Compositions and Methods for Silencing MYOC Expression

Abstract
The disclosure relates to double-stranded ribonucleic acid (dsRNA) compositions targeting MYOC, and methods of using such dsRNA compositions to alter (e.g., inhibit) expression of MYOC.
Description
SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 22, 2022 is named A108868_1490WO_SL.txt and is 596,467 bytes in size.


FIELD OF THE DISCLOSURE

The disclosure relates to the specific inhibition of the expression of the MYOC.


BACKGROUND

Glaucoma (e.g., primary open angle glaucoma (POAG)) is a major cause of irreversible vision loss in today's aging population. MYOC protein misfolding occludes its secretion from trabecular meshwork cells, leading to elevated eye pressure that in turn compresses and damages the optic nerve reducing its ability to transmit visual information to the brain, which results in vision loss. New treatments for glaucoma are needed.


SUMMARY

The present disclosure describes methods and iRNA compositions for modulating the expression of MYOC. In certain embodiments, expression of MYOC is reduced or inhibited using a MYOC-specific iRNA. Such inhibition can be useful in treating disorders related to 30 MYOC expression, such as ocular disorders (e.g., glaucoma, e.g., primary open angle glaucoma (POAG)).


Accordingly, described herein are compositions and methods that effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of MYOC, such as in a cell or in a subject (e.g., in a mammal, such as a human subject). Also described are compositions and methods for treating a disorder related to expression of MYOC, such as glaucoma (e.g., primary open angle glaucoma (POAG))


The iRNAs (e.g., dsRNAs) included in the compositions featured herein include an RNA strand (the antisense strand) having a region, e.g., a region that is 30 nucleotides or less, generally 19-24 nucleotides in length, that is substantially complementary to at least part of an mRNA transcript of MYOC (e.g., a human MYOC) (also referred to herein as a “MYOC-specific iRNA”). In some embodiments, the MYOC mRNA transcript is a human MYOC mRNA transcript, e.g., SEQ ID NO. 1 herein.


In some embodiments, the iRNA (e.g., dsRNA) described herein comprises an antisense strand having a region that is substantially complementary to a region of a human MYOC mRNA. In some embodiments, the human MYOC mRNA has the sequence NM_000261.2 (SEQ ID NO: 1). The sequence of NM_000261.2 is also herein incorporated by reference in its entirety. The reverse complement of SEQ ID NO: 1 is provided as SEQ ID NO: 2 herein.


In some aspects, the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of myocilin (MYOC), 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 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of a coding strand of human MYOC and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of a non-coding strand of human MYOC such that the sense strand is complementary to the at least 15 contiguous nucleotides in the antisense strand.


In some aspects, the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of MYOC, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 15 contiguous nucleotides in the antisense strand.


In some aspects, the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of MYOC, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from the antisense strand nucleotide sequence of duplex AD-1565804 and the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from the sense strand nucleotide sequence of duplex AD-1565804.


In some aspects, the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of MYOC, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from the antisense strand nucleotide sequence of duplex AD-1565837 and the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from the sense strand nucleotide sequence of duplex AD-1565837.


In some aspects, the present disclosure provides a human cell or tissue comprising a reduced level of MYOC mRNA or a level of MYOC protein as compared to an otherwise similar untreated cell or tissue, wherein optionally the cell or tissue is not genetically engineered (e.g., wherein the cell or tissue comprises one or more naturally arising mutations, e.g., MYOC mutations), wherein optionally the level is reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the human cell or tissue is a trabecular meshwork tissue, a ciliary body, a retinal pigment epithelium (RPE), a retinal tissue, an astrocyte, a pericyte, a Müller cell, a ganglion cell, an endothelial cell, a photoreceptor cell, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel.


The present disclosure also provides, in some aspects, a cell containing the dsRNA agent described herein.


In another aspect, provided herein is a human ocular cell, e.g., (a cell of the trabecular meshwork, a cell of the ciliary body, an RPE cell, a retinal cell, an astrocyte, a pericyte, a Müller cell, a ganglion cell, an endothelial cell, or a photoreceptor cell) comprising a reduced level of MYOC mRNA or a level of MYOC protein as compared to an otherwise similar untreated cell. In some embodiments, the level is reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.


In some aspects, the present disclosure also provides a pharmaceutical composition for inhibiting expression of a gene encoding MYOC, comprising a dsRNA agent described herein.


The present disclosure also provides, in some aspects, a method of inhibiting expression of MYOC in a cell, the method comprising:

    • (a) contacting the cell with the dsRNA agent described herein, or a pharmaceutical composition described herein; and
    • (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of MYOC, thereby inhibiting expression of the MYOC in the cell.


The present disclosure also provides, in some aspects, a method of inhibiting expression of MYOC in a cell, the method comprising:

    • (a) contacting the cell with the dsRNA agent described herein, or a pharmaceutical composition described herein; and
    • (b) maintaining the cell produced in step (a) for a time sufficient to reduce levels of MYOC mRNA, MYOC protein, or both of MYOC mRNA and protein, thereby inhibiting expression of the MYOC in the cell.


The present disclosure also provides, in some aspects, a method of inhibiting expression of MYOC in an ocular cell or tissue, the method comprising:

    • (a) contacting the cell or tissue with a dsRNA agent that binds MYOC; and
    • (b) maintaining the cell or tissue produced in step (a) for a time sufficient to reduce levels of MYOC mRNA, MYOC protein, or both of MYOC mRNA and protein, thereby inhibiting expression of MYOC in the cell or tissue.


The present disclosure also provides, in some aspects, a method of treating a subject diagnosed with MYOC-associated disorder comprising administering to the subject a therapeutically effective amount of the dsRNA agent described herein or a pharmaceutical composition described herein, thereby treating the disorder.


In any of the aspects herein, e.g., the compositions and methods above, any of the embodiments herein (e.g., below) may apply.


In some embodiments, the coding strand of human MYOC has the sequence of SEQ ID NO: 1. In some embodiments, the non-coding strand of human MYOC has the sequence of SEQ ID NO: 2.


In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.


In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 17 contiguous nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.


In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 19 contiguous nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.


In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 21 contiguous nucleotides in the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.


In some embodiments, the portion of the sense strand is a portion within a sense strand in any one of Tables 2A and 2B.


In some embodiments, the portion of the antisense strand is a portion within an antisense strand in any one of Tables 2A and 2B.


In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A and 2B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A and 2B that corresponds to the antisense sequence.


In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A and 2B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A and 2B that corresponds to the antisense sequence.


In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A and 2B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A and 2B that corresponds to the antisense sequence.


In some embodiments, the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A and 2B. In some embodiments, the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A and 2B that corresponds to the antisense sequence.


In some embodiments, the sense strand of the dsRNA agent is at least 23 nucleotides in length, e.g., 23-30 nucleotides in length.


In some embodiments, at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties. In some embodiments, the lipophilic moiety is conjugated to one or more positions in the double stranded region of the dsRNA agent. In some embodiments, the lipophilic moiety is conjugated via a linker or carrier. In some embodiments, lipophilicity of the lipophilic moiety, measured by logKow, exceeds 0.


In some embodiments, the hydrophobicity of the double-stranded RNAi agent, measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent, exceeds 0.2. In some embodiments, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.


In some embodiments, the dsRNA agent comprises at least one modified nucleotide. In some embodiments, no more than five of the sense strand nucleotides and not more than five of the nucleotides of the antisense strand are unmodified nucleotides. In some embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.


In some embodiments, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a 5′-phosphate mimic, a glycol modified nucleotide, and a 2′-O—(N-methylacetamide) modified nucleotide; and combinations thereof. In some embodiments, no more than five of the sense strand nucleotides and not more than five of the nucleotides of the antisense strand include modifications other than 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, unlocked nucleic acids (UNA) or glycerol nucleic acid (GNA).


In some embodiments, the dsRNA comprises a non-nucleotide spacer (wherein optionally the non-nucleotide spacer comprises a C3-C6 alkyl) between two of the contiguous nucleotides of the sense strand or between two of the contiguous nucleotides of the antisense strand.


In some embodiments, each strand is no more than 30 nucleotides in length. In some embodiments, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In some embodiments, at least one strand comprises a 3′ overhang of at least 2 nucleotides. In some embodiments, at least one strand comprises a 3′ overhang of 2 nucleotides.


In some embodiments, the double stranded region is 15-30 nucleotide pairs in length. In some embodiments, the double stranded region is 17-23 nucleotide pairs in length. In some embodiments, the double stranded region is 17-25 nucleotide pairs in length. In some embodiments, the double stranded region is 23-27 nucleotide pairs in length. In some embodiments, the double stranded region is 19-21 nucleotide pairs in length. In some embodiments, the double stranded region is 21-23 nucleotide pairs in length. In some embodiments, each strand has 19-30 nucleotides. In some embodiments, each strand has 19-23 nucleotides. In some embodiments, each strand has 21-23 nucleotides.


In some embodiments, the agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminus of one strand. In some embodiments, the strand is the antisense strand. In some embodiments, the strand is the sense strand.


In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand. In some embodiments, the strand is the antisense strand. In some embodiments, the strand is the sense strand.


In some embodiments, each of the 5′- and 3′-terminus of one strand comprises a phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the strand is the antisense strand.


In some embodiments, the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.


In some embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.


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


In some embodiments, the internal positions include all positions except the terminal two positions from each end of the at least one strand. In some embodiments, the internal positions include all positions except the terminal three positions from each end of the at least one strand. In some embodiments, the internal positions exclude a cleavage site region of the sense strand. In some embodiments, the internal positions include all positions except positions 9-12, counting from the 5′-end of the sense strand. In some embodiments, the internal positions include all positions except positions 11-13, counting from the 3′-end of the sense strand. In some embodiments, the internal positions exclude a cleavage site region of the antisense strand. In some embodiments, the internal positions include all positions except positions 12-14, counting from the 5′-end of the antisense strand. In some embodiments, the internal positions include all positions except positions 11-13 on the sense strand, counting from the 3′-end, and positions 12-14 on the antisense strand, counting from the 5′-end.


In some embodiments, the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5′end of each strand. In some embodiments, the one or more lipophilic moieties are conjugated to one or more of the 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, counting from the 5′-end of each strand.


In some embodiments, the positions in the double stranded region exclude a cleavage site region of the sense strand.


In some embodiments, 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 position 16 of the antisense strand. In some embodiments, the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 21, position 20, or position 15 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 20 or position 15 of the sense strand. In some embodiments, the lipophilic moiety is conjugated to position 16 of the antisense strand. In some embodiments, the lipophilic moiety is conjugated to position 6, counting from the 5′-end of the sense strand.


In some embodiments, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound. In some embodiments, the lipophilic moiety is selected from the group consisting of lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-((oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.


In some embodiments, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) or the double stranded region. In some embodiments, 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, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone.


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


In some embodiments, the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.


In some embodiments, the lipophilic moiety or targeting ligand is conjugated via a bio-cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.


In some embodiments, the 3′ end of the sense strand is protected via an end cap which is 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, and decalinyl.


In some embodiments, the dsRNA agent further comprises a targeting ligand, e.g., a ligand that targets an ocular tissue or a liver tissue. In some embodiments, the ocular tissue is a trabecular meshwork tissue, a ciliary body, a retinal tissue, a retinal pigment epithelium (RPE) or choroid tissue, e.g., a choroid vessel.


In some embodiments, the ligand is conjugated to the sense strand. In some embodiments, the ligand is conjugated to the 3′ end or the 5′ end of the sense strand. In some embodiments, the ligand is conjugated to the 3′ end of the sense strand.


In some embodiments, the ligand comprises N-acetylgalactosamine (GalNAc). In some embodiments, the targeting ligand comprises one or more GalNAc conjugates or one or more GalNAc derivatives. In some embodiments, the ligand is one or more GalNAc conjugates or one or more GalNAc derivatives are attached through a monovalent linker, or a bivalent, trivalent, or tetravalent branched linker. In some embodiments, the ligand is




embedded image


In some embodiments, the dsRNA agent is conjugated to the ligand as shown in the following schematic




embedded image


wherein X is O or S. In some embodiments, the X is O.


In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occurring at the first internucleotide linkage at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration.


In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occurring at the first and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.


In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, second and third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.


In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.


In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.


In some embodiments, the dsRNA agent further comprises a phosphate or phosphate mimic at the 5′-end of the antisense strand. In some embodiments, the phosphate mimic is a 5′-vinyl phosphonate (VP).


In various embodiments of the aforementioned dsRNA agents, the dsRNA agent targets a hotspot region of an mRNA encoding MYOC.


In another aspect, the present invention provides a dsRNA agent that targets a hotspot region of a myocilin (MYOC) mRNA.


In some embodiments, a cell described herein, e.g., a human cell, was produced by a process comprising contacting a human cell with the dsRNA agent described herein.


In some embodiments, a pharmaceutical composition described herein comprises the dsRNA agent and a lipid formulation.


In some embodiments (e.g., embodiments of the methods described herein), the cell is within a subject. In some embodiments, the subject is a human. In some embodiments, the level of MYOC mRNA is inhibited by at least 50%. In some embodiments, the level of MYOC protein is inhibited by at least 50%. In some embodiments, the expression of MYOC is inhibited by at least 50%. In some embodiments, inhibiting expression of MYOC decreases the MYOC protein level in a biological sample (e.g., an aqueous ocular fluid sample) from the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, inhibiting expression of MYOC gene decreases the MYOC mRNA level in a biological sample (e.g., an aqueous ocular fluid sample) from the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.


In some embodiments, the subject has been diagnosed with a MYOC-associated disorder.


In some embodiments, the subject meets at least one diagnostic criterion for a MYOC-associated disorder. In some embodiments, the MYOC associated disorder is glaucoma. In some embodiments, the MYOC associated disorder is primary open angle glaucoma (POAG).


In some embodiments, the ocular cell or tissue is a trabecular meshwork tissue, a ciliary body, RPE, a retinal cell, an astrocyte, a pericyte, a Müller cell, a ganglion cell, an endothelial cell, a photoreceptor cell, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel.


In some embodiments, the MYOC-associated disorder is a glaucoma. In some embodiments, the glaucoma is caused by or associated with an elevated eye pressure. In some embodiments, the glaucoma primary open angle glaucoma (POAG)).


In some embodiments, treating comprises amelioration of at least one sign or symptom of the disorder. In some embodiments, the at least one sign or symptom includes a measure of one or more of optic nerve damage, vision loss, tunnel vision, blurred vision, eye pain or presence, level, or activity of MYOC (e.g., MYOC gene, MYOC mRNA, or MYOC protein).


In some embodiments, a level of the MYOC that is higher than a reference level is indicative that the subject has glaucoma. In some embodiments, treating comprises prevention of progression of the disorder. In some embodiments, the treating comprises one or more of (a) inhibiting or reducing the expression or activity of MYOC; (b) reducing the level of misfolded MYOC protein; (c) reducing trabecular meshwork cell death; (d) decreasing intraocular pressure; or (e) increasing visual acuity.


In some embodiments, the treating results in at least a 30% mean reduction from baseline of MYOC mRNA in the trabecular meshwork tissue, ciliary body, retina, RPE, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel. In some embodiments, the treating results in at least a 60% mean reduction from baseline of MYOC mRNA in the trabecular meshwork tissue, ciliary body, retina, RPE, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel. In some embodiments, the treating results in at least a 90% mean reduction from baseline of MYOC mRNA in the trabecular meshwork tissue, ciliary body, retina, RPE, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel.


In some embodiments, after treatment the subject experiences at least an 8-week duration of knockdown following a single dose of dsRNA as assessed by MYOC protein in the retina. In some embodiments, treating results in at least a 12-week duration of knockdown following a single dose of dsRNA as assessed by MYOC protein in the retina. In some embodiments, treating results in at least a 16-week duration of knockdown following a single dose of dsRNA as assessed by MYOC protein in the retina.


In some embodiments, the subject is human.


In some embodiments, the dsRNA agent is administered at a dose of about 0.01 mg/kg to about 50 mg/kg.


In some embodiments, the dsRNA agent is administered to the subject intraocularly. In some embodiments, the intraocular administration comprises intravitreal administration, e.g., intravitreal injection; transscleral administration, e.g., transscleral injection; subconjunctival administration, e.g., subconjunctival injection; retrobulbar administration, e.g., retrobulbar injection; intracameral administration, e.g., intracameral injection, or subretinal administration, e.g., subretinal injection.


In some embodiments, the dsRNA agent is administered to the subject intravenously. In some embodiments, the dsRNA agent is administered to the subject topically.


In some embodiments, a method described herein further comprises measuring a level of MYOC (e.g., MYOC gene, MYOC mRNA, or MYOC protein) in the subject. In some embodiments, measuring the level of MYOC in the subject comprises measuring the level of MYOC protein in a biological sample from the subject (e.g., an aqueous ocular fluid sample). In some embodiments, a method described herein further comprises performing a blood test, an imaging test, or an aqueous ocular fluid biopsy (e.g., an aqueous humor tap).


In some embodiments, a method described herein further measuring a level of MYOC (e.g., MYOC gene, MYOC mRNA, or MYOC protein) in the subject is performed prior to treatment with the dsRNA agent or the pharmaceutical composition. In some embodiments, upon determination that a subject has a level of MYOC that is greater than a reference level, the dsRNA agent or the pharmaceutical composition is administered to the subject. In some embodiments, measuring level of MYOC in the subject is performed after treatment with the dsRNA agent or the pharmaceutical composition.


In some embodiments, a method described herein further comprises treating the subject with a therapy suitable for treatment or prevention of a MYOC-associated disorder, e.g., wherein the therapy comprises laser trabeculoplasty surgery, trabeculectomy surgery, a minimally invasive glaucoma surgery, or placement of a drainage tube in the eye. In some embodiments, a method described herein further comprises administering to the subject an additional agent suitable for treatment or prevention of a MYOC-associated disorder. In some embodiments, the additional agent comprises a carbonic anhydrase inhibitor, a prostaglandin, a beta blocker, an alpha-adrenergic agonist, a carbonic anhydrase inhibitor, a Rho kinase inhibitor, or a cholinergic agent, or any combination thereof. In some embodiments, the additional agent comprises an oral medication or an eye drop.


All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.





BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several features of the present disclosure.


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



FIG. 1A shows the experimental setup for testing the effect of human MYOC siRNA AD-822899 on intraocular pressure (IOP) in SAM mice comprising a humanized MY-OC locus comprising a Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with AAV2.Y3F-SAM-g4.



FIG. 1B shows intraocular pressure (IOP) in SAM mice comprising a humanized MYOC locus comprising a Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with AAV2.Y3F-SAM-g4 or nothing (naïve), and in which the AAV2.Y3F-SAM-g4-treated mice were subsequently treated with either human MYOC siRNA or a control luciferase siRNA.



FIG. 2A shows the experimental setup for testing the effect of human MYOC siRNAs AD-822899, AD-1565804, AD-1565837, AD-1193175, and AD-1565503 on intraocular pressure (IOP) in SAM mice comprising a humanized MYOC locus comprising a Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with LV-SAM-g4.



FIG. 2B shows intraocular pressure (IOP) in SAM mice comprising a humanized MYOC locus comprising a Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with LV-SAM-g4 or PBS, and in which the LV-SAM-g4-treated mice were subsequently treated with humanMYOC siRNA AD-822899, AD-1565804, AD-1565837, AD-1193175, or AD-1565503.



FIG. 2C shows qPCR results showing the percentage of human MYOC mRNA expression relative to Gapdh in SAM mice comprising a humanized MYOC locus comprising a Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with LV-SAM-g4 or PBS, and in which the LV-SAM-g4-treated mice were subsequently treated with human MYOC siRNA AD-822899, AD-1565804, AD-1565837, AD-1193175, or AD-1565503.



FIG. 2D shows RNASCOPE® analysis of human MYOC mRNA expression in eyes from SAM mice comprising a humanized MYOC locus comprising a Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with LV-SAM-g4 or PBS, and in which the LV-SAM-g4-treated mice were subsequently treated with human MYOC siRNA AD-1565804 or AD-1565837.



FIG. 3A shows the experimental setup for testing the effect of human MYOC siRNAs AD-1565804 or AD-1565837 on intraocular pressure (IOP) in SAM mice comprising a humanized MYOC locus comprising a Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with LV-SAM-g4.



FIG. 3B shows intraocular pressure (IOP) in SAM mice comprising a humanized MYOC locus comprising a Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with LV-SAM-g4 or PBS, and in which the LV-SAM-g4-treated mice were subsequently treated with human MYOC siRNA AD-1565804 or AD-1565837.



FIG. 3C shows qPCR results showing the percentage of human MYOC mRNA expression relative to Gapdh in SAM mice comprising a humanized MYOC locus comprising a Y437H mutation (SAM-MYOC mice, homozygous for each allele) treated with LV-SAM-g4 or PBS, and in which the LV-SAM-g4-treated mice were subsequently treated with human MYOC siRNA AD-1565804 or AD-1565837.



FIG. 4 shows the percent MYOC protein remaining relative to PBS in the TM at day 85 after dosing with either AD-1565837 duplex or AD-1565804 duplex in the non-human primate (NHP) model. Results are shown for two different MYOC antibodies, R&D and Abnova.



FIG. 5 depicts the percent MYOC protein remaining in the aqueous humor relative to pre-dose at days −35, 22, 50, and 85 after dosing with either AD-1565837 duplex or AD-1565804 duplex in the non-human primate (NHP) model.



FIG. 6A shows the percent MYOC protein remaining relative to PBS in the vitreous humor and ciliary body at day 85 after dosing with either AD-1565837 duplex or AD-1565804 duplex in the non-human primate (NHP) model.



FIG. 6B shows the percent MYOC protein remaining relative to PBS in the iris and sclera at day 85 after dosing with either AD-1565837 duplex or AD-1565804 duplex in the non-human primate (NHP) model.



FIG. 7 depicts the percent MYOC mRNA remaining in the aqueous humor relative to PBS at day 85 after dosing with either AD-1565837 duplex or AD-1565804 duplex in the non-human primate (NHP) model.





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


DETAILED DESCRIPTION

iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Described herein are iRNAs and methods of using them for modulating (e.g., inhibiting) the expression of MYOC. Also provided are compositions and methods for treatment of disorders related to MYOC expression, such as glaucoma (e.g., primary open angle glaucoma (POAG)).


Human MYOC is a secreted glycoprotein of approximately 57 kDa that regulates the activation of several signaling pathways in adjacent cells to control different processes including cell adhesion, cell-matrix adhesion, cytoskeleton organization, and cell migration. MYOC is typically expressed and secreted by a variety of tissues including the retina and the structures involved in aqueous humor regulation such as the trabecular meshwork tissue and the ciliary body. Aberrant MYOC is associated with glaucoma, for instance primary open angle glaucoma (POAG). Without wishing to be bound by theory, aberrant MYOC may exacerbate the pathogenesis of glaucoma, e.g., by impeding the drainage of aqueous humor consequently leading to an increased intraocular pressure.


The following description discloses how to make and use compositions containing iRNAs to modulate (e.g., inhibit) the expression of MYOC, as well as compositions and methods for treating disorders related to expression of MYOC.


In some aspects, pharmaceutical compositions containing MYOC iRNA and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of MYOC, and methods of using the pharmaceutical compositions to treat disorders related to expression of MYOC (e.g., glaucoma, e.g., primary open angle glaucoma (POAG)) are featured herein.


I. DEFINITIONS

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.


The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary from, for example, between 1% and 15% of the stated number or numerical range.


