated mRNA can be used in hybridization or amplification assays including, but not limited to, southern or northern analysis, polymerase Chain Reaction (PCR) analysis, and probe arrays. One method for determining RNA levels comprises contacting the isolated RNA with a nucleic acid molecule (probe) capable of hybridizing to GPR75 RNA. In one embodiment, the RNA is immobilized on a solid surface and contacted with the probe, for example, by transferring the isolated RNA from the gel to a membrane, such as a nitrocellulose membrane. In an alternative embodiment, in In a gene chip array, probes are immobilized on a solid surface and RNA is contacted with the probes. The skilled artisan can readily adapt known RNA detection methods for use in determining the level of GPR75 mRNA.

Alternative methods for determining the expression level of GPR75 in a sample involve, for example, nucleic acid amplification of mRNA in the sample or reverse transcriptase (to prepare cDNA) procedures such as by RT-PCR (experimental embodiments described by Mullis in 1987 in U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) proc.Natl. Sci. Usa 88:189-193), autonomous sequence replication (guard et al (1990) proc.Natl. Acad. Sci. Usa 87:1874-1878), transcriptional amplification system (Kwoh et al (1989) proc.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 method of nucleic acid amplification using techniques well known to those skilled in the art. These detection schemes are for nucleic acids if the nucleic acid molecules are present in very low amountsDetection of molecules is particularly useful. In a specific aspect of the disclosure, the expression level of GPR75 is determined by quantitative fluorescent RT-PCR (i.e., taqMan ^TM System), byLuciferase assays or by other methods recognized in the art for measuring GPR75 expression or mRNA levels.

The expression level of GPR75 mRNA can be monitored using membrane blotting (e.g., as used in hybridization assays, e.g., southern blotting, northern blotting, dot blotting, etc.) or microwells, sample tubes, gels, beads, or fibers (or any solid support comprising bound nucleic acid). 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. Determination of GPR75 expression levels may also involve the use of nucleic acid probes in solution.

In some embodiments, branched DNA (bDNA) analysis or real-time PCR (qPCR) is used to assess RNA expression levels. The use of such a PCR method is described and illustrated in the examples presented herein. These methods are also useful for the detection of GPR75 nucleic acid.

The level of GPR75 protein expression can be determined using any method known in the art for measuring protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high Performance Liquid Chromatography (HPLC), thin Layer Chromatography (TLC), super-diffusion chromatography, liquid or gel precipitant reactions, absorption spectroscopy, colorimetric analysis, spectrophotometric analysis, flow cytometry, immunodiffusion (single or secondary), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescent assay, electrochemiluminescent assay, and the like. Such assays may also be used in assays for proteins that indicate the presence or replication of GPR75 protein.

In some embodiments, the efficacy of the methods of the disclosure in the treatment of GPR 75-related diseases is assessed by a decrease in GPR75mRNA levels (e.g., by assessing blood GPR75 levels or otherwise).

In some embodiments, the efficacy of the methods of the disclosure in the treatment of GPR 75-related diseases is assessed by a decrease in GPR75mRNA levels (e.g., by assessing GPR75 levels in liver samples, by biopsy, or otherwise).

In some embodiments of the methods of the present disclosure, the RNAi agent is administered to the subject, thereby delivering the RNAi agent to a specific site in the subject. Inhibition of GPR75 expression can be assessed by measuring the level or change in level of GPR75mRNA or GPR75 protein in a sample from a specific site (e.g., liver cell) in a subject. In certain embodiments, the methods comprise clinically relevant inhibition of GPR75 expression, e.g., as demonstrated by clinically relevant results after treatment of a subject with an agent that reduces GPR75 expression.

As used herein, the term "detecting or determining the level of an analyte" is understood to mean performing this step to determine the presence or absence of a substance, e.g., protein, RNA. As used herein, a method of detecting or determining includes detecting or determining an analyte level that is lower than the detection level of the method used.

IX. methods of treating or preventing GPR75 related diseases

The disclosure also provides methods of reducing or inhibiting expression of GPR75 in a cell using an RNAi agent of the disclosure or a composition comprising an RNAi agent of the disclosure. The method comprises contacting the cell with a dsRNA of the present disclosure and maintaining the cell for a time sufficient to obtain degradation of mRNA transcripts of the GPR75 gene, thereby inhibiting expression of the GPR75 gene in the cell. The reduction in gene expression may be assessed by any method known in the art. For example, the reduction in GPR75 expression can be determined by determining the protein level of GPR75 protein using methods conventional to those of ordinary skill in the art, such as northern blotting, qRT-PCR; the reduction in GPR75 expression is determined by measuring the protein level of GPR75 protein using methods conventional to those of ordinary skill in the art, such as western blotting, immunological techniques.

In the methods of the present disclosure, the cells may be contacted in vitro or in vivo, i.e., the cells may be in a subject.

The cells suitable for treatment using the methods of the present disclosure may be any cells expressing the GPR75 gene. Cells suitable for use in the methods of the present disclosure may be mammalian cells, e.g., primate cells (e.g., human cells or non-human primate cells, e.g., monkey cells or chimpanzee cells), non-primate cells (e.g., rat cells or mouse cells). In one embodiment, the cell is a human cell, such as a human hepatocyte.

Expression of GPR75 in cells is inhibited by at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100%, i.e., below the detection level. In certain embodiments, GPR75 expression is inhibited by at least 50%.

The in vivo methods of the present disclosure may include administering to a subject a composition comprising an RNAi agent, wherein the RNAi agent comprises a nucleotide sequence complementary to at least a portion of an RNA transcript of a GPR75 gene of a mammal to be treated. When the organism to be treated is a mammal, such as a human, the composition may be administered by any means known in the art including, but not limited to, oral, intraperitoneal or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, intravitreal, subcutaneous, transdermal, airway (aerosol), intranasal, rectal and topical (including buccal and sublingual) administration. In certain embodiments, the composition is administered by intravenous infusion or injection. In certain embodiments, the composition is administered by subcutaneous injection. In certain embodiments, the composition is administered by intrathecal injection.

In some embodiments, administration is by depot injection (depot injection). Depot injections may release RNAi agents in a consistent manner over an extended period of time. Thus, depot injections may reduce the frequency of administration required to obtain a desired effect (e.g., a desired GPR75 inhibitory or therapeutic or prophylactic effect). Depot injections may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In certain embodiments, the depot injection is subcutaneous injection.

In one embodiment, the double stranded RNAi agent is administered by pulmonary system administration, e.g., intranasal administration or oral inhalation administration. Pulmonary system administration may be by syringe, dropper, aerosolization, or using a device such as a passive breath-driven or active power driven single/multi dose Dry Powder Inhaler (DPI) device.

The mode of administration may be selected depending on whether local or systemic treatment is desired or the area to be treated. The route of administration and the site of administration may be selected to enhance targeting.