The terms “or more” and “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 17 nucleotides of a 20 nucleotide nucleic acid molecule” means that 17, 18, 19, or 20 nucleotides have the indicated property. When “at least” is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.


As used herein, “or less” and “no more than” are understood as including the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with mismatches to a target site of “no more than 2 nucleotides” has a 2, 1, or 0 mismatches. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.


As used herein, “less than” is understood as not including the value adjacent to the phrase and including logical lower values or integers, as logical from context, to zero. For example, a duplex with mismatches to a target site of “less than 3 nucleotides” has 2, 1, or 0 mismatches. When “less than” is present before a series of numbers or a range, it is understood that “less than” can modify each of the numbers in the series or range.


As used herein, “more than” is understood as not including the value adjacent to the phrase and including logical higher values or integers, as logical from context, to infinity. For example, a duplex with mismatches to a target site of “more than 3 nucleotides” has 4, 5, 6, or more mismatches. When “more than” is present before a series of numbers or a range, it is understood that “more than” can modify each of the numbers in the series or range.


As used herein, “up to” as in “up to 10” is understood as up to and including 10, i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.


Ranges provided herein are understood to include all individual integer values and all subranges within the ranges.


The terms “activate,” “enhance,” “up-regulate the expression of,” “increase the expression of,” and the like, in so far as they refer to a MYOC gene, herein refer to the at least partial activation of the expression of a MYOC gene, as manifested by an increase in the amount of MYOC mRNA, which may be isolated from or detected in a first cell or group of cells in which a MYOC gene is transcribed and which has or have been treated such that the expression of a MYOC gene is increased, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).


In some embodiments, expression of a MYOC gene is activated by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA as described herein. In some embodiments, a MYOC gene is activated by at least about 60%, 70%, or 80% by administration of an iRNA featured in the disclosure. In some embodiments, expression of a MYOC gene is activated by at least about 85%, 90%, or 95% or more by administration of an iRNA as described herein. In some embodiments, the MYOC gene expression is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold or more in cells treated with an iRNA as described herein compared to the expression in an untreated cell. Activation of expression by small dsRNAs is described, for example, in Li et al., 2006 Proc. Natl. Acad Sci. U.S.A. 103:17337-42, and in US2007/0111963 and US2005/226848, each of which is incorporated herein by reference.


The terms “silence,” “inhibit expression of,” “down-regulate expression of,” “suppress expression of,” and the like, in so far as they refer to MYOC, herein refer to the at least partial suppression of the expression of MYOC, as assessed, e.g., based on MYOC mRNA expression, MYOC protein expression, or another parameter functionally linked to MYOC expression. For example, inhibition of MYOC expression may be manifested by a reduction of the amount of MYOC mRNA which may be isolated from or detected in a first cell or group of cells in which MYOC is transcribed and which has or have been treated such that the expression of MYOC is inhibited, as compared to a control. The control may be a second cell or group of cells substantially identical to the first cell or group of cells, except that the second cell or group of cells have not been so treated (control cells). The degree of inhibition is usually expressed as a percentage of a control level, e.g.,










(

mRNA


in


control


cells

)

-

(

mRNA


in


treated


cells

)



(

mRNA


in


control


cells

)


·
100


%




Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to MYOC expression, e.g., the amount of protein encoded by a MYOC gene. The reduction of a parameter functionally linked to MYOC expression may similarly be expressed as a percentage of a control level. In principle, MYOC silencing may be determined in any cell expressing MYOC, either constitutively or by genomic engineering, and by any appropriate assay.


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


The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence.


As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. In some embodiments, the region of complementarity comprises 0, 1, or 2 mismatches.


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


The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.


As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.


Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.


Complementary sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but are not limited to, G:U Wobble or Hoogstein base pairing.


The terms “complementary,” “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.


As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding a MYOC protein). For example, a polynucleotide is complementary to at least a part of a MYOC mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding MYOC. The term “complementarity” refers to the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.


As used herein, the term “region of complementarity” refers to the region of one nucleotide sequence agent that is substantially complementary to another sequence, e.g., the region of a sense sequence and corresponding antisense sequence of a dsRNA, or the antisense strand of an iRNA and a target sequence, e.g., a MYOC nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the antisense strand of the iRNA. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′- or 3′-terminus of the iRNA agent.


“Contacting,” as used herein, includes directly contacting a cell, as well as indirectly contacting a cell. For example, a cell within a subject may be contacted when a composition comprising an iRNA is administered (e.g., intraocularly, topically, or intravenously) to the subject.


“Introducing into a cell,” when referring to an iRNA, means facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; an iRNA may also be “introduced into a cell,” wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be by a β-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or known in the art. As used herein, a “disorder related to MYOC expression,” a “disease related to MYOC expression,” a “pathological process related to MYOC expression,” “a MYOC-associated disorder,” “a MYOC-associated disease,” or the like includes any condition, disorder, or disease in which MYOC expression is altered (e.g., decreased or increased relative to a reference level, e.g., a level characteristic of a non-diseased subject). In some embodiments, MYOC expression is decreased. In some embodiments, MYOC expression is increased. In some embodiments, the decrease or increase in MYOC expression is detectable in a tissue sample from the subject (e.g., in an aqueous ocular fluid sample). The decrease or increase may be assessed relative the level observed in the same individual prior to the development of the disorder or relative to other individual(s) who do not have the disorder. The decrease or increase may be limited to a particular organ, tissue, or region of the body (e.g., the eye). MYOC-associated disorders include, but are not limited to, glaucoma (e.g., primary open angle glaucoma (POAG)).


The term “glaucoma”, as used herein, means any disease of the eye that is caused by or associated with damage to the optic nerve. In some embodiments, the glaucoma is associated with elevated intraocular pressure. In some embodiments, the glaucoma is asymptomatic. In other embodiments, the glaucoma has one or more symptoms, e.g., loss of peripheral vision, tunnel vision, or blind spots. A non-limiting example of glaucoma that is treatable using methods provided herein is primary open angle glaucoma (POAG).


The term “double-stranded RNA,” “dsRNA,” or “siRNA” as used herein, refers to an iRNA that includes an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having “sense” and “antisense” orientations with respect to a target RNA. The duplex region can be of any length that permits specific degradation of a desired target RNA, e.g., through a RISC pathway, but will typically range from 9 to 36 base pairs in length, e.g., 15-30 base pairs in length. Considering a duplex between 9 and 36 base pairs, the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNAs generated in the cell by processing with Dicer and similar enzymes are generally in the range of 19-22 base pairs in length. One strand of the duplex region of a dsDNA comprises a sequence that is substantially complementary to a region of a target RNA. The two strands forming the duplex structure can be from a single RNA molecule having at least one self-complementary region, or can be formed from two or more separate RNA molecules. Where the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a “hairpin loop”) between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. In some embodiments, the two strands are connected covalently by means other than a hairpin loop, and the connecting structure is a linker.


In some embodiments, the iRNA agent may be a “single-stranded siRNA” that is introduced into a cell or organism to inhibit a target mRNA. In some embodiments, single-stranded RNAi agents can bind to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are optionally chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein (e.g., sequences provided in Tables 2A or 2B) may be used as a single-stranded siRNA as described herein and optionally as chemically modified, e.g., as described herein, e.g., by the methods described in Lima et al., (2012) Cell 150:883-894.


In some embodiments, an RNA interference agent includes a single stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) (Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleaves the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in some embodiments, the disclosure relates to a single stranded RNA that promotes the formation of a RISC complex to effect silencing of the target gene.


“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the terms “deoxyribonucleotide,” “ribonucleotide,” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of dsRNA featured in the disclosure by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the disclosure.


As used herein, the term “iRNA,” “RNAi”, “iRNA agent,” or “RNAi agent” or “RNAi molecule” refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript, e.g., via an RNA-induced silencing complex (RISC) pathway. In some embodiments, an iRNA as described herein effects inhibition of MYOC expression, e.g., in a cell or mammal. Inhibition of MYOC expression may be assessed based on a reduction in the level of MYOC mRNA or a reduction in the level of the MYOC protein.


The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound.


The term “lipophile” or “lipophilic moiety” broadly refers to any compound or chemical moiety having an affinity for lipids. One way to characterize the lipophilicity of the lipophilic moiety is by the octanol-water partition coefficient, logKow, where Kow is the ratio of a chemical's concentration in the octanol-phase to its concentration in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it may also be predicted by using coefficients attributed to the structural components of a chemical which are calculated using first-principle or empirical methods (see, for example, Tetko et al., J. Chem. Inf Comput. Sci. 41:1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measure of the tendency of the substance to prefer a non-aqueous or oily milieu rather than water (i.e. its hydrophilic/lipophilic balance). In principle, a chemical substance is lipophilic in character when its logKow exceeds 0. Typically, the lipophilic moiety possesses a logKow exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10. For instance, the logKow of 6-amino hexanol, for instance, is predicted to be approximately 0.7. Using the same method, the logKow of cholesteryl N-(hexan-6-ol) carbamate is predicted to be 10.7.


The lipophilicity of a molecule can change with respect to the functional group it carries. For instance, adding a hydroxyl group or amine group to the end of a lipophilic moiety can increase or decrease the partition coefficient (e.g., logKow) value of the lipophilic moiety.


Alternatively, the hydrophobicity of the double-stranded RNAi agent, conjugated to one or more lipophilic moieties, can be measured by its protein binding characteristics. For instance, in certain embodiments, the unbound fraction in the plasma protein binding assay of the double-stranded RNAi agent could be determined to positively correlate to the relative hydrophobicity of the double-stranded RNAi agent, which could then positively correlate to the silencing activity of the double-stranded RNAi agent.


In some embodiments, the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. An exemplary protocol of this binding assay is illustrated in detail in, e.g., PCT/US2019/031170. The hydrophobicity of the double-stranded RNAi agent, measured by fraction of unbound siRNA in the binding assay, exceeds 0.15, exceeds 0.2, exceeds 0.25, exceeds 0.3, exceeds 0.35, exceeds 0.4, exceeds 0.45, or exceeds 0.5 for an enhanced in vivo delivery of siRNA.


Accordingly, conjugating the lipophilic moieties to the internal position(s) of the double-stranded RNAi agent provides optimal hydrophobicity for the enhanced in vivo delivery of siRNA.


The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., a RNAi agent or a plasmid from which a RNAi agent is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.


As used herein, the term “modulate the expression of,” refers to an at least partial “inhibition” or partial “activation” of a gene (e.g., MYOC gene) expression in a cell treated with an iRNA composition as described herein compared to the expression of the corresponding gene in a control cell. A control cell includes an untreated cell, or a cell treated with a non-targeting control iRNA.


The skilled artisan will recognize that the term “RNA molecule” or “ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, a “ribonucleoside” includes a nucleoside base and a ribose sugar, and a “ribonucleotide” is a ribonucleoside with one, two or three phosphate moieties or analogs thereof (e.g., phosphorothioate). However, the terms “ribonucleoside” and “ribonucleotide” can be considered to be equivalent as used herein. The RNA can be modified in the nucleobase structure, in the ribose structure, or in the ribose-phosphate backbone structure, e.g., as described herein below. However, the molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex. As non-limiting examples, an RNA molecule can also include at least one modified ribonucleoside including but not limited to a 2′-O-methyl modified nucleoside, a nucleoside comprising a 5′ phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, an acyclic nucleoside, a glycol nucleotide, a 2′-deoxy-2′-fluoro modified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, or in combination, an RNA molecule can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the dsRNA molecule. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule. In some embodiments, modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA, e.g., via a RISC pathway. For clarity, it is understood that the term “iRNA” does not encompass a naturally occurring double stranded DNA molecule or a 100%/ deoxynucleoside-containing DNA molecule.


In some aspects, a modified ribonucleoside includes a deoxyribonucleoside. In such an instance, an iRNA agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double stranded portion of a dsRNA. In certain embodiments, the RNA molecule comprises a percentage of deoxyribonucleosides of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or higher (but not 100%) deoxyribonucleosides, e.g., in one or both strands.


As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) may be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′ end, 3′ end or both ends of either an antisense or sense strand of a dsRNA.


In some embodiments, the antisense strand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/or the 5′ end. In some embodiments, the sense strand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/or the 5′ end. In some embodiments, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.


As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a therapeutic agent (e.g., an iRNA) and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an agent (e.g., iRNA) effective to produce the intended pharmacological, therapeutic or preventive result. For example, in a method of treating a disorder related to MYOC expression (e.g., glaucoma, e.g., primary open angle glaucoma (POAG)), an effective amount includes an amount effective to reduce one or more symptoms associated with the disorder (e.g., an amount effective to; (a) inhibit or reduce the expression or activity of MYOC; (b) reduce the level of misfolded MYOC protein; (c) reduce trabecular meshwork cell death; (d) decrease intraocular pressure; or (e) increase visual acuity. For example, if a given clinical treatment is considered effective when there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to obtain at least a 10/6 reduction in that parameter. For example, a therapeutically effective amount of an iRNA targeting MYOC can reduce a level of MYOC mRNA or a level of MYOC protein by any measurable amount, e.g., by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.


The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Agents included in drug formulations are described further herein below.


As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 2006/0240093, 2007/0135372, and in International Application No. WO 2009/082817. These applications are incorporated herein by reference in their entirety. In some embodiments, the SNALP is a SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle.


As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.


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


A “subject in need thereof” includes a subject having, suspected of having, or at risk of developing a disorder related to MYOC expression, e.g., overexpression (e.g., glaucoma). In some embodiments, the subject has, or is suspected of having, a disorder related to MYOC expression or overexpression. In some embodiments, the subject is at risk of developing a disorder related to MYOC expression or overexpression.


As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene, e.g., MYOC, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all sub-ranges therebetween. As non-limiting examples, the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.


As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” and the like refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of any disorder or pathological process related to MYOC expression (e.g., glaucoma, e.g., primary open angle glaucoma (POAG)). The specific amount that is therapeutically effective may vary depending on factors known in the art, such as, for example, the type of disorder or pathological process, the patient's history and age, the stage of the disorder or pathological process, and the administration of other therapies.


In the context of the present disclosure, the terms “treat,” “treatment,” and the like mean to prevent, delay, relieve or alleviate at least one symptom associated with a disorder related to MYOC expression, or to slow or reverse the progression or anticipated progression of such a disorder. For example, the methods featured herein, when employed to treat a glaucoma, may serve to reduce or prevent one or more symptoms of the glaucoma, as described herein, or to reduce the risk or severity of associated conditions. Thus, unless the context clearly indicates otherwise, the terms “treat,” “treatment,” and the like are intended to encompass prophylaxis, e.g., prevention of disorders and/or symptoms of disorders related to MYOC expression. Treatment can also mean prolonging survival as compared to expected survival in the absence of treatment.


By “lower” in the context of a disease marker or symptom is meant any decrease, e.g., a statistically or clinically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. The decrease can be down to a level accepted as within the range of normal for an individual without such disorder.


As used herein, “MYOC” refers to “myocilin” the corresponding mRNA (“MYOC mRNA”), or the corresponding protein (“MYOC protein”). The sequence of a human MYOC mRNA transcript can be found at SEQ ID NO: 1.


II. IRNA AGENTS

Described herein are iRNA agents that modulate (e.g., inhibit) the expression of MYOC.


In some embodiments, the iRNA agent activates the expression of MYOC in a cell or mammal.


In some embodiments, the iRNA agent includes double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of MYOC in a cell or in a subject (e.g., in a mammal, e.g., in a human), where the dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of MYOC, and where the region of complementarity is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and where the dsRNA, upon contact with a cell expressing MYOC, inhibits the expression of MYOC, e.g., by at least 10%, 20%, 30%, 40%, or 50%.


The modulation (e.g., inhibition) of expression of MYOC can be assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by Western blot. Expression of MYOC in cell culture, such as in COS cells, ARPE-19 cells, hTERT RPE-1 cells, HeLa cells, primary hepatocytes, HepG2 cells, primary cultured cells or in a biological sample from a subject can be assayed by measuring MYOC mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by immunofluorescence analysis, using, for example, Western Blotting or flow cytometric techniques.


A dsRNA typically includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) typically includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of MYOC. The other strand (the sense strand) typically includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive.


In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, e.g., 15-30 nucleotides in length.


One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of 9 to 36, e.g., 15-30 base pairs. Thus, in some embodiments, to the extent that it becomes processed to a functional duplex of e.g., 15-30 base pairs that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in some embodiments, then, an miRNA is a dsRNA. In some embodiments, a dsRNA is not a naturally occurring miRNA. In some embodiments, an iRNA agent useful to target MYOC expression is not generated in the target cell by cleavage of a larger dsRNA.


A dsRNA as described herein may further include one or more single-stranded nucleotide overhangs. The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.


In some embodiments, MYOC is a human MYOC.


In specific embodiments, the dsRNA comprises a sense strand that comprises or consists of a sense sequence selected from the sense sequences provided in Tables 2A or 2B and an antisense strand that comprises or consists of an antisense sequence selected from the antisense sequences provided in Tables 2A or 2B.


In some aspects, a dsRNA will include at least sense and antisense nucleotide sequences, whereby the sense strand is selected from the sequences provided in Tables 2A or 2B and the corresponding antisense strand is selected from the sequences provided in Tables 2A or 2B.


In these aspects, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated by the expression of MYOC. As such, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand, and the second oligonucleotide is described as the corresponding antisense strand. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.


The skilled person is well aware that dsRNAs having a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can be effective as well.


In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 2A and 2B, dsRNAs described herein can include at least one strand of a length of minimally 19 nucleotides. It can be reasonably expected that shorter duplexes having one of the sequences of Tables 2A and 2B minus only a few nucleotides on one or both ends will be similarly effective as compared to the dsRNAs described above.


In some embodiments, the dsRNA has a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 2A or 2B.


In some embodiments, the dsRNA has an antisense sequence that comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides of an antisense sequence provided in Tables 2A or 2B and a sense sequence that comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides of a corresponding sense sequence provided in Tables 2A or 2B.


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


In some such embodiments, the dsRNA, although it comprises only a portion of the sequences provided in Tables 2A or 2B is equally effective in inhibiting a level of MYOC expression as is a dsRNA that comprises the full-length sequences provided in Tables 2A or 2B. In some embodiments, the dsRNA differs in its inhibition of a level of expression of MYOC by not more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% inhibition compared with a dsRNA comprising the full sequence disclosed herein.


In some embodiments, an iRNA described herein comprises an antisense strand comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2. In some embodiments, an iRNA described herein comprises a sense strand comprising at least 15 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.


A human MYOC mRNA may have the sequence of SEQ ID NO: 1 provided herein. Homo sapiens myocilin (MYOC), mRNA










(SEQ ID NO: 1)



GAGCCAGCAAGGCCACCCATCCAGGCACCTCTCAGCACAGCAGAGCTTTCCAGAGGAAGCCTCA






CCAAGCCTCTGCAATGAGGTTCTTCTGTGCACGTTGCTGCAGCTTTGGGCCTGAGATGCCAGCT





GTCCAGCTGCTGCTTCTGGCCTGCCTGGTGTGGGATGTGGGGGCCAGGACAGCTCAGCTCAGGA





AGGCCAATGACCAGAGTGGCCGATGCCAGTATACCTTCAGTGTGGCCAGTCCCAATGAATCCAG





CTGCCCAGAGCAGAGCCAGGCCATGTCAGTCATCCATAACTTACAGAGAGACAGCAGCACCCAA





CGCTTAGACCTGGAGGCCACCAAAGCTCGACTCAGCTCCCTGGAGAGCCTCCTCCACCAATTGA





CCTTGGACCAGGCTGCCAGGCCCCAGGAGACCCAGGAGGGGCTGCAGAGGGAGCTGGGCACCCT





GAGGCGGGAGCGGGACCAGCTGGAAACCCAAACCAGAGAGTTGGAGACTGCCTACAGCAACCTC





CTCCGAGACAAGTCAGTTCTGGAGGAAGAGAAGAAGCGACTAAGGCAAGAAAATGAGAATCTGG





CCAGGAGGTTGGAAAGCAGCAGCCAGGAGGTAGCAAGGCTGAGAAGGGGCCAGTGTCCCCAGAC





CCGAGACACTGCTCGGGCTGTGCCACCAGGCTCCAGAGAAGTTTCTACGTGGAATTTGGACACT





TTGGCCTTCCAGGAACTGAAGTCCGAGCTAACTGAAGTTCCTGCTTCCCGAATTTTGAAGGAGA





GCCCATCTGGCTATCTCAGGAGTGGAGAGGGAGACACCGGATGTGGAGAACTAGTTTGGGTAGG





AGAGCCTCTCACGCTGAGAACAGCAGAAACAATTACTGGCAAGTATGGTGTGTGGATGCGAGAC





CCCAAGCCCACCTACCCCTACACCCAGGAGACCACGTGGAGAATCGACACAGTTGGCACGGATG





TCCGCCAGGTTTTTGAGTATGACCTCATCAGCCAGTTTATGCAGGGCTACCCTTCTAAGGTTCA





CATACTGCCTAGGCCACTGGAAAGCACGGGTGCTGTGGTGTACTCGGGGAGCCTCTATTTCCAG





GGCGCTGAGTCCAGAACTGTCATAAGATATGAGCTGAATACCGAGACAGTGAAGGCTGAGAAGG





AAATCCCTGGAGCTGGCTACCACGGACAGTTCCCGTATTCTTGGGGTGGCTACACGGACATTGA





CTTGGCTGTGGATGAAGCAGGCCTCTGGGTCATTTACAGCACCGATGAGGCCAAAGGTGCCATT





GTCCTCTCCAAACTGAACCCAGAGAATCTGGAACTCGAACAAACCTGGGAGACAAACATCCGTA





AGCAGTCAGTCGCCAATGCCTTCATCATCTGTGGCACCTTGTACACCGTCAGCAGCTACACCTC





AGCAGATGCTACCGTCAACTTTGCTTATGACACAGGCACAGGTATCAGCAAGACCCTGACCATC





CCATTCAAGAACCGCTATAAGTACAGCAGCATGATTGACTACAACCCCCTGGAGAAGAAGCTCT





TTGCCTGGGACAACTTGAACATGGTCACTTATGACATCAAGCTCTCCAAGATGTGAAAAGCCTC





CAAGCTGTACAGGCAATGGCAGAAGGAGATGCTCAGGGCTCCTGGGGGGAGCAGGCTGAAGGGA





GAGCCAGCCAGCCAGGGCCCAGGCAGCTTTGACTGCTTTCCAAGTTTTCATTAATCCAGAAGGA





TGAACATGGTCACCATCTAACTATTCAGGAATTGTAGTCTGAGGGCGTAGACAATTTCATATAA





TAAATATCCTTTATCTTCTGTCAGCATTTATGGGATGTTTAATGACATAGTTCAAGTTTTCTTG





TGATTTGGGGCAAAAGCTGTAAGGCATAATAGTTTCTTCCTGAAAACCATTGCTCTTGCATGTT





ACATGGTTACCACAAGCCACAATAAAAAGCATAACTTCTAAAGGAAGCAGAATAGCTCCTCTGG





CCAGCATCGAATATAAGTAAGATGCATTTACTACAGTTGGCTTCTAATGCTTCAGATAGAATAC





AGTTGGGTCTCACATAACCCTTTACATTGTGAAATAAAATTTTCTTACCCAA






The reverse complement of SEQ ID NO: 1 is provided as SEQ ID NO: 2 herein:










(SEQ ID NO: 2)



TTGGGTAAGAAAATTTTATTTCACAATGTAAAGGGTTATGTGAGACCCAACTGTATTCTATCTG






AAGCATTAGAAGCCAACTGTAGTAAATGCATCTTACTTATATTCGATGCTGGCCAGAGGAGCTA





TTCTGCTTCCTTTAGAAGTTATGCTTTTTATTGTGGCTTGTGGTAACCATGTAACATGCAAGAG





CAATGGTTTTCAGGAAGAAACTATTATGCCTTACAGCTTTTGCCCCAAATCACAAGAAAACTTG





AACTATGTCATTAAACATCCCATAAATGCTGACAGAAGATAAAGGATATTTATTATATGAAATT





GTCTACGCCCTCAGACTACAATTCCTGAATAGTTAGATGGTGACCATGTTCATCCTTCTGGATT





AATGAAAACTTGGAAAGCAGTCAAAGCTGCCTGGGCCCTGGCTGGCTGGCTCTCCCTTCAGCCT





GCTCCCCCCAGGAGCCCTGAGCATCTCCTTCTGCCATTGCCTGTACAGCTTGGAGGCTTTTCAC





ATCTTGGAGAGCTTGATGTCATAAGTGACCATGTTCAAGTTGTCCCAGGCAAAGAGCTTCTTCT





CCAGGGGGTTGTAGTCAATCATGCTGCTGTACTTATAGCGGTTCTTGAATGGGATGGTCAGGGT





CTTGCTGATACCTGTGCCTGTGTCATAAGCAAAGTTGACGGTAGCATCTGCTGAGGTGTAGCTG





CTGACGGTGTACAAGGTGCCACAGATGATGAAGGCATTGGCGACTGACTGCTTACGGATGTTTG





TCTCCCAGGTTTGTTCGAGTTCCAGATTCTCTGGGTTCAGTTTGGAGAGGACAATGGCACCTTT





GGCCTCATCGGTGCTGTAAATGACCCAGAGGCCTGCTTCATCCACAGCCAAGTCAATGTCCGTG





TAGCCACCCCAAGAATACGGGAACTGTCCGTGGTAGCCAGCTCCAGGGATTTCCTTCTCAGCCT





TCACTGTCTCGGTATTCAGCTCATATCTTATGACAGTTCTGGACTCAGCGCCCTGGAAATAGAG





GCTCCCCGAGTACACCACAGCACCCGTGCTTTCCAGTGGCCTAGGCAGTATGTGAACCTTAGAA





GGGTAGCCCTGCATAAACTGGCTGATGAGGTCATACTCAAAAACCTGGCGGACATCCGTGCCAA





CTGTGTCGATTCTCCACGTGGTCTCCTGGGTGTAGGGGTAGGTGGGCTTGGGGTCTCGCATCCA





CACACCATACTTGCCAGTAATTGTTTCTGCTGTTCTCAGCGTGAGAGGCTCTCCTACCCAAACT





AGTTCTCCACATCCGGTGTCTCCCTCTCCACTCCTGAGATAGCCAGATGGGCTCTCCTTCAAAA





TTCGGGAAGCAGGAACTTCAGTTAGCTCGGACTTCAGTTCCTGGAAGGCCAAAGTGTCCAAATT





CCACGTAGAAACTTCTCTGGAGCCTGGTGGCACAGCCCGAGCAGTGTCTCGGGTCTGGGGACAC





TGGCCCCTTCTCAGCCTTGCTACCTCCTGGCTGCTGCTTTCCAACCTCCTGGCCAGATTCTCAT





TTTCTTGCCTTAGTCGCTTCTTCTCTTCCTCCAGAACTGACTTGTCTCGGAGGAGGTTGCTGTA





GGCAGTCTCCAACTCTCTGGTTTGGGTTTCCAGCTGGTCCCGCTCCCGCCTCAGGGTGCCCAGC





TCCCTCTGCAGCCCCTCCTGGGTCTCCTGGGGCCTGGCAGCCTGGTCCAAGGTCAATTGGTGGA





GGAGGCTCTCCAGGGAGCTGAGTCGAGCTTTGGTGGCCTCCAGGTCTAAGCGTTGGGTGCTGCT





GTCTCTCTGTAAGTTATGGATGACTGACATGGCCTGGCTCTGCTCTGGGCAGCTGGATTCATTG





GGACTGGCCACACTGAAGGTATACTGGCATCGGCCACTCTGGTCATTGGCCTTCCTGAGCTGAG





CTGTCCTGGCCCCCACATCCCACACCAGGCAGGCCAGAAGCAGCAGCTGGACAGCTGGCATCTC





AGGCCCAAAGCTGCAGCAACGTGCACAGAAGAACCTCATTGCAGAGGCTTGGTGAGGCTTCCTC





TGGAAAGCTCTGCTGTGCTGAGAGGTGCCTGGATGGGTGGCCTTGCTGGCTC






In some embodiments, an iRNA described herein includes at least 15 contiguous nucleotides from one of the sequences provided in Tables 2A and 2B, and may optionally be coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in MYOC.


While a target sequence is generally 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that may serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays described herein or known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.


Further, it is contemplated that for any sequence identified, e.g., in Tables 2A and 2B, further optimization can be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those and sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.


In some embodiments, the disclosure provides an iRNA of Table 2B that is un-modified or un-conjugated. In some embodiments, an RNAi agent of the disclosure has a nucleotide sequence as provided in Table 2A, but lacks one or more ligand or moiety shown in the table. A ligand or moiety (e.g., a lipophilic ligand or moiety) can be included in any of the positions provided in the instant application.


An iRNA as described herein can contain one or more mismatches to the target sequence. In some embodiments, an iRNA as described herein contains no more than 3 mismatches. In some embodiments, when the antisense strand of the iRNA contains mismatches to a target sequence, the area of mismatch is not located in the center of the region of complementarity. In some embodiments, when the antisense strand of the iRNA contains mismatches to the target sequence, the mismatch is restricted to be within the last 5 nucleotides from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide iRNA agent RNA strand which is complementary to a region of MYOC, the RNA strand generally does not contain any mismatch within the central 13 nucleotides. The methods described herein, or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of MYOC. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of MYOC is important, especially if the particular region of complementarity in a MYOC gene is known to have polymorphic sequence variation within the population.


In some embodiments, at least one end of a dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. In some embodiments, dsRNAs having at least one nucleotide overhang have superior inhibitory properties relative to their blunt-ended counterparts. In some embodiments, the RNA of an iRNA (e.g., a dsRNA) is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the disclosure may be synthesized and/or modified by methods well established 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, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position, or having an acyclic sugar) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in this disclosure include, but are not limited to, RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.


Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.


Representative U.S. patents that teach the preparation of the above 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; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, each of which is herein incorporated by reference.


Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.


Representative U.S. patents that teach the preparation of the above oligonucleosides 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, each of which is herein incorporated by reference.


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


Some embodiments featured in the disclosure include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as —O—P—O—CH2— ] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.


Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′ position. OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2.


In other embodiments, an iRNA agent comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides (or nucleosides). In certain embodiments, the sense strand or the antisense strand, or both sense strand and antisense strand, include less than five acyclic nucleotides per strand (e.g., four, three, two or one acyclic nucleotides per strand). The one or more acyclic nucleotides can be found, for example, in the double-stranded region, of the sense or antisense strand, or both strands; at the 5′-end, the 3′-end, both of the 5′ and 3′-ends of the sense or antisense strand, or both strands, of the iRNA agent. In some embodiments, one or more acyclic nucleotides are present at positions 1 to 8 of the sense or antisense strand, or both. In some embodiments, one or more acyclic nucleotides are found in the antisense strand at positions 4 to 10 (e.g., positions 6-8) from the 5′-end of the antisense strand. In some embodiments, the one or more acyclic nucleotides are found at one or both 3′-terminal overhangs of the iRNA agent.


The term “acyclic nucleotide” or “acyclic nucleoside” as used herein refers to any nucleotide or nucleoside having an acyclic sugar, e.g., an acyclic ribose. An exemplary acyclic nucleotide or nucleoside can include a nucleobase, e.g., a naturally occurring or a modified nucleobase (e.g., a nucleobase as described herein). In certain embodiments, a bond between any of the ribose carbons (C1, C2, C3, C4, or C5), is independently or in combination absent from the nucleotide. In some embodiments, the bond between C2-C3 carbons of the ribose ring is absent, e.g., an acyclic 2′-3′-seco-nucleotide monomer. In other embodiments, the bond between C1-C2, C3-C4, or C4-C5 is absent (e.g., a 1′-2′, 3′-4′ or 4′-5′-seco nucleotide monomer). Exemplary acyclic nucleotides are disclosed in U.S. Pat. No. 8,314,227, incorporated herein by reference in its entirely. For example, an acyclic nucleotide can include any of monomers D-J in FIGS. 1-2 of U.S. Pat. No. 8,314,227. In some embodiments, the acyclic nucleotide includes the following monomer:




embedded image


wherein Base is a nucleobase, e.g., a naturally occurring or a modified nucleobase (e.g., a nucleobase as described herein).


In certain embodiments, the acyclic nucleotide can be modified or derivatized, e.g., by coupling the acyclic nucleotide to another moiety, e.g., a ligand (e.g., a GalNAc, a cholesterol ligand), an alkyl, a polyamine, a sugar, a polypeptide, among others.


In other embodiments, the iRNA agent includes one or more acyclic nucleotides and one or more LNAs (e.g., an LNA as described herein). For example, one or more acyclic nucleotides and/or one or more LNAs can be present in the sense strand, the antisense strand, or both. The number of acyclic nucleotides in one strand can be the same or different from the number of LNAs in the opposing strand. In certain embodiments, the sense strand and/or the antisense strand comprises less than five LNAs (e.g., four, three, two or one LNAs) located in the double stranded region or a 3′-overhang. In other embodiments, one or two LNAs are located in the double stranded region or the 3′-overhang of the sense strand. Alternatively, or in combination, the sense strand and/or antisense strand comprises less than five acyclic nucleotides (e.g., four, three, two or one acyclic nucleotides) in the double-stranded region or a 3′-overhang. In some embodiments, the sense strand of the iRNA agent comprises one or two LNAs in the 3′-overhang of the sense strand, and one or two acyclic nucleotides in the double-stranded region of the antisense strand (e.g., at positions 4 to 10 (e.g., positions 6-8) from the 5′-end of the antisense strand) of the iRNA agent.


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


Other modifications include 2′-methoxy (2′-OCH3), 2′-5 aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties 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. Pat. 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, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.


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


Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch el al., Angeusandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.


Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187: 5,459,255; 5,484,908; 5,502,177: 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.


The RNA of an iRNA can also be modified to include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodiments an agent of the disclosure may include one or more locked nucleic acids (LNAs) (also referred to herein as “locked nucleotides”). In some embodiments, a locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting, e.g., the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, increase thermal stability, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).


Examples of bicyclic nucleosides for use in the polynucleotides of the disclosure include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the disclosure include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′; 4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH2OCH3)—O-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2—N(OCH3)-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′ (see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The contents of each of the foregoing are incorporated herein by reference for the methods provided therein. Representative U.S. Patents that teach the preparation of locked nucleic acids include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; 7,399,845, and 8,314,227, each of which is herein incorporated by reference in its entirety. Exemplary LNAs include but are not limited to, a 2′, 4′-C methylene bicyclo nucleotide (see for example Wengel et al., International PCT 5 Publication No. WO 00/66604 and WO 99/14226).


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).


A RNAi agent of the disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)-0-2′ bridge. In some embodiments, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.”


A RNAi agent of the disclosure may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2′ and C4′ carbons of ribose or the C3′ and —C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.


Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US 2013/0190383; and WO 2013/036868, the contents of each of which are hereby incorporated herein by reference for the methods provided therein.


In some embodiments, a RNAi agent of the disclosure comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039).


Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the contents of each of which are hereby incorporated herein by reference for the methods provided therein.


In other embodiments, the iRNA agents include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in the iRNA molecules can result in enhanced affinity and specificity to nucleic acid targets, complementary sequences, or template strands.


Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″ phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.


Other modifications of a RNAi agent of the disclosure include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic on the antisense strand of a RNAi agent. Suitable phosphate mimics are disclosed in, for example US 2012/0157511, the contents of which are incorporated herein by reference for the methods provided therein. In one embodiment, the double stranded RNAi agent of the invention further comprises a 5′-phosphate or a 5′-phosphate mimic at the 5′ nucleotide of the antisense strand. In another embodiment, the double stranded RNAi agent further comprises a 5′-phosphate mimic at the 5′ nucleotide of the antisense strand. In a specific embodiment, the 5′-phosphate mimic is a 5′-vinyl phosphonate (5′-VP). In one embodiment, the phosphate mimic is a 5′-cyclopropyl phosphonate (VP). In some embodiments, the 5′-end of the antisense strand of the double-stranded iRNA agent does not contain a 5′-vinyl phosphonate (VP).


In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a glycol modified nucleotide (GNA), e.g., Ggn, Cgn, Tgn, or Agn, a nucleotide with a 2′ phosphate, e.g., G2p, C2p, A2p or U2p, and, a vinyl-phosphonate nucleotide; and combinations thereof. In other embodiments, each of the duplexes of Tables 5 and 6 may be particularly modified to provide another double-stranded iRNA agent of the present disclosure. In one example, the 3′-terminus of each sense duplex may be modified by removing the 3′-terminal L96 ligand and exchanging the two phosphodiester internucleotide linkages between the three 3′-terminal nucleotides with phosphorothioate internucleotide linkages. That is, the three 3′-terminal nucleotides (N) of a sense sequence of the formula:

    • 5′-N1- . . . -Nn-2Nn-1NnL96 3′


      may be replaced with
    • 5′-N1- . . . -Nn-2sNn-1sNn 3′.


An RNA target may have regions, or spans of the target RNA's nucleotide sequence, which are relatively more susceptible or amenable than other regions of the RNA target to mediating cleavage of the RNA target via RNA interference induced by the binding of an RNAi agent to that region. The increased susceptibility to RNA interference within such “hotspot regions” (or simply “hotspots”) means that iRNA agents targeting the region will likely have higher efficacy in inducing iRNA interference than iRNA agents which target other regions of the target RNA. For example, without being bound by theory, the accessibility of a target region of a target RNA may influence the efficacy of iRNA agents which target that region, with some hotspot regions having increased accessibility. Secondary structures, for instance, that form in the RNA target (e.g., within or proximate to hotspot regions) may affect the ability of the iRNA agent to bind the target region and induce RNA interference.


According to certain aspects of the invention, an iRNA agent may be designed to target a hotspot region of any of the target RNAs described herein, including any identified portions of a target RNA (e.g., a particular exon). As used herein, a hotspot region may refer to an approximately 19-200, 19-150, 19-100, 19-75, 19-50, 21-200, 21-150, 21-100, 21-75, 21-50, 50-200, 50-150, 50-100, 50-75, 75-200, 75-150, 75-100, 100-200, or 100-150 nucleotide region of a target RNA sequence for which targeting using RNAi agents provides an observably higher probability of efficacious silencing relative to targeting other regions of the same target RNA. According to certain aspects of the invention, a hotspot region may comprise a limited region of the target RNA, and in some cases, a substantially limited region of the target, including for example, less than half of the length of the target RNA, such as about 5%, 10%, 15%, 20%, 25%, or 30% of the length of the target RNA. Conversely, the other regions against which a hotspot is compared may cumulatively comprise at least a majority of the length of the target RNA. For example, the other regions may cumulatively comprise at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% of the length of the target RNA.


Compared regions of the target RNA may be empirically evaluated for identification of hotspots using efficacy data obtained from in vitro or in vivo screening assays. For example, RNAi agents targeting various regions that span a target RNA may be compared for frequency of efficacious iRNA agents (e.g., the amount by which target gene expression is inhibited, such as measured by mRNA expression or protein expression) that bind each region. In general, a hotspot can be recognized by observing clustering of multiple efficacious RNAi agents that bind to a limited region of the RNA target. A hotspot may be sufficiently characterized as such by observing efficacy of iRNA agents which cumulatively span at least about 60% of the target region identified as a hotspot, such as about 70%, about 80%, about 90%, or about 95% or more of the length of the region, including both ends of the region (i.e. at least about 60%, 70%, 80%, 90%, or 95% or more of the nucleotides within the region, including the nucleotides at each end of the region, were targeted by an iRNA agent). According to some aspects of the invention, an iRNA agent which demonstrates at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% inhibition over the region (e.g., no more than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% mRNA remaining) may be identified as efficacious.


Amenability to targeting of RNA regions may also be assessed using quantitative comparison of inhibition measurements across different regions of a defined size (e.g, 25, 30, 40, 50, 60, 70, 80, 90, or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nts). For example, an average level of inhibition may be determined for each region and the averages of each region may be compared. The average level of inhibition within a hotspot region may be substantially higher than the average of averages for all evaluated regions. According to some aspects, the average level of inhibition in a hotspot region may be at least about 10%, 20%, 30%, 40%, or 50% higher than the average of averages. According to some aspects, the average level of inhibition in a hotspot region may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 1.6, 1.7, 1.8. 1.9, or 2.0 standard deviations above the average of averages. The average level of inhibition may be higher by a statistically significant (e.g., p<0.05) amount. According to some aspects, each inhibition measurement within a hotspot region may be above a threshold amount (e.g., at or below a threshold amount of mRNA remaining). According to some aspects, each inhibition measurement within the region may be substantially higher than an average of all inhibition measurements across all the measured regions. For example, each inhibition measurement in a hotspot region may be at least about 10%, 20%, 30%, 40%, or 50% higher than the average of all inhibition measurements. According to some aspects, each inhibition measurement may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 1.6, 1.7, 1.8. 1.9, or 2.0 standard deviations above the average of all inhibition measurements. Each inhibition measurement may be higher by a statistically significant (e.g., p<0.05) amount than the average of all inhibition measurements. A standard for evaluating a hotspot may comprise various combinations of the above standards where compatible (e.g., an average level of inhibition of at least about a first amount and having no inhibition measurements below a threshold level of a second amount, lesser than the first amount).


It is therefore expressly contemplated that any iRNA agent, including the specific exemplary iRNA agents described herein, which targets a hotspot region of a target RNA, may be preferably selected for inducing RNA interference of the target mRNA as targeting such a hotspot region is likely to exhibit a robust inhibitory response relative to targeting a region which is not a hotspot region. RNAi agents targeting target sequences that substantially overlap (e.g., by at least about 70%, 75%, 80%, 85%, 90%, 95% of the target sequence length) or, preferably, that reside fully within the hotspot region may be considered to target the hotspot region. Hotspot regions of the RNA target(s) of the instant invention may include any region for which the data disclosed herein demonstrates higher frequency of targeting by efficacious RNAi agents, including by any of the standards described elsewhere herein, whether or not the range(s) of such hotspot region(s) are explicitly specified.


In various embodiments, a dsRNA agent of the present invention targets a hotspot region of an mRNA encoding MYOC.


III. IRNA MOTIFS

In certain aspects of the disclosure, the double-stranded RNAi agents of the disclosure include agents with chemical modifications as disclosed, for example, in WO 2013/075035, the contents of which are incorporated herein by reference for the methods provided therein. As shown herein and in WO 2013/075035, a superior result may be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into a sense strand or antisense strand of an RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of the RNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense or antisense strand. The RNAi agent may be optionally conjugated with a lipophilic moiety or ligand, e.g., a C16 moiety or ligand, for instance on the sense strand. The RNAi agent may be optionally modified with a (S)-glycol nucleic acid (GNA) modification, for instance on one or more residues of the antisense strand. The resulting RNAi agents present superior gene silencing activity.


In some embodiments, the sense strand sequence may be represented by formula (I):





5′ np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3′  (I)


wherein:


i and j are each independently 0 or 1;


p and q are each independently 0-6;


each Na independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;


each Nb independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;


each np and nq independently represent an overhang nucleotide;


wherein Nb and Y do not have the same modification; and


XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. In some embodiments, YYY is all 2′-F modified nucleotides.


In some embodiments, the Na and/or Nb comprise modifications of alternating pattern.


In some embodiments, 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 can occur at or the vicinity of the cleavage site (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) of the sense strand, the count starting from the 1′nucleotide, from the 5′-end; or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end.


In some embodiments, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:





5′ np-Na-YYY-Nb-ZZZ-Na-nq 3′  (Ib);





5′ np-Na-XXX-Nb-YYY-Na-nq 3′  (Ic); or





5′ np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3′  (Id).


When the sense strand is represented by formula (Ib), Nb represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


When the sense strand is represented as formula (Ic), Nb represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


When the sense strand is represented as formula (Id), each Nb independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. In some embodiments, Nb is 0, 1, 2, 3, 4, 5 or 6. Each Na can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


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 may be represented by the formula:





5′ np-Na-YYY- Na-nq 3′  (Ia).


When the sense strand is represented by formula (Ia), each Na independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


In some embodiments, the antisense strand sequence of the RNAi may be represented by formula (Ie):





5′ nq′-Na′-(Z′Z′Z′)k-Nb′-Y′Y′Y′-Nb′-(X′X′X′)l-N′a-np′ 3′  (Ie)


wherein:


k and l are each independently 0 or 1;


p′ and q′ are each independently 0-6;


each Na′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;


each Nb′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

    • each np′ and nq′ independently represent an overhang nucleotide;
    • wherein Nb′ and Y′ do not have the same modification;
    • and


X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one of three identical modification on three consecutive nucleotides.


In some embodiments, the Na′ and/or Nb′ comprise modification of alternating pattern.


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′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the 1st nucleotide, from the 5′-end; or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end. In some embodiments, the Y′Y′Y′ motif occurs at positions 11, 12, 13.


In some embodiments, Y′Y′Y′ motif is all 2′-Ome modified nucleotides.


In on embodiment, k is 1 and 1 is 0, or k is 0 and 1 is 1, or both 5 k and l are 1.


The antisense strand can therefore be represented by the following formulas:





5′ nq′-Na′-Z′Z′Z′-Nb′-Y′Y′Y′-Na′np′ 3′  (Ig);





5′ nq′-Na′-Y′Y′Y′-Nb′-X′X′X′-np′ 3′  (Ih); or





5′ nq′-Na′-Z′Z′Z′-Nb′-Y′Y′Y′-Nb′-X′X′X′-Na′-np′ 3′  (Ii).


When the antisense strand is represented by formula (Ig), Nb′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


When the antisense strand is represented as formula (Ii), each Nb′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. In some embodiments, Nb is 0, 1, 2, 3, 4, 5 or 6.


In other embodiments, k is 0 and 1 is 0 and the antisense strand may be represented by the formula:





5′ np′-Na′-Y′Y′Y′-Na′-nq′ 3′  (If).


When the antisense strand is represented as formula (If), each Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


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


Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, HNA, CeNA, GNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.


In some embodiments, the sense strand of the RNAi agent may contain YYY motif occurring at 9, 10 and 11 positions of the strand when the duplex region is 21 nt, the count starting from the 1 nucleotide from the 5′-end, or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.


In some embodiments the antisense strand may Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 1st nucleotide from the 5′-end, or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.


The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with an antisense strand being represented by any one of formulas (If), (Ig), (Ih), and (Ii), respectively.


Accordingly, certain RNAi agents for use in the methods of the disclosure may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex represented by formula (Ij):





sense:5′ np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3′





antisense: 3′ np′-Na′-(X′X′X′)k-Nb′-Y′Y′Y′-Nb′-(Z′Z′Z′)i-Na′-nq′ 5′  (Ij)


wherein,


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


p, p′, q, and q′ are each independently 0-6;


each Na and Na′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;


each Nb and Nb′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;


wherein


each np′, np, nq′, and nq, each of which may or may not be present independently represents an overhang nucleotide; and


XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modification on three consecutive nucleotides.