In one aspect, the disclosure also provides a method of inhibiting the expression of the GPR75 gene in a mammal. The method comprises administering to a mammal a composition of dsrnas targeting the GPR75 gene in mammalian cells and maintaining the mammal for a time sufficient to obtain degradation of RNA transcripts of the GPR75 gene, thereby inhibiting expression of the GPR75 gene in the cells. The reduction in genome expression can be assessed by any method known in the art and described herein, such as qRT-PCR. The reduction in protein production can be assessed by any method known in the art and described herein, e.g., ELISA.

The present disclosure also provides methods of treating a subject in need thereof. The methods of treatment of the present disclosure include administering an RNAi agent of the present disclosure to a subject, e.g., a subject that would benefit from inhibiting GPR75 expression, in a therapeutically effective amount of an RNAi agent targeting the GPR75 gene or a pharmaceutical composition comprising an RNAi agent targeting the GPR75 gene.

Furthermore, the present disclosure provides methods of preventing, treating, or inhibiting the progression of a GPR 75-related disease or condition (e.g., a body weight disorder, such as obesity).

The method comprises administering to the subject a therapeutically effective amount of any RNAi agent, e.g., a dsRNA agent, or a pharmaceutical composition provided herein, thereby preventing, treating, or inhibiting the progression of a GPR 75-related disease or disorder in the subject.

The RNAi agents of the present disclosure can be administered as "free RNAi agents". The free RNAi agent is administered in the absence of the pharmaceutical composition. The naked RNAi agent can be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is Phosphate Buffered Saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted so that it is suitable for administration to a subject. In certain embodiments, the free RNAi agent can be formulated in water or physiological saline.

Alternatively, RNAi agents of the present disclosure can be administered as pharmaceutical compositions, e.g., dsRNA liposome formulations.

Subjects who would benefit from a reduction or inhibition of GPR75 gene expression are subjects with a GPR 75-related disease, subjects at risk of developing a GPR 75-related disease.

The disclosure also provides methods of using RNAi agents, or pharmaceutical compositions thereof, e.g., for treating subjects that would benefit from a reduction or inhibition of GPR75 expression, e.g., subjects having GPR 75-related diseases, in combination with other drugs or other therapeutic methods (e.g., with known drugs or known therapeutic methods, e.g., those currently used to treat such diseases). For example, in certain embodiments, RNAi agents targeting GPR75 are administered in combination with agents for treating GPR 75-related disorders, e.g., as described elsewhere herein or as known in the art. For example, additional agents and treatments suitable for treating a subject that would benefit from reduced GPR75 expression (e.g., a subject having a GPR 75-related disorder) may include agents currently used to treat symptoms of a GPR 75-related disorder.

Examples of additional therapeutic agents that may be used with the RNAi agents of the invention include, but are not limited to, diabetes therapeutic agents, diabetes complication therapeutic agents, cardiovascular disease therapeutic agents, antihyperlipidemic agents, antihypertensive or antihypertensive agents, antiobesity agents, non-alcoholic steatohepatitis (NASH) therapeutic agents, chemotherapeutic agents, immunotherapeutic agents, immunosuppressants, and the like. Such combination therapies may advantageously use lower doses of the administered therapeutic agent, thereby avoiding the possible toxicity or complications associated with various monotherapy.

Examples of agents for treating diabetes include insulin preparations (e.g., animal insulin preparations extracted from bovine or porcine pancreas; human insulin preparations synthesized by genetic engineering techniques or methods using microorganisms), insulin sensitivity enhancers and pharmaceutically acceptable salts, hydrates or solvates thereof (e.g., pioglitazone, traglizone, rosiglitazone, netoglitazone (netoglitazone), balaglitazone (balaglitazone), risaglitazone (risoglitazone), ti Sha Lieza (tesaglitazone), faglizar (faaglitazone), CLX-0921, R-483, NIP-221, NIP-223, DRF-2189, GW-7282TAK-559, T-131, RG-12525, LY-510929, DRF-519818, BMS-298585, DRF-2725, GW-1536, GI-262570, KRP-297, TZD18 (Merck), DRF-2655, etc.), alpha-glucosidase inhibitors (e.g., glibose), carboglizate (glazate), faglizate (glazate), praglizate (glabride), praglizate (glazin), acetonide, tolmide (glabride), tolmide (glabra), tolmide), etc. (tolalode) (e.g., tolalode) (e) (e.g., tolaloglinide, tolide) (e.g., 35, etc.) (glizamide) (e.g., glazin-84), glazin-84, methyl (glazin-35), etc.) (gla-35), phosphotyrosine phosphatase inhibitors (e.g., vanadate, etc.), and the like.

Examples of agents for treating diabetic complications include, but are not limited to, aldose reductase inhibitors (e.g., tolrestat), epalrestat (epalrestat), zenarestat (zenarestat), zopolrestat (zanarestat), milnacestat (ministratt), filarestat (fidareatat), SK-860, CT-112, etc.), neurotrophic factors (e.g., NGF, NT-3, BDNF, etc.), PKC inhibitors (e.g., LY-333531, etc.), advanced glycation end product (AGE) inhibitors (e.g., ALT946, p Ma Gua (pimagedine), pyridoxamine (pyradoxamine), benzoyl thiazole bromide (ALT 766), etc.), active oxygen quenchers (e.g., lipoic acid or derivatives thereof, bioflavonoids including flavones, isoflavones, procyanidins, anthocyanins, pynols, xanthophylls, lutein, vitamin E, coenzyme Q, etc.), vascular dilation (e.g., methicone, etc.), etc.

Antihyperlipidemic agents include, for example, statin-based compounds (e.g., pravastatin, simvastatin, lovastatin, atorvastatin, fluvastatin, rosuvastatin, etc.), squalene synthetase inhibitors, or fibrate (fibrate) compounds having a triglyceride lowering effect (e.g., fenofibrate, gemfibrozil, bezafibrate, clofibrate, xin Beite (sinfibrate), clinofibrate (clinofibrate), etc.), nicotinic acid, PCSK9 inhibitors, triglyceride lowering agents, or cholesterol chelators as cholesterol synthesis inhibitors.

Antihypertensives include, for example, angiotensin converting enzyme inhibitors (e.g., captopril, enalapril, delapril, benazepril, cilazapril, enalapril, fosinoprilat, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolapril, etc.) or angiotensin II antagonists (e.g., losartan, candesartan cilexetil, olmesartan, valsartan, telmisartan, irbesartan, tasosartan, telmisartan, li Pisha, fosaprepitant, etc.) or calcium channel blockers (e.g., amlodipine) or aspirin.

Non-alcoholic steatohepatitis (NASH) therapeutic agents include, for example, ursodeoxycholic acid (ursodiol), pioglitazone, orlistat, betaine, rosiglitazone.

Anti-obesity agents include, for example, central anti-obesity agents (e.g., dexfenfluramine, fenfluramine, phentermine, sibutramine, amphetamine, dexamphetamine, mazindol, phenylpropanolamine, clobetasol, etc.), gastrointestinal lipase inhibitors (e.g., orlistat, etc.), beta 3-adrenoreceptor agonists (e.g., CL-316243, SR-58111-A, UL-TG-307, SB-226552, AJ-9677, BMS-196085, etc.), peptide-based appetite suppressants (e.g., leptin, CNTF, etc.), cholecystokinin agonists (e.g., linetript (lintitre), FPL-15849, etc.), and the like.