In some embodiments, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In some embodiments, k is 0 and l is 0; or k is 1 and 1 is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.


Exemplary combinations of the sense strand and antisense strand forming a RNAi duplex include the formulas below:





5′ np-Na-YYY-Na-nq 3′





3′ np′-Na′-Y′Y′Y′-Na′nq′ 5′  (Ik)





5′ np-Na-YYY-Nb-ZZZ-Na-nq 3′





3′ np-Na′-Y′Y′Y′-Nb′-Z′Z′Z′-Na′-nq′ 5′  (Il)





5′ np-Na-XXX-Nb′-YYY-Na-nq 3′





3′np-Na′-X′X′X′-Nb′-Y′Y′Y′-Na′-nq′ 5′  (Im)





5′ np-Na-XXX-Nb-YYY- Nb-ZZZ-Na-nq 3′





3′ np-Na′-X′X′X′-Nb′-Y′Y′Y′-Nb′-Z′Z′Z′-Na′-nq′ 5′  (In)


When the RNAi agent is represented by formula (Ik), each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


When the RNAi agent is represented by formula (Il), each Nb independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


When the RNAi agent is represented as formula (Im), each Nb, Nb′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


When the RNAi agent is represented as formula (In), each Nb, Nb′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na, Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of Na, Na′, Nb and Nb′ independently comprises modifications of alternating pattern.


Each of X, Y and Z in formulas (Ij), (Ik), (Il), (Im), and (In) may be the same or different from each other.


When the RNAi agent is represented by formula (Ij), (Ik), (Il), (Im), and (In), at least one of the Y nucleotides may form a base pair with one of the Y′ nucleotides. Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y′ nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y′ nucleotides.


When the RNAi agent is represented by formula (Il) or (In), at least one of the Z nucleotides may form a base pair with one of the Z′ nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z′ nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding Z′ nucleotides.


When the RNAi agent is represented as formula (Im) or (In), at least one of the X nucleotides may form a base pair with one of the X′ nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X′ nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X′ nucleotides.


In some embodiments, the modification on the Y nucleotide is different than the modification on the Y′ nucleotide, the modification on the Z nucleotide is different than the modification on the Z′ nucleotide, and/or the modification on the X nucleotide is different than the modification on the X′ nucleotide.


In some embodiments, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications. In some embodiments, when the RNAi agent is represented by formula (In), the Na modifications are 2′-O-methyl or 2′-fluoro modifications and np′>0 and at least one np′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In some embodiments, when the RNAi agent is represented by formula (In), the Na modifications are 2′-O-methyl or 2′-fluoro modifications, np′>0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more moieties or ligands (e.g., one or more lipophilic moieties, optionally one or more C16 moieties, or one or more GalNAc moieties) attached through a bivalent or trivalent branched linker. In some embodiments, when the RNAi agent is represented by formula (In), the Na modifications are 2′-O-methyl or 2′-fluoro modifications, np′>0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more moieties or ligands (e.g., one or more lipophilic moieties, optionally one or more C16 moieties, or one or more GalNAc moieties) attached through a bivalent or trivalent branched linker.


In some embodiments, when the RNAi agent is represented by formula (IIIa), the N3 modifications are 2′-O-methyl or 2′-fluoro modifications, np′>0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more moieties or ligands (e.g., one or more lipophilic moieties, optionally one or more C16 moieties, or one or more GalNAc moieties) attached through a bivalent or trivalent branched linker.


In some embodiments, the RNAi agent is a multimer containing at least two duplexes represented by formula (Ij), (Ik), (Il), (Im), and (In), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.


In some embodiments, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (Ij), (Ik), (Il), (Im), and (In), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.


In some embodiments, two RNAi agents represented by formula (Ij), (Ik), (Il), (Im), and (In) are linked to each other at the 5′ end, and one or both of the 3′ ends and are optionally conjugated to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.


Various publications describe multimeric RNAi agents that can be used in the methods of the disclosure. Such publications include WO2007/091269, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520; and U.S. Pat. No. 7,858,769, the contents of each of which are hereby incorporated herein by reference for the methods provided therein. In certain embodiments, the RNAi agents of the disclosure may include GalNAc ligands.


As described in more detail below, the RNAi agent that contains conjugations of one or more carbohydrate moieties to a RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A 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 a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.


The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.


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


In certain specific embodiments, the RNAi agent for use in the methods of the disclosure is an agent selected from the group of agents listed in any one of Tables 2A and 2B. These agents may further comprise a ligand. The ligand can be attached to the sense strand, antisense strand or both strands, at the 3′-end, 5′-end, or both ends. For instance, the ligand may be conjugated to the sense strand, in particular, the 3′-end of the sense strand.


IV. IRNA CONJUGATES

The iRNA agents disclosed herein can be in the form of conjugates. The conjugate may be attached at any suitable location in the iRNA molecule, e.g., at the 3′ end or the 5′ end of the sense or the antisense strand. The conjugates are optionally attached via a linker.


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


In some embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In some embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Typical ligands will not take part in duplex pairing in a duplexed nucleic acid.


Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Examples of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an a helical peptide.


Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.


Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-((oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.


Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as an ocular cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.


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


In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present disclosure as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.


Ligand-conjugated oligonucleotides of the disclosure may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.


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


In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present disclosure, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.


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


A. Lipophilic Moieties

In certain embodiments, the lipophilic moiety is an aliphatic, cyclic such as alicyclic, or polycyclic such as polyalicyclic compound, such as a steroid (e.g., sterol) or a linear or branched aliphatic hydrocarbon. The lipophilic moiety may generally comprise a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may comprise various substituents or one or more heteroatoms, such as an oxygen or nitrogen atom. Such lipophilic aliphatic moieties include, without limitation, saturated or unsaturated C4-C30 hydrocarbon (e.g., C6-C18 hydrocarbon), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C10 terpenes, C15 sesquiterpenes, C20 diterpenes, C30 triterpenes, and C40 tetraterpenes), and other polyalicyclic hydrocarbons. For instance, the lipophilic moiety may contain a C4-C30 hydrocarbon chain (e.g., C4-C30 alkyl or alkenyl). In some embodiments the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain (e.g., a linear C6-C18 alkyl or alkenyl). In some embodiments, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain (e.g., a linear C16 alkyl or alkenyl).


The lipophilic moiety may be attached to the RNAi agent by any method known in the art, including via a functional grouping already present in the lipophilic moiety or introduced into the RNAi agent, such as a hydroxy group (e.g., —CO—CH2—OH). The functional groups already present in the lipophilic moiety or introduced into the RNAi agent include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.


Conjugation of the RNAi agent and the lipophilic moiety may occur, for example, through formation of an ether or a carboxylic or carbamoyl ester linkage between the hydroxy and an alkyl group R—, an alkanoyl group RCO— or a substituted carbamoyl group RNHCO—. The alkyl group R may be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chained or branched; and saturated or unsaturated). Alkyl group R may be a butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl or octadecyl group, or the like.


In some embodiments, the lipophilic moiety is conjugated to the double-stranded RNAi agent via a linker a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from the azide-alkyne cycloaddition), or carbamate.


In another embodiment, the lipophilic moiety is a steroid, such as sterol. Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentanophenanthrene ring system. Steroids include, without limitation, bile acids (e.g., cholic acid, deoxycholic acid and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol, and cationic steroids, such as cortisone. A “cholesterol derivative” refers to a compound derived from cholesterol, for example by substitution, addition or removal of substituents.


In another embodiment, the lipophilic moiety is an aromatic moiety. In this context, the term “aromatic” refers broadly to mono- and polyaromatic hydrocarbons. Aromatic groups include, without limitation, C6-C14 aryl moieties comprising one to three aromatic rings, which may be optionally substituted; “aralkyl” or “arylalkyl” groups comprising an aryl group covalently linked to an alkyl group, either of which may independently be optionally substituted or unsubstituted; and “heteroaryl” groups. As used herein, the term “heteroaryl” refers to groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14n electrons shared in a cyclic array, and having, in addition to carbon atoms, one to about three heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).


As employed herein, a “substituted” alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group is one having one to about four, preferably one to about three, more preferably one or two, non-hydrogen substituents. Suitable substituents include, without limitation, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups.


In some embodiments, the lipophilic moiety is an aralkyl group, e.g., a 2-arylpropanoyl moiety. The structural features of the aralkyl group are selected so that the lipophilic moiety will bind to at least one protein in vivo. In certain embodiments, the structural features of the aralkyl group are selected so that the lipophilic moiety binds to serum, vascular, or cellular proteins. In certain embodiments, the structural features of the aralkyl group promote binding to albumin, an immunoglobulin, a lipoprotein, α-2-macroglubulin, or α-1-glycoprotein.


In certain embodiments, the ligand is naproxen or a structural derivative of naproxen. Procedures for the synthesis of naproxen can be found in U.S. Pat. Nos. 3,904,682 and 4,009,197, which are hereby incorporated by reference in their entirety. Naproxen has the chemical name (S)-6-Methoxy-α-methyl-2-naphthaleneacetic acid and the structure is




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In certain embodiments, the ligand is ibuprofen or a structural derivative of ibuprofen. Procedures for the synthesis of ibuprofen can be found in U.S. Pat. No. 3,228,831, which is incorporated herein by reference for the methods provided therein. The structure of ibuprofen is




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Additional exemplary aralkyl groups are illustrated in U.S. Pat. No. 7,626,014, which is incorporated herein by reference for the methods provided therein.


In another embodiment, suitable lipophilic moieties include lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-((oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.


In certain embodiments, more than one lipophilic moiety can be incorporated into the double-strand RNAi agent, particularly when the lipophilic moiety has a low lipophilicity or hydrophobicity. In some embodiments, two or more lipophilic moieties are incorporated into the same strand of the double-strand RNAi agent. In some embodiments, each strand of the double-strand RNAi agent has one or more lipophilic moieties incorporated. In some embodiments, two or more lipophilic moieties are incorporated into the same position (i.e., the same nucleobase, same sugar moiety, or same internucleosidic linkage) of the double-strand RNAi agent. This can be achieved by, e.g., conjugating the two or more lipophilic moieties via a carrier, or conjugating the two or more lipophilic moieties via a branched linker, or conjugating the two or more lipophilic moieties via one or more linkers, with one or more linkers linking the lipophilic moieties consecutively.


The lipophilic moiety may be conjugated to the RNAi agent via a direct attachment to the ribosugar of the RNAi agent. Alternatively, the lipophilic moiety may be conjugated to the double-strand RNAi agent via a linker or a carrier.


In certain embodiments, the lipophilic moiety may be conjugated to the RNAi agent via one or more linkers (tethers).


In some embodiments, the lipophilic moiety is conjugated to the double-stranded RNAi agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from the azide-alkyne cycloaddition), or carbamate.


B. Lipid Conjugates

In some embodiments, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule can typically bind a serum protein, such as human serum albumin (HSA). An HSA binding ligand allows for vascular distribution of the conjugate to a target tissue. For example, the target tissue can be the eye. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.


A lipid-based ligand can be used to modulate, e.g., control (e.g., inhibit) the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.


In some embodiments, the lipid-based ligand binds HSA. For example, the ligand can bind HSA with a sufficient affinity such that distribution of the conjugate to a non-kidney tissue is enhanced. However, the affinity is typically not so strong that the HSA-ligand binding cannot be reversed.


In some embodiments, the lipid-based ligand binds HSA weakly or not at all, such that distribution of the conjugate to the kidney is enhanced. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid-based ligand.


In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells.


Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low-density lipoprotein (LDL).


C. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, such as a helical cell-permeation agent. In some embodiments, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is typically an α-helical agent, and can have a lipophilic and a lipophobic phase.


The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.


A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 3). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 4)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 5)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 6)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Typically, the peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.


An RGD peptide for use in the compositions and methods of the disclosure may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidomimetics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. In some embodiments, conjugates of this ligand target PECAM-1 or VEGF.


An RGD peptide moiety can be used to target a particular cell type, e.g., a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an dsRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver a iRNA agent to a tumor cell expressing αVß3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).


A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).


D. Carbohydrate Conjugates and Ligands

In some embodiments of the compositions and methods of the disclosure, an iRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).


In certain embodiments, the compositions and methods of the disclosure include a C16 ligand. In exemplary embodiments, the C16 ligand of the disclosure has the following structure (exemplified here below for a uracil base, yet attachment of the C16 ligand is contemplated for a nucleotide presenting any base (C, G, A, etc.) or possessing any other modification as presented herein, provided that 2′ ribo attachment is preserved) and is attached at the 2′ position of the ribo within a residue that is so modified:




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As shown above, a C16 ligand-modified residue presents a straight chain alkyl at the 2′-ribo position of an exemplary residue (here, a Uracil) that is so modified.


In some embodiments, a carbohydrate conjugate of a RNAi agent of the instant disclosure further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell permeation peptide.


Additional carbohydrate conjugates (and linkers) suitable for use in the present disclosure include those described in WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.


In certain embodiments, the compositions and methods of the disclosure include a vinyl phosphonate (VP) modification of an RNAi agent as described herein. In exemplary embodiments, a vinyl phosphonate of the disclosure has the following structure:




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A vinyl phosphonate of the instant disclosure may be attached to either the antisense or the sense strand of a dsRNA of the disclosure. In certain preferred embodiments, a vinyl phosphonate of the instant disclosure is attached to the antisense strand of a dsRNA, optionally at the 5′ end of the antisense strand of the dsRNA. The dsRNA agent can comprise a phosphorus-containing group at the 5′-end of the sense strand or antisense strand. The 5′-end phosphorus-containing group can be 5′-end phosphate (5′-P), 5′-end phosphorothioate (5′-PS), 5′-end phosphorodithioate (5′-PS2), 5′-end vinylphosphonate (5′-VP), 5′-end methylphosphonate (MePhos), or 5′-deoxy-5′-C-malonyl. When the 5′-end phosphorus-containing group is 5′-end vinylphosphonate (5′-VP), the 5′-VP can be either 5′-E-VP isomer (i.e., trans-vinylphosphonate,




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5′-Z-VP isomer (i.e., cis-vinylphosphonate,




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or mixtures thereof.


Vinyl phosphate modifications are also contemplated for the compositions and methods of the instant disclosure. An exemplary vinyl phosphate structure is:




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


In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives may be attached via a linker, e.g., a bivalent or trivalent branched linker. In some embodiments the GalNAc conjugate is conjugated to the 3′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3′ end of the sense strand) via a linker, e.g., a linker as described herein.


In some embodiments, the GalNAc conjugate is




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




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In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and shown below:




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In some embodiments, a carbohydrate conjugate for use in the compositions and methods of the disclosure is selected from the group consisting of.




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




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(Formula XXIII), when one of X or Y is an oligonucleotide, the other is a hydrogen.


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


In some embodiments, an iRNA of the disclosure is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the disclosure include, but are not limited to,




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


E. Thermally Destabilizing Modifications

In certain embodiments, a dsRNA molecule can be optimized for RNA interference by incorporating thermally destabilizing modifications in the seed region of the antisense strand (i.e., at positions 2-9 of the 5′-end of the antisense strand) to reduce or inhibit off-target gene silencing. It has been discovered that dsRNAs with an antisense strand comprising at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5′ end, of the antisense strand have reduced off-target gene silencing activity. Accordingly, in some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five, or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5′ region of the antisense strand. In some embodiments, one or more thermally destabilizing modification(s) of the duplex is/are located in positions 2-9, or preferably positions 4-8, from the 5′-end of the antisense strand. In some further embodiments, the thermally destabilizing modification(s) of the duplex is/are located at position 6, 7, or 8 from the 5′-end of the antisense strand. In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5′-end of the antisense strand. The term “thermally destabilizing modification(s)” includes modification(s) that would result with a dsRNA with a lower overall melting temperature (Tm) (preferably a Tm with one, two, three, or four degrees lower than the Tm of the dsRNA without having such modification(s). In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5, or 9 from the 5′-end of the anti sense strand.


The thermally destabilizing modifications can include, but are not limited to, abasic modification; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2′-deoxy modification or acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycol nucleic acid (GNA).


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




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Wherein R═H, Me, Et or OMe; R′═H, Me, Et or OMe; R″═H, Me, Et or OMe



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wherein B is a modified or unmodified nucleobase.


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




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wherein B is a modified or unmodified nucleobase.


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




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wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either, S or racemic.


The term “acyclic nucleotide” refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the 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 oxygen (e.g., C1′, C2′, C3′, C4′, or O4′) are independently or in combination absent from the nucleotide. In some embodiments, acyclic nucleotide is




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wherein B is a modified or unmodified nucleobase, R1 and R2 independently are H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar). The term “UNA” refers to unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomers with bonds between C1′-C4′ being removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar is removed (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are hereby incorporated by reference in their entirety). The acyclic derivative provides greater backbone flexibility without affecting the Watson-Crick pairings. The acyclic nucleotide can be linked via 2′-5′ or 3′-5′ linkage.


The term ‘GNA’ refers to glycol nucleic acid which is a polymer similar to DNA or RNA but differing in the composition of its “backbone” in that is composed of repeating glycerol units linked by phosphodiester bonds:




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The thermally destabilizing modification of the duplex can be mismatches (i.e., noncomplementary base pairs) between the thermally destabilizing nucleotide and the opposing nucleotide in the opposite strand within the dsRNA duplex. Exemplary mismatch 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 a combination thereof. Other mismatch base pairings known in the art are also amenable to the present invention. A mismatch can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., the mismatch base pairing can occur between the nucleobases from respective nucleotides independent of the modifications on the ribose sugars of the nucleotides. In certain embodiments, the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2′-deoxy nucleobase; e.g., the 2′-deoxy nucleobase is in the sense strand.


In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes nucleotides with impaired W—C H-bonding to complementary base on the target mRNA, such as:




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


The thermally destabilizing modifications may also include universal base with reduced or abolished capability to form hydrogen bonds with the opposing bases, and phosphate modifications.


In some embodiments, the thermally destabilizing modification of the duplex includes nucleotides with non-canonical bases such as, but not limited to, nucleobase modifications with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand. These nucleobase modifications have been evaluated for destabilization of the central region of the dsRNA duplex as described in WO 2010/0011895, which is herein incorporated by reference in its entirety. Exemplary nucleobase modifications are:




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In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes one or more α-nucleotide complementary to the base on the tar et mRNA, such as:




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wherein R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.


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




embedded image


The alkyl for the R group can be a C1-C6alkyl. Specific alkyls for the R group include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.


As the skilled artisan will recognize, in view of the functional role of nucleobases is defining specificity of a RNAi agent of the disclosure, while nucleobase modifications can be performed in the various manners as described herein, e.g., to introduce destabilizing modifications into a RNAi agent of the disclosure, e.g., for purpose of enhancing on-target effect relative to off-target effect, the range of modifications available and, in general, present upon RNAi agents of the disclosure tends to be much greater for non-nucleobase modifications, e.g., modifications to sugar groups or phosphate backbones of polyribonucleotides. Such modifications are described in greater detail in other sections of the instant disclosure and are expressly contemplated for RNAi agents of the disclosure, either possessing native nucleobases or modified nucleobases as described above or elsewhere herein.


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


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 still 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 the destabilizing modification. For example, the stabilizing modification can be the nucleotide at the 5′-end or the 3′-end of the destabilizing modification, i.e., at position −1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5′-end and the 3′-end of the destabilizing modification, i.e., positions −1 and +1 from the position 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 positions +1 and +2 from the position 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 limitations, a stabilizing modification in the sense strand can be present at any positions. 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 opposite or complimentary 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 stabilizing modifications at positions opposite or complimentary to 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 block of two, three, or four stabilizing modifications.


In some embodiments, the sense strand does not comprise a stabilizing modification in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.


Exemplary thermally stabilizing modifications include, but are not limited to, 2′-fluoro modifications. Other thermally stabilizing 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 limitations, the 2′-fluoro nucleotides all can be present in one strand. In some embodiments, both the sense and the antisense strands comprise at least two 2′-fluoro nucleotides. The 2′-fluoro modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the 2′-fluoro modification can occur on every nucleotide on the sense strand or antisense strand; each 2′-fluoro modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both 2′-fluoro modifications in an alternating pattern. The alternating pattern of the 2′-fluoro modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the 2′-fluoro modifications on the sense strand can have a shift relative to the alternating pattern of the 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 limitations, a 2′-fluoro modification in the antisense strand can be present at any positions. In some embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5′-end. In some other embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5′-end. In still some other embodiments, the antisense 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 the destabilizing modification. For example, the 2′-fluoro nucleotide can be the nucleotide at the 5′-end or the 3′-end of the destabilizing modification, i.e., at position −1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a 2′-fluoro nucleotide at each of the 5′-end and the 3′-end of the destabilizing modification, i.e., positions −1 and +1 from the position of the destabilizing modification.


In some embodiments, the antisense strand comprises at least two 2′-fluoro nucleotides at the 3′-end of the destabilizing modification, i.e., at positions +1 and +2 from the position 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) 2′-fluoro nucleotides. Without limitations, a 2′-fluoro modification in the sense strand can be present at any positions. In some embodiments, the antisense 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 at positions opposite or complimentary 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 positions opposite or complimentary to 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 block of two, three, or four 2′-fluoro nucleotides.


In some embodiments, the sense strand does not comprise a 2′-fluoro nucleotide in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.


In some embodiments, the dsRNA molecule of the disclosure comprises a 21 nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense, wherein the antisense strand contains at least one thermally destabilizing nucleotide, where the at least one thermally destabilizing nucleotide occurs in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), wherein one end of the dsRNA is blunt, while the other end is comprises a 2 nt overhang, 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 comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a 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 5′-end of the antisense strand. Preferably, the 2 nt overhang is at the 3′-end of the antisense.


In some embodiments, every nucleotide in the sense strand and antisense strand of the dsRNA molecule may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.


As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases, the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. E.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.


It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.


In some embodiments, each residue of the sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, or 2′-fluoro. The strands can contain more than one modification. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. It is to be understood that these modifications are in addition to the at least one thermally destabilizing modification of the duplex present in the antisense strand.


At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-deoxy, 2′-O-methyl, or 2′-fluoro modifications, acyclic nucleotides or others. In some embodiments, the sense strand and antisense strand each comprises two differently modified nucleotides selected from 2′-O-methyl or 2′-deoxy. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl nucleotide, 2′-deoxy nucleotide, 2′-deoxy-2′-fluoro nucleotide, 2′-O—N-methylacetamido (2′-O-NMA) nucleotide, a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotide, 2′-O-aminopropyl (2′-O-AP) nucleotide, or 2′-ara-F nucleotide. Again, it is to be understood that these modifications are in addition to the at least one thermally destabilizing modification of the duplex present in the antisense strand.


In some embodiments, the dsRNA molecule of the disclosure comprises modifications of an alternating pattern, particular in the B1, B2, B3, B1′, B2′, B3′, B4′ regions. The term “alternating motif” or “alternative pattern” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.


The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.


In some embodiments, the dsRNA molecule of the disclosure comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 3′-5′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 3′-5′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.


The dsRNA molecule of the disclosure may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.


In some embodiments, the dsRNA molecule comprises the phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region comprises two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3′-end of the antisense strand.


In some embodiments, the sense strand of the dsRNA molecule comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.


In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate 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 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.


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


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


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


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


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


In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.


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


In some embodiments, the dsRNA molecule of the disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within positions 1-10 of the termini position(s) of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage at one end or both ends of the sense or antisense strand.


In some embodiments, the dsRNA molecule of the disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within positions 1-10 of the internal region of the duplex of each of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked through phosphorothioate methylphosphonate internucleotide linkage at position 8-16 of the duplex region counting from the 5′-end of the sense strand; the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modification within positions 1-10 of the termini position(s).


In some embodiments, the dsRNA molecule of the disclosure further comprises one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 1-5 and one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 18-23 of the sense strand (counting from the 5′-end), and one to five phosphorothioate or methylphosphonate internucleotide linkage modification at positions 1 and 2 and one to five within positions 18-23 of the antisense strand (counting from the 5′-end).


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


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 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 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 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 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 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 within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 (counting from the 5′-end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 (counting from the 5′-end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one within position 18-23 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 within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 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 within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 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 18-23 of the antisense strand (counting from the 5′-end).


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


In some embodiments, the dsRNA molecule of the disclosure further comprises 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 the antisense strand (counting from the 5′-end).


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


In some embodiments, the dsRNA molecule of the disclosure further comprises 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 the antisense strand (counting from the 5′-end).


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


In some embodiments, the dsRNA molecule of the disclosure further comprises 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 the antisense strand (counting from the 5′-end).


In some embodiments, compound of the disclosure comprises a pattern of backbone chiral centers. In some embodiments, a common pattern of backbone chiral centers comprises at least 5 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 6 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 7 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 8 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 9 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 16 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 17 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 18 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 19 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages which are not chiral (as a non-limiting example, a phosphodiester). In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration, and no more than 8 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration, and no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration, and no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration, and no more than 4 internucleotidic linkages which are not chiral. In some embodiments, the internucleotidic linkages in the Sp configuration are optionally contiguous or not contiguous. In some embodiments, the internucleotidic linkages in the Rp configuration are optionally contiguous or not contiguous. In some embodiments, the internucleotidic linkages which are not chiral are optionally contiguous or not contiguous.


In some embodiments, compound of the disclosure comprises a block is a stereochemistry block. In some embodiments, a block is an Rp block in that each internucleotidic linkage of the block is Rp. In some embodiments, a 5′-block is an Rp block. In some embodiments, a 3′-block is an Rp block. In some embodiments, a block is an Sp block in that each internucleotidic linkage of the block is Sp. In some embodiments, a 5′-block is an Sp block. In some embodiments, a 3′-block is an Sp block. In some embodiments, provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks. In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each internucleotidic linkage in a natural phosphate linkage.


In some embodiments, compound of the disclosure comprises a 5′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block comprises 4 or more nucleoside units. In some embodiments, a 5′-block comprises 5 or more nucleoside units. In some embodiments, a 5′-block comprises 6 or more nucleoside units. In some embodiments, a 5′-block comprises 7 or more nucleoside units. In some embodiments, a 3′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block comprises 4 or more nucleoside units. In some embodiments, a 3′-block comprises 5 or more nucleoside units. In some embodiments, a 3′-block comprises 6 or more nucleoside units. In some embodiments, a 3′-block comprises 7 or more nucleoside units.


In some embodiments, compound of the disclosure comprises a type of nucleoside in a region or an oligonucleotide is followed by a specific type of internucleotidic linkage, e.g., natural phosphate linkage, modified internucleotidic linkage, Rp chiral internucleotidic linkage, Sp chiral internucleotidic linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by natural phosphate linkage (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 natural phosphate linkage (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 dsRNA molecule of the disclosure comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mismatch can occur in the overhang region or the duplex region. The base pair can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.


In some embodiments, the dsRNA molecule of the disclosure comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.


In some embodiments, the nucleotide at the 1 position within the duplex region from the 5′-end in 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 pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.


It was found that introducing 4′-modified or 5′-modified nucleotide to the 3′-end of a phosphodiester (PO), phosphorothioate (PS), or phosphorodithioate (PS2) linkage of a dinucleotide at any position of single stranded or double stranded oligonucleotide can exert steric effect to the internucleotide linkage and, hence, protecting or stabilizing it against nucleases.


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


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


In some embodiments, 5′-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 5′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 5′-alkylated nucleoside is 5′-methyl nucleoside. The 5′-methyl can be either racemic or chirally pure R or S isomer.


In some embodiments, 4′-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 4′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4′-alkylated nucleoside is 4′-methyl nucleoside. The 4′-methyl can be either racemic or chirally pure R or S isomer.


In some embodiments, 4′-O-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 5′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4′-O-alkylated nucleoside is 4′-O-methyl nucleoside. The 4′-O-methyl can be either racemic or chirally pure R or S isomer.


In some embodiments, the dsRNA molecule of the disclosure can comprise 2′-5′ linkages (with 2′-H, 2′-OH, and 2′-OMe and with P═O or P═S). For example, the 2′-5′ linkages modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.


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


Various publications describe multimeric siRNA which can all be used with the dsRNA of the disclosure. Such publications include WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/11768 6, WO2009/014887, and WO2011/031520 which are hereby incorporated by their entirely.


In some embodiments dsRNA molecules of the disclosure are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R═alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, 5′-alkenylphosphonates (i.e. vinyl, substituted vinyl), (OH)2(O)P-5′-CH2—), 5′-alkyletherphosphonates (R═alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-). In one example, the modification can in placed in the antisense strand of a dsRNA molecule.


F. Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.


Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In some embodiments, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.


In some embodiments, a dsRNA of the disclosure is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXI)-(XXXIV):




embedded image


wherein:


q2A, q2B, q3A, q3B, q4A, q4B q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;


P2A p2B, p3A, P3B, P4A, P4B, P5A, P5B, P5C, T2A, T2B, T3A, T3B, T4A, T4B, T5B, T5C are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O) CH2, CH2NH or CH2O;


Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, Q5C are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R′)═C(R″), C≡C or C(O);


R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, R5C are each independently for each occurrence absent, NH, O, S CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), —C(O)—CH(Ra)—NH—, CO, CH═N—O,




embedded image


L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B and L5C represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and Ra is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):




embedded image


wherein L5A, L5B and L5C represent a monosaccharide, such as GalNAc derivative.


Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.


A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a some embodiments, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).


Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.


A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a suitable pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.


A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted.


In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In some embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).


i. Redox Cleavable Linking Groups


In some embodiments, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.


ii. Phosphate-Based Cleavable Linking Groups


In some embodiments, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. 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—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—. In some embodiments, phosphate-based linking groups are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —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—, —O—P(S)(H)—S—. In some embodiments, a phosphate-based linking group is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.


iii. Acid Cleavable Linking Groups


In some embodiments, a 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 acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). In some embodiments, the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.


IV. ESTER-BASED CLEAVABLE LINKING GROUPS

In some embodiments, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.


V. PEPTIDE-BASED CLEAVABLE LINKING GROUPS

In some embodiments, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide-based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula -NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above. Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which is herein incorporated by reference.


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


“Chimeric” iRNA compounds, or “chimeras,” in the context of the present disclosure, are iRNA compounds, e.g., dsRNAs, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.


In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are 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., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad Sci., 1992, 660:306; Manoharan et al., Bioorg. Med Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.


V. DELIVERY OF IRNA

The delivery of an iRNA to a subject in need thereof can be achieved in a number of different ways. li vivo delivery can be performed directly by administering a composition comprising an iRNA, e.g. a dsRNA, to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.


A. Direct Delivery

In general, any method of delivering a nucleic acid molecule can be adapted for use with an iRNA (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). However, there are three factors that are important to consider in order to successfully deliver an iRNA molecule in vivo: (a) biological stability of the delivered molecule, (2) preventing non-specific effects, and (3) accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example by direct injection or implantation into a tissue (as a non-limiting example, the eye) or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that may otherwise be harmed by the agent or that may degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ., et al (2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al(2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, ER., et al (2004) Proc. Natl. Acad Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, KA., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo.


Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to other groups, e.g., a lipid or carbohydrate group as described herein. Such conjugates can be used to target iRNA to particular cells, e.g., liver cells, e.g., hepatocytes. For example, GalNAc conjugates or lipid (e.g., LNP) formulations can be used to target iRNA to particular cells, e.g., liver cells, e.g., hepatocytes.


iRNA molecules can also be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O., et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic- iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328: Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.


B. Vector Encoded iRNAs


In another aspect, iRNA targeting MYOC can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).


The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively, each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In some embodiments, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.


An iRNA expression vector is typically a DNA plasmid or viral vector. An expression vector compatible with eukaryotic cells, e.g., with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors contain convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.


An iRNA expression plasmid can be transfected into a target cell as a complex with a cationic lipid carrier (e.g., Oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the disclosure. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.


Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno- associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.


Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.


Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.


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


Adenoviruses are also contemplated for use in delivery of iRNAs. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3.499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991): Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing an iRNA featured in the disclosure, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.


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


Another typical viral vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.


The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.


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


VI. PHARMACEUTICAL COMPOSITIONS CONTAINING IRNA

In some embodiments, the disclosure provides pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the iRNA is useful for treating a disease or disorder related to the expression or activity of MYOC (e.g., glaucoma, e.g., primary open angle glaucoma (POAG)). Such pharmaceutical compositions are formulated based on the mode of delivery. In some embodiments, compositions can be formulated for localized delivery, e.g., by intraocular delivery (e.g., intravitreal administration, e.g., intravitreal injection; transscleral administration, e.g., transscleral injection; subconjunctival administration, e.g., subconjunctival injection; retrobulbar administration, e.g., retrobulbar injection; intracameral administration, e.g., intracameral injection; or subretinal administration, e.g., subretinal injection). In other embodiments, compositions can be formulated for topical delivery. In another example, compositions can be formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. In some embodiments, a composition provided herein (e.g., a composition comprising a GalNAc conjugate or an LNP formulation) is formulated for intravenous delivery.


The pharmaceutical compositions featured herein are administered in a dosage sufficient to inhibit expression of MYOC. In general, a suitable dose of iRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day. The pharmaceutical composition may be administered once daily, or the iRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as can be used with the agents of the present disclosure. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.


The effect of a single dose on MYOC levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5-day intervals, or at not more than 1, 2, 3, 4, 12, 24, or 36-week intervals.


The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the disclosure can be made using conventional methodologies or on the basis of in vivo testing using a suitable animal model.


A suitable animal model, e.g., a mouse or a cynomolgus monkey, e.g., an animal containing a transgene expressing human MYOC, can be used to determine the therapeutically effective dose and/or an effective dosage regimen administration of MYOC siRNA.


The present disclosure also includes pharmaceutical compositions and formulations that include the iRNA compounds featured herein. The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be local (e.g., by intraocular injection), topical (e.g., by an eye drop solution), or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal, or intraventricular administration.


Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the iRNAs featured in the disclosure are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the disclosure may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.


A. Liposomal Formulations

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present disclosure, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.


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


In order to traverse intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.


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


Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.


Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.


Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis


Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).


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


One major type of liposomal composition includes phospholipids other than naturally derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.


Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).


Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).


Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).


Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).


Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety. Ilium et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Aca, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.). Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.


A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.


Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-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 emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (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 find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.


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


If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.


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


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


B. Nucleic Acid Lipid Particles

In some embodiments, a MYOC dsRNA featured in the disclosure is fully encapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present disclosure typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid- lipid particles of the present disclosure are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.


In some embodiments, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.


The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.


In some embodiments, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.


In some embodiments, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.


The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.


The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or a PEG- distearyloxypropyl (C]8). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about mol % or about 2 mol % of the total lipid present in the particle.


In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.


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


LNP01

In some embodiments, the lipidoid ND98·4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is herein incorporated by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (e.g., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.




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LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.


Additional exemplary lipid-dsRNA formulations are provided in the following table.









TABLE 4







Exemplary lipid formulations











cationic lipid/non-cationic




lipid/cholesterol/PEG-lipid conjugate



Cationic Lipid
Lipid:siRNA ratio













SNALP
1,2-Dilinolenyloxy-N,N-
DLinDMA/DPPC/Cholesterol/PEG-



dimethylaminopropane (DLinDMA)
cDMA




(57.1/7.1/34.4/1.4)




lipid:siRNA~7:1


S-XTC
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DPPC/Cholesterol/PEG-cDMA



[1,3]-dioxolane (XTC)
57.1/7.1/34.4/1.4




lipid:siRNA~7:1


LNP05
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG



[1,3]-dioxolane (XTC)
57.5/7.5/31.5/3.5




lipid:siRNA~6:1


LNP06
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG



[1,3]-dioxolane (XTC)
57.5/7.5/31.5/3.5




lipid:siRNA~11:1


LNP07
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG



[1,3]-dioxolane (XTC)
60/7.5/31/1.5,




lipid:siRNA~6:1


LNP08
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG



[1,3]-dioxolane (XTC)
60/7.5/31/1.5,




lipid:siRNA~11:1


LNP09
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG



[1,3]-dioxolane (XTC)
50/10/38.5/1.5




Lipid:siRNA 10:1


LNP10
(3aR,5s,6aS)-N,N-dimethyl-2,2-
ALN100/DSPC/Cholesterol/PEG-DMG



di((9Z,12Z)-octadeca-9,12-
50/10/38.5/1.5



dienyl)tetrahydro-3aH-
Lipid:siRNA 10:1



cyclopenta[d][1,3]dioxol-5-amine



(ALN100)


LNP11
(6Z,9Z,28Z,31Z)-heptatriaconta-
MC-3/DSPC/Cholesterol/PEG-DMG



6,9,28,31-tetraen-19-yl 4-
50/10/38.5/1.5



(dimethylamino)butanoate (MC3)
Lipid:siRNA 10:1


LNP12
1,1′-(2-(4-(2-((2-(bis(2-
C12-200/DSPC/Cholesterol/PEG-DMG



hydroxydodecyl)amino)ethyl)(2-
50/10/38.5/1.5



hydroxydodecyl)amino)ethyl)piperazin-
Lipid:siRNA 10:1



1-yl)ethylazanediyl)didodecan-2-ol



(C12-200)


LNP13
XTC
XTC/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 33:1


LNP14
MC3
MC3/DSPC/Chol/PEG-DMG




40/15/40/5




Lipid:siRNA: 11:1


LNP15
MC3
MC3/DSPC/Chol/PEG-DSG/GalNAc-




PEG-DSG




50/10/35/4.5/0.5




Lipid:siRNA: 11:1


LNP16
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 7:1


LNP17
MC3
MC3/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 10:1


LNP18
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 12:1


LNP19
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/35/5




Lipid:siRNA: 8:1


LNP20
MC3
MC3/DSPC/Chol/PEG-DPG




50/10/38.5/1.5




Lipid:siRNA: 10:1


LNP21
C12-200
C12-200/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 7:1


LNP22
XTC
XTC/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 10:1










DSPC: distearoylphosphatidylcholine


DPPC: dipalmitoylphosphatidylcholine


PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000)


PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000)


PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)


SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.


XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. 61/185,712, filed Jun. 10, 2009: U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference.


MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, and International Application No. PCT/US10/28224, filed Jun. 10, 2010, which are hereby incorporated by reference.


ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.


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


C. Synthesis of Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles featured in the disclosure may be prepared by known organic synthesis techniques. All substituents are as defined below unless indicated otherwise.


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


“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.


“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.


“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.


“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.


The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —ORx, —NRxRy, —NRx(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy.


“Halogen” means fluoro, chloro, bromo and iodo.


In some embodiments, the methods featured in the disclosure may require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this disclosure are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.


Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles featured in the disclosure are formulated using a cationic lipid of formula A:




embedded image


where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.




embedded image


Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.




embedded image


Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.


Synthesis of MC3

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


Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:




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Synthesis of 515:

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).


Synthesis of 516:

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with IN HCl solution (1×100 mL) and saturated NaHCO3solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H]− 232.3 (96.94%).


Synthesis of 517A and 517B:

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (˜3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (1x 50 mL). Organic phase was dried over an·Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: −6 g crude


517A —Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS −[M+H]−266.3, [M+NH4+]−283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.


Synthesis of 518:

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


General Procedure for the Synthesis of Compound 519:

A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6, Found 654.6.


Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.


Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the disclosure are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the disclosure may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.


Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intravitreal, subretinal, transscleral, subconjunctival, retrobulbar, intracameral, 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 generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.


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


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


D. Additional Formulations

i. Emulsions


The compositions of the present disclosure may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1p m in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, 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., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise, a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.


Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).


Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, 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 typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).


Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.


A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, 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).


Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.


Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.


The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, 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 base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.


In some embodiments of the present disclosure, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically, microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotopically clear dispersions of two immiscible liquids that are stabilized by interfacial films 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 commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).


The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, 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). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.


Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, 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, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.


Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present disclosure will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.


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


ii. Penetration Enhancers


In some embodiments, the present disclosure employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.


Penetration enhancers may be classified as belonging to one of five broad categories, 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). Each of the above-mentioned classes of penetration enhancers are described below in greater detail.


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


Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, M A, 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).


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


Chelating Agents: Chelating agents, as used in connection with the present disclosure, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present disclosure, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of β-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, M A, 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).


Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).


Agents that enhance uptake of iRNAs at the cellular level may 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. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, CA), Lipofectamine™ (Invitrogen; Carlsbad, CA), RNAiMAX (Invitrogen; Carlsbad, CA), Oligofectamine™ (Invitrogen; Carlsbad, CA), Optifect™ (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, WI), TransFast™ Transfection Reagent (Promega; Madison, WI), Tfx™-20 Reagent (Promega; Madison, WI), Tfx™-50 Reagent (Promega; Madison, WI), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass™ D1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec™/LipoGen™ (Invivogen; San Diego, CA, USA), PerFectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, CA, USA), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), or HiFect™ (B-Bridge International, Mountain View, CA, USA), among others.


Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.


iii. Carriers


Certain compositions of the present disclosure also incorporate carrier compounds in the formulation. As used herein, “carrier compound” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).


iv. Excipients


In contrast to a carrier compound, a pharmaceutical carrier or excipient may comprise, e.g., a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).


Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.


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


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


v. Other Components


The compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions, e.g., at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.


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


In some embodiments, pharmaceutical compositions featured in the disclosure include (a) one or more iRNA compounds and (b) one or more biologic agents which function by a non-RNAi mechanism. Examples of such biologic agents include agents that interfere with an interaction of MYOC and at least one MYOC binding partner.


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


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 compositions featured in the disclosure lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.


In addition to their administration, as discussed above, the iRNAs featured in the disclosure can be administered in combination with other known agents effective in treatment of diseases or disorders related to MYOC expression (e.g., glaucoma, e.g., primary open angle glaucoma (POAG)). In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.


VII. METHODS OF TREATING DISORDERS RELATED TO EXPRESSION OF MYOC

The present disclosure relates to the use of an iRNA targeting MYOC to inhibit MYOC expression and/or to treat a disease, disorder, or pathological process that is related to MYOC expression (e.g., glaucoma, e.g., primary open angle glaucoma (POAG)).


In some aspects, a method of treatment of a disorder related to expression of MYOC is provided, the method comprising administering an iRNA (e.g., a dsRNA) disclosed herein to a subject in need thereof. In some embodiments, the iRNA inhibits (decreases) MYOC expression.


In some embodiments, the subject is an animal that serves as a model for a disorder related to MYOC expression, e.g., glaucoma, e.g., primary open angle glaucoma (POAG). .


A. Glaucoma

In some embodiments, the disorder related to MYOC expression is glaucoma. A non-limiting example of glaucoma that is treatable using the method described herein includes primary open angle glaucoma (POAG).


Clinical and pathological features of glaucoma include, but are not limited to, vision loss, a reduction in visual acuity (e.g., halos around lights and blurriness)) and decreased leakage of aqueous humor from the eye.


In some embodiments, the subject with glaucoma is less than 18 years old. In some embodiments, the subject with glaucoma is an adult. In some embodiments, the subject with glaucoma is more than 60 years old. In some embodiments, the subject has, or is identified as having, elevated levels of MYOC mRNA or protein relative to a reference level (e.g., a level of MYOC that is greater than a reference level).


In some embodiments, glaucoma is diagnosed using analysis of a sample from the subject (e.g., an aqueous ocular fluid sample). In some embodiments, the sample is analyzed using a method selected from one or more of: fluorescent in situ hybridization (FISH), immunohistochemistry, MYOC immunoassay, electron microscopy, laser microdissection, and mass spectrometry. In some embodiments, glaucoma is diagnosed using any suitable diagnostic test or technique, e.g., Goldmann Applanation Tonometry, measurement of central corneal thickness (CCT), automated static threshold perimetry (e.g. Humphrey field analysis), Van Herick technique, gonioscopy, ultrasound biomicroscopy and anterior segment optical coherence tomography (AS-OCT), angiography (e.g., fluorescein angiography or indocyanine green angiography), electroretinography, ultrasonography, pachymetry, optical coherence tomography (OCT), computed tomography (CT) and magnetic resonance imaging (MRI), tonometry, color vision testing, visual field testing, slit-lamp examination, ophthalmoscopy, and physical examination (e.g., to assess visual acuity (e.g., by fundoscopy or optical coherence tomography (OCT)).


B. Combination Therapies

In some embodiments, an iRNA (e.g., a dsRNA) disclosed herein is administered in combination with a second therapy (e.g., one or more additional therapies) known to be effective in treating a disorder related to MYOC expression (glaucoma, e.g., primary open angle glaucoma (POAG)) or a symptom of such a disorder. The iRNA may be administered before, after, or concurrent with the second therapy. In some embodiments, the iRNA is administered before the second therapy. In some embodiments, the iRNA is administered after the second therapy. In some embodiments, the iRNA is administered concurrent with the second therapy.


The second therapy may be an additional therapeutic agent. The iRNA and the additional therapeutic agent can be administered in combination in the same composition or the additional therapeutic agent can be administered as part of a separate composition.


In some embodiments, the second therapy is a non-iRNA therapeutic agent that is effective to treat the disorder or symptoms of the disorder.


In some embodiments, the iRNA is administered in conjunction with a therapy.


Exemplary combination therapies include, but are not limited to, laser trabeculoplasty surgery, trabeculectomy surgery, a minimally invasive glaucoma surgery, placement of a drainage tube in the eye, oral medication or eye drops.


Administration Dosages, Routes, and Timing

A subject (e.g., a human subject, e.g., a patient) can be administered a therapeutic amount of iRNA. The therapeutic amount can be, e.g., 0.05-50 mg/kg.


In some embodiments, the iRNA is formulated for delivery to a target organ, e.g., to the eye.


In some embodiments, the iRNA is formulated as a lipid formulation, e.g., an LNP formulation as described herein. In some such embodiments, the therapeutic amount is 0.05-5 mg/kg dsRNA. In some embodiments, the lipid formulation, e.g., LNP formulation, is administered intravenously.


In some embodiments, the iRNA is in the form of a GalNAc conjugate e.g., as described herein. In some such embodiments, the therapeutic amount is 0.5-50 mg dsRNA. In some embodiments, the e.g., GalNAc conjugate is administered subcutaneously.


In some embodiments, the administration is repeated, for example, on a regular basis, such as, daily, biweekly (i.e., every two weeks) for one month, two months, three months, four months, six months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. In some embodiments, the iRNA agent is administered in two or more doses. In some embodiments, the number or amount of subsequent doses is dependent on the achievement of a desired effect, e.g., to(a) inhibit or reduce the expression or activity of MYOC; (b) reduce the level of misfolded MYOC protein; (c) reduce trabecular meshwork cell death; (d) decrease intraocular pressure; or (e) increase visual acuity, or the achievement of a therapeutic or prophylactic effect, e.g., reduction or prevention of one or more symptoms associated with the disorder.