Chemotherapeutic agents include, for example, alkylating agents (e.g., cyclophosphamide, ifosfamide, etc.), metabolic antagonists (e.g., methotrexate, 5-fluorouracil, etc.), anticancer antibiotics (e.g., mitomycin, doxorubicin, etc.), plant-derived anticancer agents (e.g., vincristine, vindesine, taxol, etc.), cisplatin, carboplatin, etoposide, etc. Among these, 5-fluorouracil derivatives such as fluoroiron (furvulon) and neofluoroiron (neoftulon) are preferable.

Immunotherapeutic agents include, for example, microbial or bacterial components (e.g., muramyl dipeptide derivatives, streptokinase (picibanil), etc.), polysaccharides having immunopotentiating activity (e.g., lentinan, schizophyllan (sizofilan), coriolus versicolor polysaccharide (krestin), etc.), cytokines obtained by genetic engineering techniques (e.g., interferons, interleukins (IL), etc.), colony stimulating factors (e.g., granulocyte colony stimulating factor, erythropoietin, etc.), and the like. In one embodiment, the immunotherapeutic agent is IL-1, IL-2, IL-12, and the like.

Immunosuppressants include, for example, calcineurin inhibitors/immunophilin modulators such as cyclosporine (mountain, gengraf, neomountain), tacrolimus (privater, FK 506), ASM 981, sirolimus (RAPA, rapamycin, rapamicine) or its derivative SDZ-RAD, glucocorticoids (prednisone, prednisolone, methylprednisolone, dexamethasone, etc.), purine synthesis inhibitors (mycophenolate, MMF, cellCept (R), azathioprine, cyclophosphamide), interleukin antagonists (basiliximab), daclizumab (daclizumab), deoxyarginin), lymphocyte depleting agents such as anti-thymocyte globulin (thymus globulin, lymphoglobulin), anti-CD 3 antibodies (OKT 3), etc.

The RNAi agent and the additional therapeutic agent can be administered simultaneously or in the same combination, e.g., by intrathecal administration, or the additional therapeutic agent can be administered as part of separate compositions or at separate times, or by other methods known in the art or described herein.

In one embodiment, the method comprises administering a composition described herein such that expression of the target GPR75 gene is reduced for at least one month. In some embodiments, expression is reduced for at least 2 months, 3 months, or 6 months.

In certain embodiments, administration comprises a rapid-acting dose administered at a higher frequency, e.g., at a frequency of once daily, twice weekly, once weekly, for an initial dosing period, e.g., 2 to 4 doses.

In some embodiments, RNAi agents useful in the methods and compositions described herein specifically target RNA (primary RNA or processing RNA) of the target GPR75 gene. Compositions and methods for inhibiting the expression of these genes using RNAi agents can be prepared and practiced as described herein.

Administration of dsRNA according to the methods of the present disclosure can result in a reduction in the severity, sign, symptom, or marker of GPR 75-related diseases or disorders in patients suffering from such diseases or disorders. In some embodiments, administration of the dsRNA results in a decrease in blood glucose levels in a subject having a GPR 75-related disorder. In other embodiments, administration of the dsRNA results in a decrease in blood lipid levels in a subject having a GPR 75-related disorder. "reduced" in this context means a statistically significant reduction or clinically significant reduction in such levels. The reduction may be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or about 100%.

Efficacy of treatment or prevention of a disease can be assessed, for example, by measuring disease progression, disease remission, symptom severity, pain relief, quality of life, the dosage of drug required to maintain therapeutic effect, the level of disease markers, or any other measurable parameter of a given disease being treated or pertinently prevented. It is fully within the ability of one skilled in the art to monitor the efficacy of treatment or prophylaxis by measuring any one of these parameters or any combination of parameters. It is fully within the ability of one skilled in the art to monitor the efficacy of treatment or prophylaxis by measuring any one of these parameters or any combination of parameters. With respect to administration of RNAi agents targeting GPR75, or pharmaceutical compositions thereof, an "effective against" GPR 75-related disorder is one in which administration in a clinically appropriate manner produces a beneficial effect on at least a portion of statistically significant patients, such as an improvement in symptoms, cure, disease reduction, longevity prolongation, improvement in quality of life, or other effects generally recognized as positive by physicians familiar with the treatment of GPR 75-related disorders and related etiologies.

The therapeutic or prophylactic effect is apparent when there is a statistically significant improvement in one or more parameters of the disease state, or when there is no exacerbation or development of the intended symptoms. For example, a favorable change in at least 10%, e.g., at least 20%, 30%, 40%, 50% or more, of the measurable parameters of the disease may be indicative of effective treatment. Experimental animal models of a given disease known in the art may also be used to determine the efficacy of a given RNAi agent drug or pharmaceutical formulation. When experimental animal models are used, the efficacy of the treatment is demonstrated when a statistically significant decrease in markers or symptoms is observed.

Alternatively, efficacy may be measured by a decrease in disease severity as determined by one of skill in the diagnostic arts based on clinically acceptable disease severity stratification criteria. Any positive change resulting in, for example, a reduction in disease severity, measured using a suitable scale, represents adequate treatment with an RNAi agent or RNAi agent formulation as described herein.

A therapeutic amount of dsRNA, for example, about 0.01mg/kg to about 200mg/kg, may be administered to a subject.

RNAi agents can be administered intrathecally by intravitreal injection or intravenous infusion over a period of time. In certain embodiments, treatment may be performed in a less frequent manner after the initial treatment regimen. Administration of RNAi agents can reduce GPR75 levels, for example, by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least about 99% or more in cells, tissues, blood, CSF samples, or other compartments of a patient. In one embodiment, administration of an RNAi agent can reduce GPR75 levels, for example, by at least 50% in cells, tissues, blood, CSF samples, or other compartments of a patient.

A smaller dose, e.g., 5% infusion response, may be administered to the patient and side effects, e.g., allergic reactions, monitored prior to administration of the full dose of RNAi agent. In another example, a patient may be monitored for undesired immunostimulatory effects, such as an increase in cytokine (e.g., TNF- α or INF- α) levels.

Alternatively, the RNAi agent can be administered orally, pulmonary, intravenously (i.e., by intravenous injection), or subcutaneously (i.e., by subcutaneous injection). One or more injections may be used to deliver a desired, e.g., monthly, dose of RNAi agent to a subject. The injection may be repeated over a period of time. The administration may be repeated periodically. In certain embodiments, treatment may be performed in a less frequent manner after the initial treatment regimen. Repeated dosing regimens may include periodic administration of a therapeutic amount of the RNAi agent, e.g., once a month or extended to once a quarter, twice a year, once a year. In certain embodiments, the RNAi agent is administered about once a month to about once a quarter (i.e., about once every three months).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test the RNAi agents and methods presented herein, suitable methods and materials are described below. All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

An informal sequence listing is presented herein and forms a part of the presented description.

The invention is further illustrated by the following examples, which should not be construed as limiting. All references, patents, and published patent applications cited in this specification are herein incorporated by reference in their entirety as well as the sequence listing and drawings.

Examples

Example 1: iRNA synthesis

Reagent source

If the source of the reagents is not specifically set forth herein, such reagents may be obtained from any molecular biological reagent provider, with quality/purity standards consistent with molecular biological applications.

SiRNA design

The selection of siRNA designs targeting the human G protein coupled receptor 75 (GPR 75) gene (human NCBI refseqID: NM-006794.4NCBI GeneID:1) was designed using custom R and Python scripts. Human NM-006794.4 REFSEQ mRNA is 2094 bases in length.

A detailed list of a set of unmodified siRNA sense and antisense strand sequences targeting GPR75 is shown in table 2.

A detailed list of a set of modified siRNA sense and antisense strand sequences targeting GPR75 is shown in table 3.

It should be understood that throughout this application, a duplex name without a decimal equivalent to a duplex name with a decimal that is only the lot number of the duplex. For example, AD-1230521 is equivalent to AD-1230521.

siRNA synthesis

The siRNA is synthesized and annealed using conventional methods known in the art. Briefly, siRNA sequences were synthesized on a 1. Mu. Mol scale by phosphoramidite chemical synthesis on a solid support using a Mermade 192 synthesizer (BioAutomation). The solid support is a controlled pore glass loaded with custom GalNAc ligands (3' -GalNAc conjugates), universal solid supports (AM Chemicals) or first nucleotides of interestAuxiliary synthesis reagents and standards 2-cyanoethyl phosphoramidite monomer (2 ' -deoxy-2 ' -fluoro, 2' -O-methyl, RNA, DNA) was obtained from Thermo-Fisher (Milwaukee, WI), honene (china), or Chemgenes (Wilmington, MA, USA). Additional phosphoramidite monomers are obtained from commercial suppliers, internally prepared, or obtained from various CMOs using custom synthesis. Acetonitrile or acetonitrile at 9:1: phosphoramidite was prepared at a concentration of 100mM in DMF and coupled using 5-ethylsulfanyl-1H-tetrazole (ETT, 0.25M in acetonitrile)The reaction time was 400 seconds. Phosphorothioate linkages were produced in anhydrous acetonitrile/pyridine (9:1 v/v) using 100mM 3- ((dimethylamino-methylene) amino) -3H-1,2, 4-dithiazole-3-thione (DDTT, available from Chemgenes (Wilmington, mass., USA)) solution. The oxidation reaction time was 5 minutes. All sequences were synthesized with the final DMT group removed ("DMT removal").

After completion of the solid phase synthesis, the solid supported oligonucleotides were treated with 300 μl of methylamine (40% aqueous solution) in 96-well plates at room temperature for about 2 hours to cleave from the solid support, followed by removal of all other base labile protecting groups. For sequences containing any natural ribonucleotide bond (2' -OH) protected by a tert-butyldimethylsilyl (TBDMS) group, a second deprotection step was performed using tea.3hf (triethylamine trihydrofluoride). 200. Mu.L of dimethyl sulfoxide (DMSO) and 300. Mu.L of TEA.3HF were added to each oligonucleotide solution in aqueous methylamine and the solution was incubated at 60℃for about 30 minutes. After the incubation, the plate was brought to room temperature and the crude oligonucleotides were precipitated by adding 1mL of 9:1 acetonitrile to ethanol or 1:1 ethanol to isopropanol. The plates were then centrifuged at 4 ℃ for 45 minutes and the supernatant carefully poured off with the aid of a multichannel pipette. The oligonucleotide pellet was resuspended in 20mM NaOAc and subsequently desalted using a HiTrap size exclusion column (5mL,GE Healthcare) on an Agilent LC system equipped with an autosampler, UV detector, conductivity meter and fraction collector. Desalted samples were collected in 96-well plates and then analyzed by LC-MS and UV spectrophotometry, respectively, to confirm the identity and quantification of the material.

Single strand duplex formation was performed on a Tecan liquid handling robot. In 96-well plates, sense and antisense single strands were mixed in equimolar ratio to achieve a final concentration of 10 μm in 1PBS, the plates were sealed, incubated at 100 ℃ for 10 minutes, after which they were allowed to slowly return to room temperature over a period of 2 to 3 hours. The concentration and identity of each duplex was confirmed and then used in an in vitro screening assay.

Example 2: in vitro screening of siRNA duplex

Cell culture and transfection

Cells are cultured according to standard methods and transfected with the iRNA duplex of interest. For example, primary Human Hepatocytes (PHH) are transfected by: in each single well of 384 well plates, 7.5 single Opti-MEM per well plus 0.1-M RNAiMAX (Invitrogen, carlsbad Calif. cat# 13778-150) was added to each siRNA duplex of 2.5 sb. The cells were then incubated for 15 minutes at room temperature. Then will contain about 1.5X10 ⁴ mu.L of medium for each cell was added to the siRNA mixture described above. Cells were incubated for 24 hours and then RNA purification was performed. Single dose experiments were performed at 10nM, 1nM and 0.1nM

Total RNA isolation Using DYNABEADS mRNA isolation kit

Total RNA isolation was performed using DYNABEADS. Briefly, cells were lysed in 10 μl of lysis/binding buffer containing 3 μl of beads per well and mixed on an electrostatic oscillator for 10 min. The washing step was performed automatically on Biotek EL406 using a magnetic plate support. The beads were washed once in buffer a (in 3 μl), once in buffer B, twice in buffer E, and a pipetting step between each two washes. After the last aspiration, all 12 μl of RT mix was added to each well, as described below.

cDNA Synthesis

For cDNA synthesis, 1.5. Mu.l of 10 x buffer, 0.6. Mu.l of 10 x dNTPs, 1.5. Mu.l of random primers, 0.75. Mu.l of reverse transcriptase, 0.75. Mu.l of RNase inhibitor and 9.9. Mu.l of H were used in each reaction ₂ The main mix of O was added to each well. The plates were sealed, shaken on an electrostatic oscillator for 10 minutes, and then incubated at 37 ℃ for 2 hours. Subsequently, the plate was shaken at 80℃for 8 minutes.

Real-time PCR

In each well of 384 well plates (Roche cat# 04887301001), 2. Mu.l of cDNA was added to a master mix containing 0.5. Mu.l of human GAPDH TaqMan probe (4326317E), 0.5. Mu.l of human GPR75, 2. Mu.l of nuclease free water and 5. Mu.l of Lightcycle 480 probe master mix (Roche cat# 04887301001). Real-time PCR was performed in the LightCycler480 real-time PCR system (Roche).

To calculate the relative fold change, the data were analyzed using the ΔΔct method and normalized to the assay performed with 10nM AD-1955 transfected cells or mock transfected cells. Calculation of IC using 4-parameter fitting model of XLFit ₅₀ And normalized to cells transfected with AD-1955 or mock transfected cells. The sense and antisense sequences of AD-1955 are: sense cuuagcaguacuucgadtsdt (SEQ ID NO: 13) and antisense ucgaagacuagcguaaagdtsdt (SEQ ID NO: 14).