In some embodiments, the iRNA agent is administered according to a schedule. For example, the iRNA agent may be administered once per week, twice per week, three times per week, four times per week, or five times per week. In some embodiments, the schedule involves regularly spaced administrations, e.g., hourly, every four hours, every six hours, every eight hours, every twelve hours, daily, every 2 days, every 3 days, every 4 days, every 5 days, weekly, biweekly, or monthly. In some embodiments, the iRNA agent is administered at the frequency required to achieve a desired effect.


In some embodiments, the schedule involves closely spaced administrations followed by a longer period of time during which the agent is not administered. For example, the schedule may involve an initial set of doses that are administered in a relatively short period of time (e.g., about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, or about every 72 hours) followed by a longer time period (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks) during which the iRNA agent is not administered. In some embodiments, the iRNA agent is initially administered hourly and is later administered at a longer interval (e.g., daily, weekly, biweekly, or monthly). In some embodiments, the iRNA agent is initially administered daily and is later administered at a longer interval (e.g., weekly, biweekly, or monthly). In certain embodiments, the longer interval increases overtime or is determined based on the achievement of a desired effect.


Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion dose, and monitored for adverse effects, such as an allergic reaction, or for elevated lipid levels or blood pressure. In another example, the patient can be monitored for unwanted effects.


VIII. METHODS FOR MODULATING EXPRESSION OF MYOC

In some aspects, the disclosure provides a method for modulating (e.g., inhibiting or activating) the expression of MYOC, e.g., in a cell, in a tissue, or in a subject. In some embodiments, the cell or tissue is ex vivo, in vitro, or in vivo. In some embodiments, the cell or tissue is in the eye (e.g., a trabecular meshwork tissue, a ciliary body, a retinal pigment epithelium (RPE), a retinal tissue, an astrocyte, a pericyte, a Müller cell, a ganglion cell, an endothelial cell, a photoreceptor cell, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel). In some embodiments, the cell or tissue is in a subject (e.g., a mammal, such as, for example, a human). In some embodiments, the subject (e.g., the human) is at risk, or is diagnosed with a disorder related to expression of MYOC expression, as described herein.


In some embodiments, the method includes contacting the cell with an iRNA as described herein, in an amount effective to decrease the expression of MYOC in the cell. In some embodiments, contacting a cell with an RNAi agent includes contacting a cell in vitro with the RNAi agent or contacting a cell in vivo with the RNAi agent. In some embodiments, the RNAi agent is put into physical contact with the cell by the individual performing the method, or the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell. Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area, e.g., ocular tissue. For example, the RNAi agent may contain or be coupled to a ligand, e.g., a lipophilic moiety or moieties as described below and further detailed, e.g., in PCT/US2019/031170 which is incorporated herein by reference in its entirety, including the passages therein describing lipophilic moieties, that directs or otherwise stabilizes the RNAi agent at a site of interest. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.


The expression of MYOC may be assessed based on the level of expression of MYOC mRNA, MYOC protein, or the level of another parameter functionally linked to the level of expression of MYOC. In some embodiments, the expression of MYOC is inhibited by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, the iRNA has an IC50 in the range of 0.001-0.01 nM, 0.001-0.10 nM, 0.001-1.0 nM, 0.001-10 nM, 0.01-0.05 nM, 0.01-0.50 nM, 0.02-0.60 nM, 0.01-1.0 nM, 0.01-1.5 nM, 0.01-10 nM. The IC50 value may be normalized relative to an appropriate control value, e.g., the IC50 of a non-targeting iRNA.


In some embodiments, the method includes introducing into the cell or tissue an iRNA as described herein and maintaining the cell or tissue for a time sufficient to obtain degradation of the mRNA transcript of MYOC, thereby inhibiting the expression of MYOC in the cell or tissue.


In some embodiments, the method includes administering a composition described herein, e.g., a composition comprising an iRNA that binds MYOC, to the mammal such that expression of the target MYOC is decreased, such as for an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, or four weeks or longer. In some embodiments, the decrease in expression of MYOC is detectable within 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours of the first administration.


In some embodiments, the method includes administering a composition as described herein to a mammal such that expression of the target MYOC is increased by e.g., at least 10% compared to an untreated animal. In some embodiments, the activation of MYOC occurs over an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, four weeks, or more. Without wishing to be bound by theory, an iRNA can activate MYOC expression by stabilizing the MYOC mRNA transcript, interacting with a promoter in the genome, or inhibiting an inhibitor of MYOC expression.


The iRNAs useful for the methods and compositions featured in the disclosure specifically target RNAs (primary or processed) of MYOC. Compositions and methods for inhibiting the expression of MYOC using iRNAs can be prepared and performed as described elsewhere herein.


In some embodiments, the method includes administering a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of MYOC of the subject, e.g., the mammal, e.g., the human, to be treated. The composition may be administered by any appropriate means known in the art including, but not limited to ocular (e.g., intraocular), topical, and intravenous administration.


In certain embodiments, the composition is administered intraocularly (e.g., by intravitreal administration, e.g., intravitreal injection; transscleral administration, e.g., transscleral injection; subconjunctival administration, e.g., subconjunctival injection; retrobulbar administration, e.g., retrobulbar injection; intracameral administration, e.g., intracameral injection; or subretinal administration, e.g., subretinal injection. In other embodiments, the composition is administered topically. In other embodiments, the composition is administered by intravenous infusion or injection.


In certain embodiments, the composition is administered by intravenous infusion or injection. In some such embodiments, the composition comprises a lipid formulated siRNA (e.g., an LNP formulation, such as an LNP 11 formulation) for intravenous infusion.


Unless otherwise defined, 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 disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned 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.


SPECIFIC EMBODIMENTS





    • 1. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of myocilin (MYOC), 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 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of a coding strand of human MYOC and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of a non-coding strand of human MYOC such that the sense strand is complementary to the at least 15 contiguous nucleotides in the antisense strand.

    • 2. The dsRNA agent of embodiment 1, wherein the coding strand of human MYOC comprises the sequence SEQ ID NO: 1.

    • 3. The dsRNA agent of embodiment 1 or 2, wherein the non-coding strand of human MYOC comprises the sequence of SEQ ID NO: 2.

    • 4. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of MYOC, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 15 contiguous nucleotides in the antisense strand.

    • 5. The dsRNA agent of embodiment 4, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

    • 5a. The dsRNA agent of any of the preceding embodiments, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from the antisense strand nucleotide sequence of duplex AD-1565804 and the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from the sense strand nucleotide sequence of duplex AD-1565804.

    • 5b. The dsRNA agent of any of the preceding embodiments, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from the antisense strand nucleotide sequence of duplex AD-1565837 and the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from the sense strand nucleotide sequence of duplex AD-1565837.

    • 6. The dsRNA of any of the preceding embodiments, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 17 contiguous nucleotides in the antisense strand.

    • 7. The dsRNA of embodiment 6, wherein the sense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

    • 8. The dsRNA of any of the preceding embodiments, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 19 contiguous nucleotides in the antisense strand.

    • 9. The dsRNA of embodiment 8, wherein the sense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

    • 10. The dsRNA of any of the preceding embodiments, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of nucleotide sequence of SEQ ID NO: 2 such that the sense strand is complementary to the at least 21 contiguous nucleotides in the antisense strand.

    • 11. The dsRNA of embodiment 10, wherein the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, or 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO: 1.

    • 12. The dsRNA agent of any one of the preceding embodiments, wherein the portion of the sense strand is a portion within a sense strand in any one of Tables 2A and 2B.

    • 13. The dsRNA agent of any one of the preceding embodiments, wherein the portion of the antisense strand is a portion within an antisense strand in any one of Tables 2A and 2B.

    • 14. The dsRNA agent of any of the preceding embodiments, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A and 2B.

    • 15. The dsRNA agent of any of the preceding embodiments, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A and 2B that corresponds to the antisense sequence.

    • 16. The dsRNA agent of any of the preceding embodiments, wherein the antisense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A and 2B.

    • 17. The dsRNA agent of any of the preceding embodiments, wherein the sense strand comprises a nucleotide sequence comprising at least 17 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A and 2B that corresponds to the antisense sequence.

    • 18. The dsRNA agent of any of the preceding embodiments, wherein the antisense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A and 2B.

    • 19. The dsRNA agent of any of the preceding embodiments, wherein the sense strand comprises a nucleotide sequence comprising at least 19 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A and 2B that corresponds to the antisense sequence.

    • 20. The dsRNA agent of any of the preceding embodiments, wherein the antisense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0,1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A and 2B.

    • 21. The dsRNA agent of any of the preceding embodiments, wherein the sense strand comprises a nucleotide sequence comprising at least 21 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A and 2B that corresponds to the antisense sequence.

    • 22. The dsRNA agent of any of the preceding embodiments, wherein the sense strand is at least 23 nucleotides in length, e.g., 23-30 nucleotides in length.

    • 23. The dsRNA agent of any of the preceding embodiments, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties.

    • 24. The dsRNA agent of embodiment 23, wherein the lipophilic moiety is conjugated to one or more positions in the double stranded region of the dsRNA agent.

    • 25. The dsRNA agent of embodiment 23 or 24, wherein the lipophilic moiety is conjugated via a linker or carrier.

    • 26. The dsRNA agent of any one of embodiments 23-25, wherein lipophilicity of the lipophilic moiety, measured by logKow, exceeds 0.

    • 27. The dsRNA agent of any one of the preceding embodiments, wherein the hydrophobicity of the double-stranded RNAi agent, measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent, exceeds 0.2.

    • 28. The dsRNA agent of embodiment 27, wherein the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

    • 29. The dsRNA agent of any of the preceding embodiments, wherein the dsRNA agent comprises at least one modified nucleotide.

    • 30. The dsRNA agent of embodiment 29, wherein no more than five of the sense strand nucleotides and not more than five of the nucleotides of the antisense strand are unmodified nucleotides.

    • 31. The dsRNA agent of embodiment 29, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

    • 32. The dsRNA agent of any one of embodiments 29-31, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a 5′-phosphate mimic, a glycol modified nucleotide, and a 2-O—(N-methylacetamide) modified nucleotide; and combinations thereof.

    • 33. The dsRNA agent of any of embodiments 29-31, wherein no more than five of the sense strand nucleotides and not more than five of the nucleotides of the antisense strand include modifications other than 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, unlocked nucleic acids (UNA) or glycerol nucleic acid (GNA).

    • 34. The dsRNA agent of any of the preceding embodiments, which comprises a non-nucleotide spacer (wherein optionally the non-nucleotide spacer comprises a C3-C6 alkyl) between two of the contiguous nucleotides of the sense strand or between two of the contiguous nucleotides of the antisense strand.

    • 35. The dsRNA agent of any of the preceding embodiments, wherein each strand is no more than 30 nucleotides in length.

    • 36. The dsRNA agent of any of the preceding embodiments, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide.

    • 37. The dsRNA agent of any of the preceding embodiments, wherein at least one strand comprises a 3′ overhang of at least 2 nucleotides.

    • 38. The dsRNA agent of any of the preceding embodiments, wherein the double stranded region is 15-30 nucleotide pairs in length.

    • 39. The dsRNA agent of embodiment 38, wherein the double stranded region is 17-23 nucleotide pairs in length.

    • 40. The dsRNA agent of embodiment 38, wherein the double stranded region is 17-25 nucleotide pairs in length.

    • 41. The dsRNA agent of embodiment 38, wherein the double stranded region is 23-27 nucleotide pairs in length.

    • 42. The dsRNA agent of embodiment 38, wherein the double stranded region is 19-21 nucleotide pairs in length.

    • 43. The dsRNA agent of embodiment 38, wherein the double stranded region is 21-23 nucleotide pairs in length.

    • 44. The dsRNA agent of any of the preceding embodiments, wherein each strand has 19-30 nucleotides.

    • 45. The dsRNA agent of any of the preceding embodiments, wherein each strand has 19-23 nucleotides.

    • 46. The dsRNA agent of any of the preceding embodiments, wherein each strand has 21-23 nucleotides.

    • 47. The dsRNA agent of any of the preceding embodiments, wherein the agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

    • 48. The dsRNA agent of embodiment 47, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminus of one strand.

    • 49. The dsRNA agent of embodiment 48, wherein the strand is the antisense strand.

    • 50. The dsRNA agent of embodiment 48, wherein the strand is the sense strand.

    • 51. The dsRNA agent of embodiment 47, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand.

    • 52. The dsRNA agent of embodiment 51, wherein the strand is the antisense strand.

    • 53. The dsRNA agent of embodiment 51, wherein the strand is the sense strand.

    • 54. The dsRNA agent of embodiment 47, wherein each of the 5′- and 3′-terminus of one strand comprises a phosphorothioate or methylphosphonate internucleotide linkage.

    • 55. The dsRNA agent of embodiment 54, wherein the strand is the antisense strand.

    • 56. The dsRNA agent of any of the preceding embodiments, wherein the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

    • 57. The dsRNA agent of embodiment 54, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

    • 58. The dsRNA agent of any one of embodiments 23-57, wherein one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand.

    • 59. The dsRNA agent of embodiment 58, wherein the one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand via a linker or carrier.

    • 60. The dsRNA agent of embodiment 59, wherein the internal positions include all positions except the terminal two positions from each end of the at least one strand.

    • 61. The dsRNA agent of embodiment 59, wherein the internal positions include all positions except the terminal three positions from each end of the at least one strand.

    • 62. The dsRNA agent of any one of embodiments 59-61, wherein the internal positions exclude a cleavage site region of the sense strand.

    • 63. The dsRNA agent of embodiment 62, wherein the internal positions include all positions except positions 9-12, counting from the 5′-end of the sense strand.

    • 64. The dsRNA agent of embodiment 62, wherein the internal positions include all positions except positions 11-13, counting from the 3′-end of the sense strand.

    • 65. The dsRNA agent of any one of embodiments 59-61, wherein the internal positions exclude a cleavage site region of the antisense strand.

    • 66. The dsRNA agent of embodiment 65, wherein the internal positions include all positions except positions 12-14, counting from the 5′-end of the antisense strand.

    • 67. The dsRNA agent of any one of embodiments 59-61, wherein the internal positions include all positions except positions 11-13 on the sense strand, counting from the 3′-end, and positions 12-14 on the antisense strand, counting from the 5′-end.

    • 68. The dsRNA agent of any one of embodiments 23-67, wherein the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5′end of each strand.

    • 69. The dsRNA agent of embodiment 68, wherein the one or more lipophilic moieties are conjugated to one or more of the 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, counting from the 5′-end of each strand.

    • 70. The dsRNA agent of embodiment 24, wherein the positions in the double stranded region exclude a cleavage site region of the sense strand.

    • 71. The dsRNA agent of any one of embodiments 23-70, 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 position 16 of the antisense strand.

    • 72. The dsRNA agent of embodiment 71, wherein the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense strand.

    • 73. The dsRNA agent of embodiment 71, wherein the lipophilic moiety is conjugated to position 21, position 20, or position 15 of the sense strand.

    • 74. The dsRNA agent of embodiment 71, wherein the lipophilic moiety is conjugated to position 20 or position 15 of the sense strand.

    • 75. The dsRNA agent of embodiment 71, wherein the lipophilic moiety is conjugated to position 16 of the antisense strand.

    • 76. The dsRNA agent of embodiment 71, wherein the lipophilic moiety is conjugated to position 6, counting from the 5′-end of the sense strand.

    • 77. The dsRNA agent of any one of embodiments 23-76, wherein the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

    • 78. The dsRNA agent of embodiment 77, wherein the lipophilic moiety is selected from the group consisting of lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

    • 79. The dsRNA agent of embodiment 78, wherein the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

    • 80. The dsRNA agent of embodiment 79, wherein the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain.

    • 81. The dsRNA agent of embodiment 79, wherein the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

    • 82. The dsRNA agent of any one of embodiments 23-81, wherein the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) or the double stranded region.

    • 83. The dsRNA agent of embodiment 82, 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, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone.

    • 84. The dsRNA agent of any one of embodiments 23-81, wherein the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.

    • 85. The double-stranded iRNA agent of any one of embodiments 23-84, wherein the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.

    • 86. The dsRNA agent of any one of embodiments 23-85, wherein the lipophilic moiety is conjugated via a bio-cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

    • 87. The dsRNA agent of any one of embodiments 23-86, wherein the 3′ end of the sense strand is protected via an end cap which is 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, and decalinyl.

    • 88. The dsRNA agent of any one of embodiments 23-87, further comprising a targeting ligand, e.g., a ligand that targets an ocular tissue or a liver tissue.

    • 89. The dsRNA agent of embodiment 88, wherein the ligand is conjugated to the sense strand.

    • 90. The dsRNA agent of embodiment 88 or 89, wherein the ligand is conjugated to the 3′ end or the 5′ end of the sense strand.

    • 91. The dsRNA agent of embodiment 88 or 89, wherein the ligand is conjugated to the 3′ end of the sense strand.

    • 92. The dsRNA agent of any one of embodiments 88-91, wherein the ocular tissue is a trabecular meshwork tissue, a ciliary body, a retinal tissue, a retinal pigment epithelium (RPE) or choroid tissue, e.g., a choroid vessel.

    • 93. The dsRNA agent of any one of embodiments 88-91, wherein the targeting ligand comprises N-acetylgalactosamine (GalNAc).

    • 94. The dsRNA agent of any one of embodiments 88-91, wherein the targeting ligand is one or more GalNAc conjugates or one or more or GalNAc derivatives.

    • 95. The dsRNA agent of embodiment 94, wherein the one or more GalNAc conjugates or one or more GalNAc derivatives are attached through a monovalent linker, or a bivalent, trivalent, or tetravalent branched linker.

    • 96. The dsRNA agent of embodiment 94, wherein the ligand is







embedded image




    • 97. The dsRNA agent of embodiment 96, wherein the dsRNA agent is conjugated to the ligand as shown in the following schematic







embedded image




    •  wherein X is O or S.

    • 98. The dsRNA agent of embodiment 97, wherein the X is O.

    • 99. The dsRNA agent of any one of embodiments 1-98, further comprising a terminal, chiral modification occurring at the first internucleotide linkage at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,

    • a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and

    • a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration.

    • 100. The dsRNA agent of any one of embodiments 1-98, further comprising

    • a terminal, chiral modification occurring at the first and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,

    • a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and

    • a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

    • 101. The dsRNA agent of any one of embodiments 1-98, further comprising

    • a terminal, chiral modification occurring at the first, second and third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,

    • a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and

    • a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

    • 102. The dsRNA agent of any one of embodiments 1-98, further comprising

    • a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,

    • a terminal, chiral modification occurring at the third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration,

    • a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and

    • a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

    • 103. The dsRNA agent of any one of embodiments 1-98, further comprising

    • a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,

    • a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and

    • a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

    • 104. The dsRNA agent of any one of embodiments 1-103, further comprising a phosphate or phosphate mimic at the 5′-end of the antisense strand.

    • 105. The dsRNA agent of embodiment 104, wherein the phosphate mimic is a 5′-vinyl phosphonate (VP).

    • 106. A cell containing the dsRNA agent of any one of embodiments 1-105.

    • 107. A human ocular cell, e.g., (a cell of the trabecular meshwork, a cell of the ciliary body, an RPE cell, an astrocyte, a pericyte, a Müller cell, a ganglion cell, an endothelial cell, or a photoreceptor cell) comprising a reduced level of MYOC mRNA or a level of MYOC protein as compared to an otherwise similar untreated cell, wherein optionally the level is reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

    • 108. The human cell of embodiment 107, which was produced by a process comprising contacting a human cell with the dsRNA agent of any one of embodiments 1-94.

    • 109. A pharmaceutical composition for inhibiting expression of MYOC, comprising the dsRNA agent of any one of embodiments 1-105.

    • 110. A pharmaceutical composition comprising the dsRNA agent of any one of embodiments 1-105 and a lipid formulation.

    • 111. A method of inhibiting expression of MYOC in a cell, the method comprising:
      • (a) contacting the cell with the dsRNA agent of any one of embodiments 1-105, or a pharmaceutical composition of embodiment 109 or 110; and
      • (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of MYOC, thereby inhibiting expression of MYOC in the cell.

    • 112. A method of inhibiting expression of MYOC in a cell, the method comprising:
      • (a) contacting the cell with the dsRNA agent of any one of embodiments 1-105, or a pharmaceutical composition of embodiment 109 or 110. and
      • (b) maintaining the cell produced in step (a) for a time sufficient to reduce levels of MYOC mRNA, MYOC protein, or both of MYOC mRNA and protein, thereby inhibiting expression of MYOC in the cell.

    • 113. The method of embodiment 111 or 112, wherein the cell is within a subject.

    • 114. The method of embodiment 113, wherein the subject is a human.

    • 115. The method of any one of embodiments 111-114, wherein the level of MYOC mRNA is inhibited by at least 50%.

    • 116. The method of any one of embodiments 111-114, wherein the level of MYOC protein is inhibited by at least 50%.

    • 117. The method of embodiment 114-116, wherein inhibiting expression of MYOC decreases a MYOC protein level in a biological sample (e.g., an aqueous ocular fluid sample) from the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

    • 118. The method of any one of embodiments 114-117, wherein the subject has been diagnosed with a MYOC-associated disorder, e.g., glaucoma, e.g., primary open angle glaucoma (POAG).

    • 119. A method of inhibiting expression of MYOC in an ocular cell or tissue, the method comprising:

    • (a) contacting the cell or tissue with a dsRNA agent that binds MYOC; and

    • (b) maintaining the cell or tissue produced in step (a) for a time sufficient to reduce levels of MYOC mRNA, MYOC protein, or both of MYOC mRNA and protein, thereby inhibiting expression of MYOC in the cell or tissue.

    • 120. The method of embodiment 119, wherein the ocular cell or tissue comprises a trabecular meshwork tissue, a ciliary body, an RPE cell, a retinal tissue, an astrocyte, a pericyte, a Müller cell, a ganglion cell, an endothelial cell, a photoreceptor cell, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel.

    • 120a. A method of reducing intraocular pressure in a subject, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of embodiments 1-105 or a pharmaceutical composition of embodiment 109 or 110, thereby reducing intraocular pressure in the subject.

    • 120b. A method of limiting an increase in intraocular pressure, or maintaining a constant intraocular pressure, in a subject, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of embodiments 1-105 or a pharmaceutical composition of embodiment 109 or 110, thereby limiting the increase in intraocular pressure, or maintaining a constant intraocular pressure in the subject.

    • 121. A method of treating a subject having, or diagnosed with having, a MYOC-associated disorder comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of embodiments 1-105 or a pharmaceutical composition of embodiment 109 or 110, thereby treating the disorder.

    • 122. The method of embodiment 118 or 121, wherein the MYOC-associated disorder is glaucoma.

    • 122a. A method of treating a subject having glaucoma, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of embodiments 1-105 or a pharmaceutical composition of embodiment 109 or 110, thereby treating the glaucoma.

    • 123. The method of embodiment 122 or 122a, wherein glaucoma is selected from the group consisting of primary open angle glaucoma (POAG).

    • 124. The method of any one of embodiments 121-123, wherein treating comprises amelioration of at least one sign or symptom of the disorder.

    • 125. The method of embodiment 124, wherein at least one sign or symptom of glaucoma comprises a measure of one or more of optic nerve damage, vision loss, tunnel vision, blurred vision, eye pain or presence, level, or activity of MYOC (e.g., MYOC gene, MYOC mRNA, or MYOC protein).