In vitro dual luciferase and endogenous screening assays

The Hepa1-6 cells were transfected by: mu.L of siRNA duplex and 75ng of human GPR75 plasmid were added per well and 100.5 siRNA selection assay G plus 0.5 siRNA selection assay GuAAGdTsdT was used (Invitrogen, carlsbad Calif. cat# 13778-150) and then incubated for 15 minutes at room temperature. The mixture was then added to the cells, which were resuspended in 35 μl fresh complete medium. Transfected cells were incubated at 5% CO ₂ Is incubated at 37 ℃. Single dose experiments were performed at 10 nM.

24 hours after siRNA and psiCHECK2 plasmid transfection; firefly (transfection control) luciferase and Renilla (fusion with GPR75 target sequence) luciferase were measured. First, the medium is removed from the cells. Then by adding 75. Mu.L of the same medium volume to each wellLuciferase reagents and mixing to measure firefly luciferase activity. The mixture was incubated at room temperature for 30 minutes, and then luciferin (500 nm) was measured on Spectramax (Molecular Devices) to detect firefly luciferase signal. By adding 75. Mu.L of room temperature +.>Stop&Reagent(s)To measure renilla luciferase activity and incubating the plates for 10-15 minutes and then measuring luminescence again to determine renilla luciferase signal. / > Stop&The reagent quenches the firefly luciferase signal and maintains luminescence of the renilla luciferase reaction. siRNA activity was determined by normalizing the renilla (GPR 75) signal in each well relative to the firefly (control) signal. The magnitude of siRNA activity was then assessed relative to cells transfected with the same vector but not treated with siRNA or treated with non-targeted siRNA. All transfections were performed with n=4.

Total RNA isolation Using DYNABEADS mRNA isolation kit

RNA was isolated using an automated protocol on a Bio Tek-EL406 platform using DYNABEAD (Invitrogen, cat# 61012). Briefly, 70. Mu.L of lysis/binding buffer and 10. Mu.L of lysis buffer containing 3. Mu.L of magnetic beads were added to the cell-containing plates. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature, then the magnetic beads were captured and the supernatant removed. The RNA bound to the magnetic beads was then washed twice with 150. Mu.L of wash buffer A and once with wash buffer B. The beads were then washed with 150 μl of elution buffer, and the supernatant was recaptured and removed.

cDNA Synthesis Using ABI high Capacity cDNA reverse transcription kit (Applied Biosystems, foster City, calif., cat # 4368813)

In each reaction, a sample containing 1. Mu.L of 10 Xbuffer, 0.4. Mu.L of 25 XdNTP, 1. Mu.L of 10 Xrandom primer, 0.5. Mu.L of reverse transcriptase, 0.5. Mu.L of RNase inhibitor and 6.6. Mu.L of H was used ₂ 10. Mu.L of the master mix of O was added to the isolated RNA. The plates were sealed, mixed, then incubated on an electromagnetic shaker for 10 minutes at room temperature, then incubated for 2 hours at 37 ℃.

Real-time PCR

In each well of a 384 well plate (Roche cat# 04887301001), 2. Mu.L of cDNA was added to a master mix containing 0.5. Mu.L of human or mouse GAPDH TaqMan probe (ThermoFisher cat# 4352934E or 4351309) and 0.5. Mu.L of a suitable GPR75 probe (commercially available, e.g., from ThermoFisher) and 5. Mu.L of the Lightcycle 480 probe master mix (Roche cat# 04887301001). Real-time PCR was performed in the LightCycler480 real-time PCR system (Roche). Each duplex was tested (n=4) and the data normalized to cells transfected with non-targeted control siRNA. To calculate the relative fold change, the real-time data was analyzed using the ΔΔct method and normalized to the assay performed with cells transfected with non-targeted control siRNA.

Table 4 shows the results of in vitro screening in Hepa1-6 cells with subsets of dsRNA agents listed in tables 2 and 3.

Table 1 abbreviations for nucleotide monomers used in the nucleic acid sequence notation. It will be appreciated that when present in the oligonucleotide, these monomers are linked to each other by a 5'-3' -phosphodiester linkage; and it will be appreciated that when the nucleotide contains a 2' -fluoro modification, the fluoro group replaces the hydroxy group at that position in the parent nucleotide (i.e., it is a 2' -deoxy-2 ' -fluoro nucleotide).

TABLE 4 in vitro Single dose screening in Hepa1-6 cells

Example 3 in vivo screening of dsRNA duplex in mice

In vivo evaluation of siRNA molecules targeting the GPR75 gene identified from the in vitro studies described above was performed.

For example, the ability of siRNA molecules to reduce GPR75 expression in transgenic mice overexpressing human GPR75 can be assessed. Alternatively, or in addition, an animal model of a suitable weight disorder such as obesity may also be used. Some examples of useful models of weight disorders include leptin deficiency (ob/ob) mice, leptin receptor deficiency (db/db) mice, and non-obese diabetic (NOD) mice (King a.br J pharmacol.,2012,166 (3): 877-894); diet-induced C57BL/6J mouse model (Vedova MD, et al, nutr Metab insists.2016; 9:93-102); or a diet-induced ob/ob mouse model (Tolbol KS et al World J Gastroenterol 2018, 2:179). Many mouse models are available from Jackson laboratories or Charles River.

The ability of the selected dsRNA agents designed and analyzed in example 1 to reduce GPR75 expression levels and treat body weight disorders (e.g., obesity) in these animal models was evaluated.

Briefly, littermates are subcutaneously or intrathecally administered a single 0.1mg/kg, 1mg/kg, 10mg/kg or 30mg/kg dose of dsRNA agent or placebo of interest. Animals were monitored daily for body weight. Two weeks after administration, animals were sacrificed and blood and tissue samples were collected, including cerebral cortex, spinal cord, liver, spleen, and cervical lymph nodes. Uptake of dsRNA in liver cells and/or neuronal cells and expression levels of target genes in brain in treated mice were measured. The expression level of GPR75 was further assessed by mouse in situ hybridization. The body weight, blood glucose and blood lipid levels at the time of sacrifice were further assessed.

Example 4 in vivo screening of dsRNA duplex in obese mice

To evaluate the effect of GPR 75-targeted duplex on reducing GPR75 mRNA levels in vivo, diet-induced obese mice were administered a single 150 μg dose of duplex AD-1480250, AD-1481248, AD-1481278, or AD-1481773 or control by intraventricular injection on day 0. On day 21 post-administration, animals were sacrificed, cerebral cortex samples were collected, and GPR75 mRNA levels were quantified by qPCR as described above.

Table 5 provides the unmodified nucleotide sequence of the duplex and table 6 provides the modified nucleotide sequence of the duplex. The unmodified sense and antisense strand nucleotide sequences of the control duplex are: 5'-GGGAGUCAAAGUUCUGUUUGA-3' and 5' -UCAAACAGAACUUUGACUCCCAU-3. The modified sense and antisense strand nucleotide sequences of the control duplex are: 5 '-gssgag (Uhd) CfaAfAfGfuuguuusgsa-3' and 5 '-VPusCfsaaaCfaGfAfuuUfgAfcucccsasa-3'.

As shown in FIG. 1, administration of AD-1480250, AD-1481248, AD-1481278, or AD-1481773 resulted in an effective decrease in Gpr75 expression in the brain, ranging from 0.63 to 0.87-fold the expression levels observed in administration of control siRNA.

Informal sequence listing

SEQ ID NO:1

NM-006794.4 human G protein-coupled receptor 75 (GPR 75), mRNA

SEQ ID NO:2

NM-175490.4 mouse G protein-coupled receptor 75 (GPR 75), mRNA

SEQ ID NO:3

NM-001109096.1 rat G protein-coupled receptor 75 (GPR 75), mRNA

SEQ ID NO:4

NM-001204509.2 macaque G protein-coupled receptor 75 (GPR 75), mRNA

SEQ ID NO:5

Reverse complement of SEQ ID NO. 1

SEQ ID NO:6

Reverse complement of SEQ ID NO. 2

SEQ ID NO:7

Reverse complement of SEQ ID NO. 3

SEQ ID NO:8

Reverse complement of SEQ ID NO. 4

Equivalents (Eq.)

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, embodiments and methods equivalent to the specific embodiments and methods described herein. Such equivalent embodiments and methods are intended to be within the scope of the following claims.

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a G protein coupled receptor 75 (GPR 75) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having at least 90% nucleotide sequence identity to a portion of any one of SEQ ID NO:1 to SEQ ID NO:4 or to a portion of any one of SEQ ID NO:1 to SEQ ID NO:4, and wherein the sense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0, 1, 2 or 3 mismatches to a corresponding portion of any one of SEQ ID NO:5 to SEQ ID NO:8 or to a portion of any one of SEQ ID NO:5 to SEQ ID NO:8, and wherein the sense strand or the antisense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides having 0, 1, 2 or 3 mismatches to a portion of any one of SEQ ID NO: 8.

2. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a G protein-coupled receptor 75 (GPR 75) in a cell, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region of complementarity to a portion of an mRNA encoding a GPR75 gene (any one of SEQ ID NOs: 1 to 4), wherein each strand is independently 14 to 30 nucleotides in length; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

3. A double stranded RNAi agent for inhibiting expression of a G protein-coupled receptor 75 (GPR 75) gene in a cell, said double stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any antisense nucleotide sequence in any one of tables 2, 3, 5, and 6, wherein each strand is independently 14 to 30 nucleotides in length; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

4. The dsRNA agent of any one of claims 1-3, wherein the sense strand or the antisense strand is a sense strand or an antisense strand selected from the group consisting of any of the sense strands and antisense strands in any one of tables 2, 3, 5, and 6.

5. A double stranded RNAi agent for inhibiting expression of a G protein-coupled receptor 75 (GPR 75) gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence identical to SEQ ID NO:1, nucleotide 38-60, 50-72, 148-181, 153-175, 159-181, 228-250, 240-262, 341-363, 341-368, 346-368, 369-396, 369-391, 374-396, 388-410, 414-436, 424-461, 424-446, 424-451, 434-456, 439-461, 429-451, 457-504, 462-491, 482-504, 469-491, 457-479, 462-584, 475-497, 469-491, 509-537, 509-531, 515-537, 544-576, 544-566, 549-571, 580-607, 580-602, 773-607, 595-617, 615-647, 615-637, 620-642, 620-446, 625-647, 773-806, 773-795, 806-784, 872-872, 837-872, 1083-537, 515-537, 544-576, 544-566, 585-1088, 1085-1083-872, 1085, 1083-872-1088, and/or the like 1097-1119, 1238-1260, 1268-1290, 1284-1393, 1284-1306, 1292-1393, 1292-1314, 1292-1383, 1292-1314, 1301-1323, 1307-1383, 1307-1342, 1307-1329, 1313-1335, 1371-1393, 1351-1373, 1320-1342, 1336-1358, 1345-1367, 1351-1373, 1361-1383, 1366-1388, 1393-1415, 1422-1463, 1422-1444, 1441-1463, 1487-1526, 1487-1509, 1493-1526, 1493-1515, 1498-1520, 1504-1526, 1515-1571, 1515-1557, 1515-1543, 1515-1537, 1521-1543, 1530-1552, 1555-1545-1547, 1560-1562, 1569-1589, 1581-1586. 1564-1586, 1583-1629, 1583-1605, 1588-1610, 1595-1617, 1600-1629, 1600-1622, 1607-1629, 1624-1646, 1635-1657, 1672-1721, 1672-1710, 1677-1699, 1699-1721, 1672-1699, 1688-1710, 1672-1694, 1683-1705, 1693-1714, 1732-1754, 1744-1798, 1751-1773, 1758-1780, 177-1789, 1776-1798, 1790-1818, 1790-1812, 1796-1818, 1808-1856, 1808-1848, 1808-1830, 1826-1848, 1814-1836, 1819-1841, 1834-1856, 1877-2082, 77-1899, 1882-1882, 1882-1962, 1882-1965, 1782-1882, 181923-1923, 1923-1923 Nucleotide sequence comprising at least one nucleotide sequence of nucleotides corresponding to nucleotide sequence of at least 15 consecutive nucleotides from SEQ 15 of at least one of nucleotides 1887-1909, 1898-1920, 1903-1925, 1908-1930, 1913-1935, 1913-1950, 1921-1943, 1928-1950, 1933-1955, 1941-1963, 1946-1968, 1953-1985, 1953-2082, 1953-1975, 1938-1985, 1974-1996, 1974-2065, 1974-2082, 1974-2002, 1980-2002, 1985-2007, 1990-2012, 1990-2033, 1999-2021, 2005-2033, 2005-2027, 2011-2033, 2017-2039, 2025-2055, 2025-1967, 2043-2080, 2033-2065, 2033-2055, 2053-2055, 2054-2052, and 2054-2052; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

6. The dsRNA agent of any one of claims 1-5, wherein both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties.

7. The dsRNA agent of any one of claims 1-6, wherein the lipophilic moiety is conjugated to one or more positions in a double stranded region of the dsRNA agent.

8. The dsRNA agent of any one of claims 1-7, wherein the lipophilic moiety is conjugated by a linker or carrier.

9. The dsRNA agent of any one of claims 1-8, wherein the lipophilicity of the lipophilic moiety measured by logKow exceeds 0.

10. The dsRNA agent of any one of claims 1-9, wherein the hydrophobicity of the double stranded RNAi agent measured by unbound fraction in a plasma protein binding assay of the double stranded RNAi agent is greater than 0.2.

11. The dsRNA agent of claim 10, wherein the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin.

12. The dsRNA agent of any one of claims 1-11, wherein the dsRNA agent comprises at least one modified nucleotide.

13. The dsRNA agent of claim 12, wherein no more than 5 of the sense strand nucleotides and no more than 5 of the antisense strand nucleotides are unmodified nucleotides.