    • 126. The method of any one of embodiments 121-123, where treating comprises prevention of progression of the disorder.

    • 127. The method of any one of embodiments 124-126, wherein the treating comprises one or more of (a) inhibiting or reducing the expression or activity of MYOC; (b) reducing the level of misfolded MYOC protein; (c) reducing trabecular meshwork cell death; (d) decreasing intraocular pressure; or (e) increasing visual acuity.

    • 128. The method of embodiment 127, wherein the treating results in at least a 30% mean reduction from baseline of MYOC mRNA in the trabecular meshwork tissue, ciliary body, retina, RPE, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel.

    • 129. The method of embodiment 128 wherein the treating results in at least a 60% mean reduction from baseline of MYOC mRNA in the trabecular meshwork tissue, ciliary body, retina, RPE, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel.

    • 130. The method of embodiment 129, wherein the treating results in at least a 90% mean reduction from baseline of MYOC mRNA in the trabecular meshwork tissue, ciliary body, retina, RPE, a retinal blood vessel (e.g., including endothelial cells and vascular smooth muscle cells), or choroid tissue, e.g., a choroid vessel.

    • 131. The method of any one of embodiments 124-129, wherein after treatment the subject experiences at least an 8-week duration of knockdown following a single dose of dsRNA as assessed by MYOC protein in the retina.

    • 132. The method of embodiment 131, wherein treating results in at least a 12-week duration of knockdown following a single dose of dsRNA as assessed by MYOC protein in the retina.

    • 133. The method of embodiment 132, wherein treating results in at least a 16-week duration of knockdown following a single dose of dsRNA as assessed by MYOC protein in the retina.

    • 134. The method of any of embodiments 113-133, wherein the subject is human.

    • 135. The method of any one of embodiments 114-134, wherein the dsRNA agent is administered at a dose of about 0.01 mg/kg to about 50 mg/kg.

    • 136. The method of any one of embodiments 114-135, wherein the dsRNA agent is administered to the subject intraocularly, intravenously, or topically.

    • 137. The method of embodiment 136, wherein the intraocular administration comprises intravitreal administration (e.g., intravitreal injection), transscleral administration (e.g., transscleral injection), subconjunctival administration (e.g., subconjunctival injection), retrobulbar administration (e.g., retrobulbar injection), intracameral administration (e.g., intracameral injection), or subretinal administration (e.g., subretinal injection).

    • 138. The method of any one of embodiments 114-137, further comprising measuring level of MYOC (e.g., MYOC gene, MYOC mRNA, or MYOC protein) in the subject.

    • 139. The method of embodiment 138, where measuring the level of MYOC in the subject comprises measuring the level of MYOC gene, MYOC protein or MYOC mRNA in a biological sample from the subject (e.g., an aqueous ocular fluid sample).

    • 140. The method of any one of embodiments 114-139, further comprising performing a blood test, an imaging test, or an aqueous ocular fluid biopsy.

    • 141. The method of any one of embodiments 138-140, wherein measuring level of MYOC (e.g., MYOC gene, MYOC mRNA, or MYOC protein) in the subject is performed prior to treatment with the dsRNA agent or the pharmaceutical composition.

    • 142. The method of embodiment 141, wherein, upon determination that a subject has a level of MYOC (e.g., MYOC gene, MYOC mRNA, or MYOC protein) that is greater than a reference level, the dsRNA agent or the pharmaceutical composition is administered to the subject.

    • 143. The method of any one of embodiments 139-142, wherein measuring level of MYOC (e.g., MYOC gene, MYOC mRNA, or MYOC protein) in the subject is performed after treatment with the dsRNA agent or the pharmaceutical composition.

    • 144. The method of any one of embodiments 121-143, further comprising administering to the subject an additional agent and/or therapy suitable for treatment or prevention of an MYOC-associated disorder.

    • 145. The method of embodiment 144, wherein the additional agent and/or therapy comprises one or more of a photodynamic therapy, photocoagulation therapy, a steroid, a non-steroidal anti-inflammatory agent, an anti-MYOC agent, and/or a vitrectomy.





EXAMPLES
Example 1. MYOC siRNA

Nucleic acid sequences provided herein are represented using standard nomenclature. See the abbreviations of Table 1.









TABLE 1







Abbreviations of nucleotide monomers used in nucleic acid


sequence representation It will be understood that these monomers,


when present in an oligonucleotide, are mutually


linked by 5′-3′-phosphodiester bonds.








Abbreviation
Nucleotide(s)





A
Adenosine-3′-phosphate


Ab
beta-L-adenosine-3′-phosphate


Abs
beta-L-adenosine-3′-phosphorothioate


Af
2′-fluoroadenosine-3′-phosphate


Afs
2′-fluoroadenosine-3′-phosphorothioate


(Ahd)
2′-O-hexadecyl-adenosine-3′-phosphate


(Ahds)
2′-O-hexadecyl-adenosine-3′-phosphorothioate


As
adenosine-3′-phosphorothioate


(A2p)
adenosine 2′-phosphate


(A2ps)
adenosine-2′-phosphorothioate


C
cytidine-3′-phosphate


Cb
beta-L-cytidine-3′-phosphate


Cbs
beta-L-cytidine-3′-phosphorothioate


Cf
2′-fluorocytidine-3′-phosphate


Cfs
2′-fluorocytidine-3′-phosphorothioate


(Chd)
2′-O-hexadecyl-cytidine-3′-phosphate


(Chds)
2′-O-hexadecyl-cytidine-3′-phosphorothioate


Cs
cytidine-3′-phosphorothioate


(C2p)
cytosine 2′-phosphate


(C2ps)
cytidine-2′-phosphorothioate


G
guanosine-3′-phosphate


Gb
beta-L-guanosine-3′-phosphate


Gbs
beta-L-guanosine-3′-phosphorothioate


Gf
2′-fluoroguanosine-3′-phosphate


Gfs
2′-fluoroguanosine-3′-phosphorothioate


(Ghd)
2′-O-hexadecyl-guanosine-3′-phosphate


(Ghds)
2′-O-hexadecyl-guanosine-3′-phosphorothioate


Gs
guanosine-3′-phosphorothioate


(G2p)
guanosine 2′-phosphate


(G2ps)
guanosine-2-phosphorothioate


T
5′-methyluridine-3′-phosphate


Tb
beta-L-thymidine-3′-phosphate


Tbs
beta-L-thymidine-3′-phosphorothioate


Tf
2′-fluoro-5-methyluridine-3′-phosphate


Tfs
2′-fluoro-5-methyluridine-3′-phosphorothioate


Tgn
thymidine-glycol nucleic acid (GNA) S-Isomer


Agn
adenosine-glycol nucleic acid (GNA) S-Isomer


Con
cytidine-glycol nucleic acid (GNA) S-Isomer


Ggn
guanosine-glycol nucleic acid (GNA) S-Isomer


Ts
5-methyluridine-3′-phosphorothioate


(T2p)
thymidine 2′-phosphate


U
Uridine-3′-phosphate


Ub
beta-L-uridine-3′-phosphate


Ubs
beta-L-uridine-3′-phosphorothioate


Uf
2′-fluorouridine-3′-phosphate


Ufs
2′-fluorouridine-3′-phosphorothioate


(Uhd)
2′-O-hexadecyl-uridine-3′-phosphate


(Uhds)
2′-O-hexadecyl-uridine-3′-phosphorothioate


Us
uridine-3′-phosphorothioate


(U2p)
uracil 2′-phosphate


(U2ps)
uridine-2-phosphorothioate


N
any nucleotide (G, A, C, T or U)


VP
Vinyl phosphonate


a
2′-O-methyladenosine-3′-phosphate


as
2′-O-methyladenosine-3′-phosphorothioate


C
2′-O-methylcytidine-3′-phosphate


CS
2′-O-methylcytidine-3′-phosphorothioate


g
2′-O-methylguanosine-3′-phosphate


gs
2′-O-methylguanosine-3′-phosphorothioate


t
2′-O-methyl-5-methyluridine-3′-phosphate


ts
2′-O-methyl-5-methyluridine-3′-phosphorothioate


u
2′-O-methyluridine-3′-phosphate


us
2′-O-methyluridine-3′-phosphorothioate


dA
2′-deoxyadenosine-3′-phosphate


dAs
2′-deoxyadenosine-3′-phosphorothioate


dC
2′-deoxycytidine-3′-phosphate


dCs
2′-deoxycytidine-3′-phosphorothioate


dG
2′-deoxyguanosine-3′-phosphate


dGs
2′-deoxyguanosine-3′-phosphorothioate


dT
2′-deoxythymidine


dTs
2′-deoxythymidine-3′-phosphorothioate


dU
2′-deoxyuridine


s
phosphorothioate linkage


L961
N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol Hyp-(GalNAc-alkyl)3


(Aeo)
2′-O-methoxyethyladenosine-3′-phosphate


(Aeos)
2′-O-methoxyethyladenosine-3′-phosphorothioate


(Geo)
2′-O-methoxyethylguanosine-3′-phosphate


(Geos)
2′-O-methoxyethylguanosine-3′-phosphorothioate


(Teo)
2′-O-methoxyethyl-5-methyluridine-3′-phosphate


(Teos)
2′-O-methoxyethyl-5-methyluridine-3′-phosphorothioate


(m5Ceo)
2′-O-methoxyethyl-5-methylcytidine-3′-phosphate


(m5Ceos)
2′-O-methoxyethyl-5-methylcytidine-3′-phosphorothioate






1The chemical structure of L96 is as follows:





embedded image








Experimental Methods
Bioinformatics
Transcripts

Four sets of siRNAs targeting the human MYOC, “myocilin” (human: NCBI refseqID NM_000261.2; NCBI GeneID: 4653) were generated. The human NM_000261.2 REFSEQ mRNA has a length of 2100 bases. Pairs of oligos were generated using bioinformatic methods and ranked, and exemplary pairs of oligos are shown in Table 2A and Table 2B. Modified sequences are presented in Table 2A. Unmodified sequences are presented in Table 2B.









TABLE 2A







Exemplary Human MYOC siRNA Modified Single Strands and Duplex Sequences


Column 1 indicates duplex name and the number following the decimal point in a duplex name merely refers to a batch production


number. Column 2 indicates the name of the sense sequence. Column 3 indicates the sequence ID for the sequence of column 4.


Column 4 provides the modified sequence of a sense strand suitable for use in a duplex described herein. Column 5 indicates the


antisense sequence name. Column 6 indicates the sequence ID for the sequence of column 7. Column 7 provides the sequence of a


modified antisense strand suitable for use in a duplex described herein, e.g., a duplex comprising the sense sequence in the same row


of the table. Column 8 indicates the position in the target mRNA (NM_000261.2) that is complementary to the antisense strand of


Column 7. Column 9 indicated the sequence ID for the sequence of column 8.




















Seq ID






Sense
Seq ID

Antisense
NO:

mRNA target



Duplex
sequence
NO:
Sense sequence
sequence
(anti
Antisense sequence
sequence in
SEQ ID NO:


Name
name
(sense)
(5′-3′)
name
sense)
(5′-3′)
NM_000261.2
(mRNA target)


