14. The dsRNA agent of claim 12, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand comprise modifications.

15. The dsRNA agent of any one of claims 12-14, wherein at least one of the modified nucleotides is selected from the group of: deoxynucleotides, 3 '-terminal deoxythymine (dT) nucleotides, 2' -O-methyl modified nucleotides, 2 '-fluoro-modified nucleotides, 2' -deoxymodified nucleotides, locked nucleotides, unlocked nucleotides, conformational nucleotides, restrictive ethyl nucleotides, abasic nucleotides, 2 '-amino modified nucleotides, 2' -O-allyl modified nucleotides, 2 '-C-alkyl modified nucleotides, 2' -methoxyethyl modified nucleotides, 2 '-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising a non-natural base, tetrahydropyran modified nucleotides, 1, 5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides comprising a 5' -phosphorothioate group, nucleotides comprising a 5 '-methylphosphonate group, nucleotides comprising a 5' phosphate or nucleotides comprising a 5 'phosphate mimetic, nucleotides comprising a vinylphosphonate, nucleotides comprising an adenosine-diol nucleic acid (GNA), nucleotides comprising a thymine-diol nucleic acid (GNA) S-isomer nucleotides, nucleotides comprising a 2' -methyl-phosphorothioate, 2 '-hydroxy-2' -methyl-phosphate, 2 '-hydroxy-2' -phosphoguanosine, 2 '-hydroxy-2' -phospho, 3 '-hydroxy-2' -phospho-nucleotide, 2 '-hydroxy-methyl-2' -phospho-oxo-acid, guanylate 2 '-O-hexadecyl-cytidine-3' -phosphate nucleotide, 2 '-O-hexadecyl-adenosine-3' -phosphate nucleotide, 2 '-O-hexadecyl-guanosine-3' -phosphate nucleotide, 2 '-O-hexadecyl-uridine-3' -phosphate nucleotide, 5 '-Vinyl Phosphonate (VP), 2' -deoxyadenosine-3 '-phosphate nucleotide, 2' -deoxycytidine-3 '-phosphate nucleotide, 2' -deoxyguanosine-3 '-phosphate nucleotide, 2' -deoxythymidine-3 '-phosphate nucleotide, 2' -deoxyuridine nucleotide, and terminal nucleotide linked to cholesterol derivative and a lauric acid didecarboxamide group; and combinations thereof.

16. The dsRNA agent of claim 15, wherein the modified nucleotide is selected from the group consisting of: 2 '-deoxy-2' -fluoro modified nucleotides, 2 '-deoxy modified nucleotides, 3' -terminal deoxythymine nucleotides (dT), locked nucleotides, abasic nucleotides, 2 '-amino modified nucleotides, 2' -alkyl modified nucleotides, morpholino nucleotides, phosphoramidates and nucleotides comprising a non-natural base.

17. The dsRNA agent of claim 15, wherein the modified nucleotide comprises a short sequence of 3' -terminal deoxythymine nucleotides (dT).

18. The dsRNA agent of claim 15, wherein the modification on the nucleotide is a 2' -O-methyl modification, a 2' -deoxy modification, a 2' fluoro modification, a 5' -Vinyl Phosphonate (VP) modification, and a 2' -O hexadecyl nucleotide modification.

19. The dsRNA agent of claim 15, further comprising at least one phosphorothioate internucleotide linkage.

20. The dsRNA agent of claim 19, wherein the dsRNA agent comprises 6 to 8 phosphorothioate internucleotide linkages.

21. The dsRNA agent of any one of claims 1-20, wherein each strand is no more than 30 nucleotides in length.

22. The dsRNA agent of any one of claims 1-21, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide.

23. The dsRNA agent of any one of claims 1-21, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

24. The dsRNA agent of any one of claims 1-23, wherein the double stranded region is 15 to 30 nucleotide pairs in length.

25. The dsRNA agent of claim 24, wherein the double stranded region is 17 to 23 nucleotide pairs in length.

26. The dsRNA agent of claim 24, wherein the double stranded region is 17 to 25 nucleotide pairs in length.

27. The dsRNA agent of claim 24, wherein the double stranded region is 23 to 27 nucleotide pairs in length.

28. The dsRNA agent of claim 24, wherein the double stranded region is 19 to 21 nucleotide pairs in length.

29. The dsRNA agent of claim 24, wherein the double stranded region is 21 to 23 nucleotide pairs in length.

30. The dsRNA agent of any one of claims 1-29, wherein each strand has 19 to 30 nucleotides.

31. The dsRNA agent of any one of claims 1-29, wherein each strand has 19 to 23 nucleotides.

32. The dsRNA agent of any one of claims 1-29, wherein each strand has 21 to 23 nucleotides.

33. The dsRNA agent of any one of claims 1-32, wherein one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand.

34. The dsRNA agent of claim 33, wherein the one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand by a linker or carrier.

35. The dsRNA agent of claim 34, wherein the internal positions comprise all positions except for two positions from the end of each end of the at least one strand.

36. The dsRNA agent of claim 34, wherein the internal positions comprise all positions except for three positions from the end of each end of the at least one strand.

37. The dsRNA agent of claims 34-36, wherein the internal position does not comprise a cleavage site region of the sense strand.

38. The dsRNA agent of claim 37, wherein the internal positions comprise all positions except positions 9 to 12 counted from the 5' end of the sense strand.

39. The dsRNA agent of claim 37, wherein the internal positions comprise all positions except positions 11 to 13 counted from the 3' end of the sense strand.

40. The dsRNA agent of claims 34-36, wherein the internal position does not comprise a cleavage site region of the antisense strand.

41. The dsRNA agent of claim 40, wherein the internal positions comprise all positions except positions 12 to 14 counted from the 5' end of the antisense strand.

42. The dsRNA agent of any one of claims 34-36, wherein the internal positions comprise all positions except positions 11 to 13 on the sense strand counted from the 3 'end and positions 12 to 14 on the antisense strand counted from the 5' end.

43. The dsRNA agent of any one of claims 1-42, wherein the one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of: positions 4 to 8 and positions 13 to 18 on the sense strand and positions 6 to 10 and positions 15 to 18 on the antisense strand counted from the 5' end of each strand.

44. The dsRNA agent of claim 43, wherein the one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of: positions 5, 6, 7, 15 and 17 on the sense strand and positions 15 and 17 on the antisense strand counted from the 5' end of each strand.

45. The dsRNA agent of claim 7, wherein the position in the double-stranded region does not comprise a cleavage site region of the sense strand.

46. The dsRNA agent of any one of claims 1-45, wherein the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand, or to position 16 of the antisense strand.

47. The dsRNA agent of claim 46, wherein the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1 or position 7 of the sense strand.

48. The dsRNA agent of claim 46, wherein the lipophilic moiety is conjugated to position 21, position 20 or position 15 of the sense strand.

49. The dsRNA agent of claim 46, wherein the lipophilic moiety is conjugated to position 20 or position 15 of the sense strand.

50. The dsRNA agent of claim 46, wherein the lipophilic moiety is conjugated to position 16 of the antisense strand.

51. The dsRNA agent of any one of claims 1-50, wherein the lipophilic moiety is an aliphatic compound, a cycloaliphatic compound, or a polycycloaliphatic compound.

52. The dsRNA agent of claim 51, wherein the lipophilic moiety is selected from the group consisting of: lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranyloxy hexanol, hexadecyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) cholanic acid, dimethoxytrityl, or phenoxazine.

53. The dsRNA agent of claim 52, wherein the lipophilic moiety comprises a saturated C4-C30 hydrocarbon chain or an unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of: hydroxyl, amine, carboxylic acid, sulfonate, phosphoric acid, thiol, azide, and alkyne.

54. The dsRNA agent of claim 53, wherein the lipophilic moiety comprises a saturated C6-C18 hydrocarbon chain or an unsaturated C6-C18 hydrocarbon chain.

55. The dsRNA agent of claim 53, wherein the lipophilic moiety comprises a saturated C16 hydrocarbon chain or an unsaturated C16 hydrocarbon chain.

56. The dsRNA agent of claim 55, wherein the saturated or unsaturated C16 hydrocarbon chain is conjugated to position 6 counted from the 5' end of the chain.

57. The dsRNA agent of any one of claims 1-54, wherein the lipophilic moiety is conjugated by a carrier that replaces one or more nucleotides in the double-stranded region or the one or more internal positions.

58. The dsRNA agent of claim 56, wherein the carrier is a cyclic group selected from the group consisting of: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, decalinyl; or the carrier is an acyclic moiety based on a serinol backbone or a diethanolamine backbone.

59. The dsRNA agent of any one of claims 1-54, wherein the lipophilic moiety is conjugated to the double stranded iRNA agent by a linker comprising an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, product of a click reaction, or carbamate.

60. The double stranded iRNA agent of any one of claims 1-59, wherein the lipophilic moiety is conjugated to a nucleobase, a sugar moiety or an internucleoside linkage.

61. The dsRNA agent of any one of claims 1-60, wherein the lipophilic moiety or targeting ligand is conjugated through a bio-cleavable linker selected from the group consisting of: DNA, RNA, disulfides, amides, galactosamine, glucosamine, glucose, galactose, mannose functionalized mono-or oligosaccharides, and combinations thereof.

62. The dsRNA agent of any one of claims 1-61, wherein the 3' end of the sense strand is protected by an end cap, said end cap being a cyclic group having an amine, said cyclic group being selected from the group consisting of: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, decalinyl.

63. The dsRNA agent of any one of claims 1-62, further comprising a targeting ligand that targets liver tissue.

64. The dsRNA agent of claim 63, wherein the targeting ligand is a GalNAc conjugate.

65. The dsRNA agent of any one of claims 1-64, further comprising:

Terminal chiral modification of a phosphorus atom of the linkage having the Sp configuration occurring at the first internucleotide linkage at the 3' -end of the antisense strand,

terminal chiral modification of a phosphorus atom of the linkage having Rp configuration, occurring at the first internucleotide linkage at the 5' -end of the antisense strand, and

terminal chiral modification of a phosphorus atom of a linkage having either the Rp configuration or the Sp configuration occurs at the first internucleotide linkage at the 5' end of the sense strand.

66. The dsRNA agent of any one of claims 1-64, further comprising:

terminal chiral modification of a phosphorus atom of the linkage having an Sp configuration occurring at the first and second internucleotide linkages of the 3' -end of said antisense strand,

67. The dsRNA agent of any one of claims 1-64, further comprising:

terminal chiral modification of a phosphorus atom of the linkage having an Sp configuration occurring at the first, second and third internucleotide linkages of the 3' -end of said antisense strand,

68. The dsRNA agent of any one of claims 1-64, further comprising:

terminal chiral modification of the phosphorus atom of the linkage with Rp configuration at the third internucleotide linkage at the 3' -end of the antisense strand,

69. The dsRNA agent of any one of claims 1-64, further comprising:

terminal chiral modification of a phosphorus atom of a linkage having an Sp configuration occurring at the first and second internucleotide linkages of the 3' -end of said antisense strand,

Terminal chiral modification of a phosphorus atom of the linkage having Rp configuration occurring at the first and second internucleotide linkages at the 5' end of the antisense strand, and

70. The dsRNA agent of any one of claims 1-69, further comprising a phosphate or phosphate mimetic at the 5' end of the antisense strand.

71. The dsRNA agent of claim 70, wherein the phosphate mimetic is 5' -Vinylphosphonate (VP).

72. The dsRNA agent of any one of claims 1-69, wherein the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair.

73. The dsRNA agent of any one of claims 1-69, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

74. A cell comprising the dsRNA agent of any one of claims 1-73.

75. A pharmaceutical composition for inhibiting GPR75 gene expression comprising the dsRNA agent of any one of claims 1-73.

76. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1-73 and a lipid formulation.

77. A device for oral inhalation administration comprising the dsRNA agent of any one of claims 1-73.

78. The device of claim 77, wherein said device is selected from the group consisting of a nebulizer, a metered dose inhaler, and a dry powder inhaler.

79. A method of inhibiting expression of a GPR75 gene in a cell, the method comprising:

(a) Contacting the cell with the dsRNA agent of any one of claims 1-73, or the pharmaceutical composition of claim 75 or 76, or the device of claim 77 or 78; and

(b) Maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the GPR75 gene, thereby inhibiting expression of the GPR75 gene in the cells.

80. The method of claim 79, wherein the cell is in a subject.

81. The method of claim 80, wherein the subject is a human.

82. The method of any one of claims 77-81, wherein expression of the GPR75 gene is inhibited by at least 50%.

83. A method of treating a subject having or at risk of developing a GPR 75-related disorder of G protein coupled receptor 75- (GPR 75-) comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1-73, or the pharmaceutical composition of claim 75 or 76, or the device of claim 77 or 78, thereby treating the subject.

84. The method of claim 83, wherein the subject is a human.

85. The method of claim 84, wherein the GPR 75-related disease is a weight disorder.

86. The method of claim 85, wherein the weight disorder is obesity.

87. The method of any one of claims 83-86, wherein treating comprises ameliorating at least one sign or symptom of the disease.

88. The method of any one of claims 83-87, wherein administering the dsRNA agent results in a decrease in blood glucose level of the subject.

89. The method of any one of claims 83-88, wherein the dsRNA agent is administered to the subject at a dose of about 0.01mg/kg to about 50 mg/kg.

90. The method of any one of claims 83-89, wherein the dsRNA agent is administered intrathecally to the subject.

91. The method of any one of claims 83-89, wherein the dsRNA agent is administered to the subject subcutaneously.

92. The method of any one of claims 83-91, further comprising administering to the subject an additional agent or therapy suitable for treating or preventing a GRP 75-related disorder.

93. The method of claim 92, wherein the additional therapeutic agent is selected from the group consisting of: a therapeutic agent for diabetes, a therapeutic agent for diabetic complications, a therapeutic agent for cardiovascular disease, an anti-hyperlipidemia agent, a antihypertensive or antihypertensive agent, an anti-obesity agent, a therapeutic agent for nonalcoholic steatohepatitis (NASH), a chemotherapeutic agent, an immunotherapeutic agent, an immunosuppressant, an anti-inflammatory agent, an anti-steatosis agent, and any combination of the foregoing agents.