AD-
A-
7
csuscuc(Ahd)GfcAf
A-
451
VPusAfsadGc(Tgn)cug
ACCUCUCAGCACAGC
895


1565444.1
2893965.1

CfAfgcagagcususa
2893966.1

cuguGfcUfgagagsgsu
AGAGCUUU






AD-
A-
8
csuscuc(Ahd)GfcAf
A-
452
VPusAfsagcdTc(Tgn)gc
ACCUCUCAGCACAGC
896


1565445.1
2893965.1

CfAfgcagagcususa
2893967.1

uguGfcUfgagagsgsu
AGAGCUUU






AD-
A-
9
csuscuc(Ahd)GfcAf
A-
453
VPusAfsagdCu(C2p)ug
ACCUCUCAGCACAGC
897


1565692.1
2893965.1

CfAfgcagagcususa
2894431.1

cuguGfcUfgagagsgsu
AGAGCUUU






AD-
A-
10
uscsucag(Chd)aCfAf
A-
454
VPusAfsaadGc(Tgn)cu
CCUCUCAGCACAGCA
898


1565446.1
2893968.1

Gfcagagcuususa
2893969.1

gcugUfgCfugagasgsg
GAGCUUUC






AD-
A-
11
csasgcag(Ahd)gCfUf
A-
455
VPusUfsccdTc(Tgn)gga
CACAGCAGAGCUUUC
899


1565447.1
2893970.1

Ufuccagaggsasa
2893971.1

aagCfuCfugcugsusg
CAGAGGAA






AD-
A-
12
asasug(Ahd)gGfuUf
A-
456
VPusGfsudGc(Agn)cag
GCAAUGAGGUUCUUC
900


1565448.1
2893972.1

CfUfucugugcascsa
2893973.1

aagaAfcCfucauusgsc
UGUGCACG






AD-
A-
13
asasug(Ahd)gGfuUf
A-
457
VPusGfsugdCa(C2p)ag
GCAAUGAGGUUCUUC
901


1565693.1
2893972.1

CfUfucugugcascsa
2894432.1

aagaAfcCfucauusgsc
UGUGCACG






AD-
A-
14
asusgagg(Uhd)uCfU
A-
458
VPusCfsgudGc(Agn)ca
CAAUGAGGUUCUUCU
902


1565449.1
2893974.1

fUfcugugcacsgsa
2893975.1

gaagAfaCfcucaususg
GUGCACGU






AD-
A-
15
usgsagg(Uhd)UfcUf
A-
459
VPusAfscgudGc(Agn)c
AAUGAGGUUCUUCU
903


1565450.1
2893976.1

UfCfugugcacgsusa
2893977.1

agaaGfaAfccucasusu
GUGCACGUU






AD-
A-
16
usgsagg(Uhd)UfcUf
A-
460
VPusAfscgdTg(C2p)ac
AAUGAGGUUCUUCU
904


1565694.1
2893976.1

UfCfugugcacgsusa
2894433.1

agaaGfaAfccucasusu
GUGCACGUU






AD-
A-
17
gsasggu(Uhd)CfuUf
A-
461
VPusAfsacdGu(G2p)ca
AUGAGGUUCUUCUG
905


1565695.1
2894434.1

CfUfgugcacgususa
2894435.1

cagaAfgAfaccucsasu
UGCACGUUG






AD-
A-
18
asgsguu(Chd)UfuCf
A-
462
VPusCfsaacGfuGfCfac
UGAGGUUCUUCUGU
906


1193175.5
2058874.1

UfGfugcacguusgsa
1801665.1

agAfaGfaaccuscsa
GCACGUUGC






AD-
A-
19
asgsguu(Chd)uuCfU
A-
463
VPusdCsaadCgdTgcac
UGAGGUUCUUCUGU
907


1565793.1
2893978.1

fGfugcacguusgsa
2894582.1

dAgAfagaaccuscsg
GCACGUUGC






AD-
A-
20
asgsguu(Chd)UfuCf
A-
464
VPusCfsaadCg(Tgn)gc
UGAGGUUCUUCUGU
908


1565795.1
2058874.1

UfGfugcacguusgsa
2894585.1

acagAfaGfaaccuscsg
GCACGUUGC






AD-
A-
21
asgsguu(Chd)UfuCf
A-
465
VPudCsaadCg(Tgn)gca
UGAGGUUCUUCUGU
909


1565796.1
2058874.1

UfGfugcacguusgsa
2894586.1

cdAgAfagaaccuscsg
GCACGUUGC






AD-
A-
22
asgsgua(Chd)UfuCf
A-
466
VPusCfsaadCg(Tgn)gc
UGAGGUUCUUCUGU
910


1565797.1
2894587.1

UfGfugcacguusgsa
2894588.1

acagAfaGfuaccuscsg
GCACGUUGC






AD-
A-
23
asgsgau(Chd)UfuCf
A-
467
VPusCfsaadCg(Tgn)gc
UGAGGUUCUUCUGU
911


1565798.1
2894589.1

UfGfugcacguusgsa
2894590.1

acagAfaGfauccuscsg
GCACGUUGC






AD-
A-
24
asgscuu(Chd)UfuCf
A-
468
VPusCfsaadCg(Tgn)gc
UGAGGUUCUUCUGU
912


1565799.1
2894591.1

UfGfugcacguusgsa
2894592.1

acagAfaGfaagcuscsg
GCACGUUGC






AD-
A-
25
asgsguu(Chd)uuCfU
A-
469
VPusdCsaadCgdTgcac
UGAGGUUCUUCUGU
913


1565451.1
2893978.1

fGfugcacguusgsa
2893979.1

dAgAfagaaccuscsa
GCACGUUGC






AD-
A-
26
gsgsuuc(Uhd)ucUfG
A-
470
VPusdGscadAcdGugca
GAGGUUCUUCUGUG
914


1565452.1
2893980.1

fUfgcacguugscsa
2893981.1

dCaGfaagaaccsusc
CACGUUGCU






AD-
A-
27
gsgsuuc(Uhd)UfcUf
A-
471
VPusGfscadAc(G2p)ug
GAGGUUCUUCUGUG
915


1565696.1
2894436.1

GfUfgcacguugscsa
2894437.1

cacaGfaAfgaaccsusc
CACGUUGCU






AD-
A-
28
gsusucu(Uhd)CfUfG
A-
472
VPusdCsaadCgdTgcac
AGGUUCUUCUGUGCA
916


1565794.1
2894583.1

fugcacguusgsa
2894584.1

dAgAfagaacscsu
CGUUGC






AD-
A-
29
gsusucu(Uhd)cuGfU
A-
473
VPusdAsgcdAadCgugc
AGGUUCUUCUGUGCA
917


1565453.1
2893982.1

fGfcacguugcsusa
2893983.1

dAcAfgaagaacscsu
CGUUGCUG






AD-
A-
30
ususcuu(Chd)ugUfG
A-
474
VPusdCsagdCadAcgug
GGUUCUUCUGUGCAC
918


1565454.1
2893984.1

fCfacguugcusgsa
2893985.1

dCaCfagaagaascsc
GUUGCUGC






AD-
A-
31
uscsuuc(Uhd)GfuGf
A-
475
VPusGfscadGc(Agn)ac
GUUCUUCUGUGCACG
919


1565455.1
2893986.1

CfAfcguugcugscsa
2893987.1

gugcAfcAfgaagasasc
UUGCUGCA






AD-
A-
32
uscsuuc(Uhd)guGfC
A-
476
VPusdGscadGcdAacgu
GUUCUUCUGUGCACG
920


1565456.1
2893988.1

fAfcguugcugscsa
2893989.1

dGcAfcagaagasasc
UUGCUGCA






AD-
A-
33
csusucug(Uhd)gCfA
A-
477
VPusUfsgcadGc(Agn)a
UUCUUCUGUGCACGU
921


1565457.1
2893990.1

fCfguugcugcsasa
2893991.1

cgugCfaCfagaagsasa
UGCUGCAG






AD-
A-
34
csusucug(Uhd)gCfA
A-
478
VPusUfsgcdAg(C2p)aa
UUCUUCUGUGCACGU
922


1565697.1
2893990.1

fCfguugcugcsasa
2894438.1

cgugCfaCfagaagsasa
UGCUGCAG






AD-
A-
35
ususcug(Uhd)GfcAf
A-
479
VPusCfsugdCa(G2p)ca
UCUUCUGUGCACGUU
923


1565698.1
2894439.1

CfGfuugcugcasgsa
2894440.1

acguGfcAfcagaasgsa
GCUGCAGC






AD-
A-
36
uscsugug(Chd)aCfG
A-
480
VPusGfscudGc(Agn)gc
CUUCUGUGCACGUUG
924


1565458.1
2893992.1

fUfugcugcagscsa
2893993.1

aacgUfgCfacagasasg
CUGCAGCU






AD-
A-
37
csusgug(Chd)AfcGf
A
481
VPusAfsgcudGc(Agn)g
UUCUGUGCACGUUGC
925


1565459.1
2893994.1

UfUfgcugcagcsusa
2893995.1

caacGfuGfcacagsasa
UGCAGCUU






AD-
A-
38
csusgug(Chd)AfcGf
A-
482
VPusAfsgcdTg(C2p)ag
UUCUGUGCACGUUGC
926


1565699.1
2893994.1

UfUfgcugcagcsusa
2894441.1

caacGfuGfcacagsasa
UGCAGCUU






AD-
A-
39
usgsugc(Ahd)CfgUf
A-
483
VPusAfsagdCu(G2p)ca
UCUGUGCACGUUGCU
927


1565700.1
2894442.1

UfGfcugcagcususa
2894443.1

gcaaCfgUfgcacasgsa
GCAGCUUU






AD-
A-
40
gsusgca(Chd)GfuUf
A-
484
VPusAfsaadGc(Tgn)gc
CUGUGCACGUUGCUG
928


1565460.1
2893996.1

GfCfugcagcuususa
2893997.1

agcaAfcGfugcacsasg
CAGCUUUG






AD-
A-
41
gscsacg(Uhd)ugCfU
A-
485
VPusdCscadAadGcugc
GUGCACGUUGCUGCA
929


1565461.1
2893998.1

fGfcagcuuugsgsa
2893999.1

dAgCfaacgugcsasc
GCUUUGGG






AD-
A-
42
csascgu(Uhd)gcUfG
A-
486
VPusdCsccdAadAgcug
UGCACGUUGCUGCAG
930


1565462.1
2894000.1

fCfagcuuuggsgsa
2894001.1

dCaGfcaacgugscsa
CUUUGGGC






AD-
A-
43
gsasgaug(Chd)cAfG
A-
487
VPusAfsgcdTg(G2p)ac
CUGAGAUGCCAGCUG
931


1565701.1
2894444.1

fCfuguccagcsusa
2894445.1

agcuGfgCfaucucsasg
UCCAGCUG






AD-
A-
44
gsasugc(Chd)AfgCf
A-
488
VPusGfscadGc(Tgn)gg
GAGAUGCCAGCUGUC
932


1565463.1
2894002.1

UfGfuccagcugscsa
2894003.1

acagCfuGfgcaucsusc
CAGCUGCU






AD-
A-
45
csusguc(Chd)agCfUf
A-
489
VPusdCsagdAadGcagc
AGCUGUCCAGCUGCU
933


1565464.1
2894004.1

Gfcugcuucusgsa
2894005.1

dAgCfuggacagscsu
GCUUCUGG






AD-
A-
46
cscsagc(Uhd)GfcUf
A-
490
VPusAfsggdCc(Agn)ga
GUCCAGCUGCUGCUU
934


1565465.1
2894006.1

GfCfuucuggccsusa
2894007.1

agcaGfcAfgcuggsasc
CUGGCCUG






AD-
A-
47
csusggc(Chd)UfgCfC
A-
491
VPusUfscdCc(Agn)cac
UUCUGGCCUGCCUGG
935


1565466.1
2894008.1

fUfggugugggsasa
2894009.1

caggCfaGfgccagsasa
UGUGGGAU






AD-
A-
48
csusggc(Chd)UfgCfC
A-
492
VPusUfscccdAc(Agn)cc
UUCUGGCCUGCCUGG
936


1565467.1
2894008.1

fUfggugugggsasa
2894010.1

aggCfaGfgccagsasa
UGUGGGAU






AD-
A-
49
csusggc(Chd)UfgCfC
A-
493
VPusUfsccdCa(C2p)ac
UUCUGGCCUGCCUGG
937


1565702.1
2894008.1

fUfggugugggsasa
2894446.1

caggCfaGfgccagsasa
UGUGGGAU






AD-
A-
50
usgsgcc(Uhd)GfcCf
A-
494
VPusAfsucdCc(Agn)ca
UCUGGCCUGCCUGGU
938


1565468.1
2894011.1

UfGfgugugggasusa
2894012.1

ccagGfcAfggccasgsa
GUGGGAUG






AD-
A-
51
gscscug(Chd)CfuGf
A-
495
VPusAfscadTcdAcacac
UGGCCUGCCUGGUGU
939


1565469.1
2894013.1

GfUfgugugaugsusa
2894014.1

cAfgGfcaggcscsa
GGGAUGUG






AD-
A-
52
gscscug(Chd)CfuGf
A-
496
VPusAfscadTc(C2p)cac
UGGCCUGCCUGGUGU
940


1565703.1
2894447.1

GfUfgugggaugsusa
2894448.1

accAfgGfcaggcscsa
GGGAUGUG






AD-
A-
53
asgsag(Uhd)gGfcCf
A-
497
VPusAfsuadCu(G2p)gc
CCAGAGUGGCCGAUG
941


1565704.1
2894449.1

GfAfugccaguasusa
2894450.1

aucgGfcCfacucusgsg
CCAGUAUA






AD-
A-
54
asgsug(Uhd)gGfcCf
A-
498
VPusUfscadTu(G2p)gg
UCAGUGUGGCCAGUC
942


1565705.1
2894451.1

AfGfucccaaugsasa
2894452.1

acugGfcCfacacusgsa
CCAAUGAA






AD-
A-
55
gsusgugg(Chd)cAfG
A-
499
VPusUfsucdAu(Tgn)gg
CAGUGUGGCCAGUCC
943


1565470.1
2894015.1

fUfcccaaugasasa
2894016.1

gacuGfgCfcacacsusg
CAAUGAAU






AD-
A-
56
gsusgugg(Chd)cAfG
A-
500
VPusdTsucdAudTggga
CAGUGUGGCCAGUCC
944


1565471.1
2894015.1

fUfcccaaugasasa
2894017.1

dCuGfgccacacsusg
CAAUGAAU






AD-
A-
57
gscscagg(Chd)cAfUf
A-
501
VPusGfsadTg(Agn)cug
GAGCCAGGCCAUGUC
945


1565472.1
2894018.1

Gfucagucauscsa
2894019.1

acauGfgCfcuggcsusc
AGUCAUCC






AD-
A-
58
gscscagg(Chd)cAfUf
A-
502
VPusGfsaugdAc(Tgn)g
GAGCCAGGCCAUGUC
946


1565473.1
2894018.1

Gfucagucauscsa
2894020.1

acauGfgCfcuggcsusc
AGUCAUCC






AD-
A-
59
gscscagg(Chd)cAfUf
A-
503
VPusGfsaudGa(C2p)ug
GAGCCAGGCCAUGUC
947


1565706.1
2894018.1

Gfucagucauscsa
2894453.1

acauGfgCfcuggcsusc
AGUCAUCC






AD-
A-
60
gsgscca(Uhd)GfuCf
A-
504
VPusUfsaudGg(Agn)ug
CAGGCCAUGUCAGUC
948


1565474.1
2894021.1

AfGfucauccausasa
2894022.1

acugAfcAfuggccsusg
AUCCAUAA






AD-
A-
61
gsascc(Uhd)gGfaGf
A-
505
VPusGfscudTu(G2p)gu
UAGACCUGGAGGCCA
949


1565707.1
2894454.1

GfCfcaccaaagscsa
2894455.1

ggccUfcCfaggucsusa
CCAAAGCU






AD-
A-
62
csusgg(Ahd)gGfcCf
A-
506
VPusCfsgadGc(Tgn)uu
ACCUGGAGGCCACCA
950


1565475.1
2894023.1

AfCfcaaagcucsgsa
2894024.1

ggugGfcCfuccagsgsu
AAGCUCGA






AD-
A-
63
gsasaac(Chd)CfaAf
A-
507
VPusAfsacdTc(Tgn)cug
UGGAAACCCAAACCA
951


1565476.1
2894025.1

AfCfcagagagususa
2894026.1

guuUfgGfguuucscsa
GAGAGUUG






AD-
A-
64
csasgca(Ahd)CfcUfC
A-
508
VPusUfsgdTc(Tgn)cgg
UACAGCAACCUCCUCC
952


1565477.1
2894027.1

fCfuccgagacsasa
2894028.1

aggaGfgUfugcugsusa
GAGACAA






AD-
A-
65
csasgca(Ahd)CfcUfC
A-
509
VPusUfsgudCu(C2p)gg
UACAGCAACCUCCUCC
953


1565708.1
2894027.1

fCfuccgagacsasa
2894456.1

aggaGfgUfugcugsusa
GAGACAA






AD-
A-
66
gscsaac(Chd)UfcCf
A-
510
VPusCfsudTg(Tgn)cuc
CAGCAACCUCCUCCGA
954


1565478.1
2894029.1

UfCfcgagacaasgsa
2894030.1

ggagGfaGfguugcsusg
GACAAGU






AD-
A-
67
gscsaac(Chd)UfcCf
A-
511
VPusCfsuugdTc(Tgn)c
CAGCAACCUCCUCCGA
955


1565479.1
2894029.1

UfCfcgagacaasgsa
2894031.1

ggagGfaGfguugcsusg
GACAAGU






AD-
A-
68
gscsaac(Chd)UfcCf
A-
512
VPusCfsuudGu(C2p)uc
CAGCAACCUCCUCCGA
956


1565709.1
2894029.1

UfCfcgagacaasgsa
2894457.1

ggagGfaGfguugcsusg
GACAAGU






AD-
A-
69
asgsaca(Ahd)guCfAf
A-
513
VPusdCscudCcdAgaac
CGAGACAAGUCAGUU
957


1565480.1
2894032.1

Gfuucuggagsgsa
2894033.1

dTgAfcuugucuscsg
CUGGAGGA






AD-
A-
70
csasagu(Chd)AfgUf
A-
514
VPusCfsuudCc(Tgn)cc
GACAAGUCAGUUCUG
958


1565481.1
2894034.1

UfCfuggaggaasgsa
2894035.1

agaaCfuGfacuugsusc
GAGGAAGA






AD-
A-
71
gsuscag(Uhd)UfcUf
A-
515
VPusUfscudCu(Tgn)cc
AAGUCAGUUCUGGAG
959


1565482.1
2894036.1

GfGfaggaagagsasa
2894037.1

uccaGfaAfcugacsusu
GAAGAGAA






AD-
A-
72
asgsaau(Chd)ugGfC
A-
516
VPusdCsaadCcdTccug
UGAGAAUCUGGCCAG
960


1565483.1
2894038.1

fCfaggagguusgsa
2894039.1

dGcCfagauucuscsa
GAGGUUGG






AD-
A-
73
usgsgcc(Ahd)GfgAf
A-
517
VPusGfscdTu(Tgn)cca
UCUGGCCAGGAGGUU
961


1565484.1
2894040.1

GfGfuuggaaagscsa
2894041.1

accuCfcUfggccasgsa
GGAAAGCA






AD-
A-
74
usgsgcc(Ahd)GfgAf
A-
518
VPusGfscudTu(C2p)ca
UCUGGCCAGGAGGUU
962


1565710.1
2894040.1

GfGfuuggaaagscsa
2894458.1

accuCfcUfggccasgsa
GGAAAGCA






AD-
A-
75
csasgcc(Ahd)GfgAf
A-
519
VPusGfsccdTu(G2p)cu
AGCAGCCAGGAGGUA
963


1565711.1
2894459.1

GfGfuagcaaggscsa
2894460.1

accuCfcUfggcugscsu
GCAAGGCU






AD-
A-
76
usgscca(Chd)CfaGf
A-
520
VPusUfsucdTc(Tgn)gg
UGUGCCACCAGGCUC
964


1565485.1
2894042.1

GfCfuccagagasasa
2894043.1

agccUfgGfuggcascsa
CAGAGAAG






AD-
A-
77
asasuu(Uhd)gGfaCf
A-
521
VPusAfsaggdCc(Agn)a
GGAAUUUGGACACUU
965


1565486.1
2894044.1

AfCfuuuggccususa
2894045.1

agugUfcCfaaauuscsc
UGGCCUUC






AD-
A-
78
asasuu(Uhd)gGfaCf
A-
522
VPusAfsagdGc(C2p)aa
GGAAUUUGGACACUU
966


1565712.1
2894044.1

AfCfuuuggccususa
2894461.1

agugUfcCfaaauuscsc
UGGCCUUC






AD-
A-
79
ascsacu(Uhd)UfgGf
A-
523
VPusUfsucdCu(G2p)ga
GGACACUUUGGCCUU
967


1565713.1
2894462.1

CfCfuuccaggasasa
2894463.1

aggcCfaAfaguguscsc
CCAGGAAC






AD-
A-
80
csusuugg(Chd)cUfU
A-
524
VPusdCsagdTudCcugg
CACUUUGGCCUUCCA
968


1565487.1
2894046.1

fCfcaggaacusgsa
2894047.1

dAaGfgccaaagsusg
GGAACUGA






AD-
A-
81
asgsgaa(Chd)UfgAf
A-
525
VPusUfsagdCu(C2p)gg
CCAGGAACUGAAGUC
969


1565714.1
2894048.1

AfGfuccgagcusasa
2894464.1

acuuCfaGfuuccusgsg
CGAGCUAA






AD-
A-
82
asgsgaa(Chd)UfgAf
A-
526
VPusUfsadGc(Tgn)cgg
CCAGGAACUGAAGUC
970


1565488.1
2894048.1

AfGfuccgagcusasa
2894049.1

acuuCfaGfuuccusgsg
CGAGCUAA






AD-
A-
83
gsasagu(Chd)CfgAf
A-
527
VPusCfsuudCa(G2p)uu
CUGAAGUCCGAGCUA
971


1565715.1
2894465.1

GfCfuaacugaasgsa
2894466.1

agcuCfgGfacuucsasg
ACUGAAGU






AD-
A-
84
gscsuaa(Chd)UfgAf
A-
528
VPusAfsagdCa(G2p)ga
GAGCUAACUGAAGUU
972


1565716.1
2894467.1

AfGfuuccugcususa
2894468.1

acuuCfaGfuuagcsusc
CCUGCUUC






AD-
A-
85
csusaac(Uhd)GfaAf
A-
529
VPusGfsaadGc(Agn)gg
AGCUAACUGAAGUUC
973


1565489.1
2894050.1

GfUfuccugcuuscsa
2894051.1

aacuUfcAfguuagscsu
CUGCUUCC






AD-
A-
86
gsasagu(Uhd)CfcUf
A-
530
VPusAfsuudCg(G2p)ga
CUGAAGUUCCUGCUU
974


1565717.1
2894469.1

GfCfuucccgaasusa
2894470.1

agcaGfgAfacuucsasg
CCCGAAUU






AD-
A-
87
gsasgaa(Chd)UfaGf
A-
531
VPusUfsccdTa(C2p)cc
UGGAGAACUAGUUU
975


1565718.1
2894052.1

UfUfuggguaggsasa
2894471.1

aaacUfaGfuucucscsa
GGGUAGGAG






AD-
A-
88
gsasgaa(Chd)UfaGf
A-
532
VPusUfscdCu(Agn)ccc
UGGAGAACUAGUUU
976


1565490.1
2894052.1

UfUfuggguaggsasa
2894053.1

aaacUfaGfuucucscsa
GGGUAGGAG






AD-
A-
89
ascsuag(Uhd)UfuGf
A-
533
VPusGfscudCu(C2p)cu
GAACUAGUUUGGGU
977


1565719.1
2894054.1

GfGfuaggagagscsa
2894472.1

acccAfaAfcuagususc
AGGAGAGCC






AD-
A-
90
ascsuag(Uhd)UfuGf
A-
534
VPusGfscdTc(Tgn)cuac
GAACUAGUUUGGGU
978


1565491.1
2894054.1

GfGfuaggagagscsa
2894055.1

ccAfaAfcuagususc
AGGAGAGC






AD-
A-
91
gsgsgu(Ahd)gGfaGf
A-
535
VPusCfsgudGa(G2p)ag
UUGGGUAGGAGAGCC
979


1565720.1
2894473.1

AfGfccucucacsgsa
2894474.1

gcucUfcCfuacccsasa
UCUCACGC






AD-
A-
92
gsusagg(Ahd)GfaGf
A-
536
VPusAfsgcdGu(G2p)ag
GGGUAGGAGAGCCUC
980


1565721.1
2894475.1

CfCfucucacgcsusa
2894476.1

aggcUfcUfccuacscsc
UCACGCUG






AD-
A-
93
gsusagg(Ahd)gaGfC
A-
537
VPusdAsgcdGudGagag
GGGUAGGAGAGCCUC
981


1565492.1
2894056.1

fCfucucacgcsusa
2894057.1

dGcUfcuccuacscsc
UCACGCUG






AD-
A-
94
usasggag(Ahd)gCfCf
A-
538
VPusdCsagdCgdTgaga
GGUAGGAGAGCCUCU
982


1565493.1
2894058.1

Ufcucacgcusgsa
2894059.1

dGgCfucuccuascsc
CACGCUGA






AD-
A-
95
asgsgag(Ahd)GfcCf
A-
539
VPusUfscadGc(G2p)ug
GUAGGAGAGCCUCUC
983


1565722.1
2894477.1

UfCfucacgcugsasa
2894478.1

agagGfcUfcuccusasc
ACGCUGAG






AD-
A-
96
gscscuc(Uhd)CfaCf
A-
540
VPusCfsugdTu(C2p)uc
GAGCCUCUCACGCUG
984


1565723.1
2894060.1

GfCfugagaacasgsa
2894479.1

agcgUfgAfgaggcsusc
AGAACAGC






AD-
A-
97
gscscuc(Uhd)CfaCf
A-
541
VPusCfsudGu(Tgn)cuc
GAGCCUCUCACGCUG
985


1565494.1
2894060.1

GfCfugagaacasgsa
2894061.1

agcgUfgAfgaggcsusc
AGAACAGC






AD-
A-
98
gscscuc(Uhd)CfaCf
A-
542
VPusCfsugudTc(Tgn)c
GAGCCUCUCACGCUG
986


1565495.1
2894060.1

GfCfugagaacasgsa
2894062.1

agcgUfgAfgaggcsusc
AGAACAGC






AD-
A-
99
csascgc(Uhd)GfaGf
A-
543
VPusUfsuudCu(G2p)c
CUCACGCUGAGAACA
987


1565724.1
2894480.1

AfAfcagcagaasasa
2894481.1

uguucUfcAfgcgugsasg
GCAGAAAC






AD-
A-
100
ascsgcug(Ahd)gAfAf
A-
544
VPusGfsuudTc(Tgn)gc
UCACGCUGAGAACAG
988


1565496.1
2894063.1

Cfagcagaaascsa
2894064.1

uguuCfuCfagcgusgsa
CAGAAACA






AD-
A-
101
csgscug(Ahd)GfaAf
A-
545
VPusUfsgudTu(C2p)ug
CACGCUGAGAACAGC
989


1565725.1
2894065.1

CfAfgcagaaacsasa
2894482.1

cuguUfcUfcagcgsusg
AGAAACAA






AD-
A-
102
csgscug(Ahd)GfaAf
A-
546
VPusUfsgdTu(Tgn)cug
CACGCUGAGAACAGC
990


1565497.1
2894065.1

CfAfgcagaaacsasa
2894066.1

cuguUfcUfcagcgsusg
AGAAACAA






AD-
A-
103
gscsugag(Ahd)aCfAf
A-
547
VPusUfsugdTu(Tgn)cu
ACGCUGAGAACAGCA
991


1565498.1
2894067.1

Gfcagaaacasasa
2894068.1

gcugUfuCfucagcsgsu
GAAACAAU






AD-
A-
104
csusgag(Ahd)AfcAf
A-
548
VPusAfsuudGu(Tgn)uc
CGCUGAGAACAGCAG
992


1565499.1
2894069.1

GfCfagaaacaasusa
2894070.1

ugcuGfuUfcucagscsg
AAACAAUU






AD-
A-
105
usgsaga(Ahd)CfaGf
A-
549
VPusAfsaudTg(Tgn)uu
GCUGAGAACAGCAGA
993


1565500.1
2894071.1

CfAfgaaacaaususa
2894072.1

cugcUfgUfucucasgsc
AACAAUUA






AD-
A-
106
gsasgaa(Chd)AfgCf
A-
550
VPusUfsaadTu(G2p)uu
CUGAGAACAGCAGAA
994


1565726.1
2894483.1

AfGfaaacaauusasa
2894484.1

ucugCfuGfuucucsasg
ACAAUUAC






AD-
A-
107
asgsaac(Ahd)GfcAf
A-
551
VPusGfsuadAu(Tgn)gu
UGAGAACAGCAGAAA
995


1565501.1
2894073.1

GfAfaacaauuascsa
2894074.1

uucuGfcUfguucuscsa
CAAUUACU






AD-
A-
108
asgsaac(Ahd)gcAfGf
A-
552
VPusdGsuadAudTguu
UGAGAACAGCAGAAA
996


1565502.1
2894075.1

Afaacaauuascsa
2894076.1

udCuGfcuguucuscsa
CAAUUACU






AD-
A-
109
gsasacag(Chd)aGfAf
A-
553
VPusdAsgudAadTuguu
GAGAACAGCAGAAAC
997


1565503.1
2894077.1

Afacaauuacsusa
2894078.1

dTcUfgcuguucsusc
AAUUACUG






AD-
A-
110
asascag(Chd)AfgAf
A-
554
VPusCfsagdTa(Agn)uu
AGAACAGCAGAAACA
998


1565801
1991726.1

AfAfcaauuacusgsa
2894595.1

guuuCfuGfcuguuscsu
AUUACUGG






AD
A-
111
asascag(Chd)AfgAf
A-
555
VPusCfsagdTa(A2p)uu
AGAACAGCAGAAACA
999


1565802.1
1991726.1

AfAfcaauuacusgsa
2894596.1

guuuCfuGfcuguuscsu
AUUACUGG






AD-
A-
112
asascac(Chd)AfgAfA
A-
556
VPusdCsagdTadAuugu
AGAACAGCAGAAACA
1000


1565803.1
2894597.1

fAfcaauuacusgsa
2894598.1

dTuCfugguguuscsu
AUUACUGG






AD-
A-
113
asascug(Chd)AfgAf
A-
557
VPusdCsagdTadAuugu
AGAACAGCAGAAACA
1001


1565804.1
2894599.1

AfAfcaauuacusgsa
2894600.1

dTuCfugcaguuscsu
AUUACUGG






AD-
A-
114
asasgag(Chd)AfgAf
A-
558
VPusdCsagdTadAuugu
AGAACAGCAGAAACA
1002


1565805.1
2894601.1

AfAfcaauuacusgsa
2894602.1

dTuCfugcucuuscsu
AUUACUGG






AD-
A-
115
asascag(Chd)AfgAf
A-
559
VPusCfsaguAfaUfUfgu
AGAACAGCAGAAACA
1003


1073418.5
1991726.1

AfAfcaauuacusgsa
1802980.1

uuCfuGfcuguuscsu
AUUACUGG






AD-
A-
116
asascag(Chd)agAfAf
A-
560
VPusdCsagdTadAuugu
AGAACAGCAGAAACA
1004


1565504.1
2894079.1

Afcaauuacusgsa
2894080.1

dTuCfugcuguuscsu
AUUACUGG






AD-
A-
117
asascag(Chd)agAfAf
A-
561
VPusdCsagdTadAuugu
AGAACAGCAGAAACA
1005


1565504.2
2894079.1

Afcaauuacusgsa
2894080.1

dTuCfugcuguuscsu
AUUACUGG






AD-
A-
118
ascsagc(Ahd)gaAfAf
A-
562
VPusdCscadGudAauug
GAACAGCAGAAACAA
1006


1565505.1
2894081.1

Cfaauuacugsgsa
2894082.1

dTuUfcugcugususc
UUACUGGC






AD-
A-
119
csasgc(Ahd)gAfAfAf
A-
563
VPusdCsagdTadAuugu
AACAGCAGAAACAAU
1007


1565800.1
2894593.1

caauuacusgsa
2894594.1

dTuCfugcugsusu
UACUGG






AD-
A-
120
csasgcag(Ahd)aAfCf
A-
564
VPusGfsccdAg(Tgn)aa
AACAGCAGAAACAAU
1008


1565506.1
2894083.1

Afauuacuggscsa
2894084.1

uuguUfuCfugcugsusu
UACUGGCA






AD-
A-
121
asgscag(Ahd)AfaCf
A-
565
VPusUfsgcdCa(G2p)ua
ACAGCAGAAACAAUU
1009


1565727.1
2894485.1

AfAfuuacuggcsasa
2894486.1

auugUfuUfcugcusgsu
ACUGGCAA






AD-
A-
122
gscsaga(Ahd)AfcAf
A-
566
VPusUfsugdCc(Agn)gu
CAGCAGAAACAAUUA
1010


1565507.1
2894085.1

AfUfuacuggcasasa
2894086.1

aauuGfuUfucugcsusg
CUGGCAAG






AD-
A-
123
csasgaa(Ahd)CfaAf
A-
567
VPusCfsuudGc(C2p)ag
AGCAGAAACAAUUAC
1011


1565728.1
1577510.1

UfUfacuggcaasgsa
2894487.1

uaauUfgUfuucugscsu
UGGCAAGU






AD-
A-
124
csasgaa(Ahd)CfaAf
A-
568
VPusCfsuugdCc(Agn)g
AGCAGAAACAAUUAC
1012


1565508.1
1577510.1

UfUfacuggcaasgsa
2894087.1

uaauUfgUfuucugscsu
UGGCAAGU






AD-
A
125
asgsaaa(Chd)AfaUf
A-
569
VPusAfscudTgdAcagu
GCAGAAACAAUUACU
1013


1565509.1
2894088.1

UfAfcugucaagsusa
2894089.1

aaUfuGfuuucusgsc
GGCAAGUA






AD-
A-
126
gsasaac(Ahd)AfuUf
A-
570
VPusUfsacdTu(G2p)cc
CAGAAACAAUUACUG
1014


1565730.1
2894490.1

AfCfuggcaagusasa
2894491.1

aguaAfuUfguuucsusg
GCAAGUAU






AD-
A-
127
asasaca(Ahd)UfuAf
A-
571
VPusAfsuadCu(Tgn)gc
AGAAACAAUUACUGG
1015


1565510.1
2894090.1

CfUfggcaaguasusa
2894091.1

caguAfaUfuguuuscsu
CAAGUAUG






AD-
A-
128
asascaa(Uhd)UfaCf
A-
572
VPusCfsaudAc(Tgn)ug
GAAACAAUUACUGGC
1016


1565511.1
2894092.1

UfGfgcaaguausgsa
2894093.1

ccagUfaAfuuguususc
AAGUAUGG






AD-
A-
129
asascaa(Uhd)uaCfU
A-
573
VPusdCsaudAcdTugcc
GAAACAAUUACUGGC
1017


1565512.1
2894094.1

fGfgcaaguausgsa
2894095.1

dAgUfaauuguususc
AAGUAUGG






AD-
A-
130
gsgscaag(Uhd)aUfG
A-
574
VPusAfsucdCa(C2p)ac
CUGGCAAGUAUGGUG
1018


1565731.1
2894096.1

fGfuguguggasusa
2894492.1

accaUfaCfuugccsasg
UGUGGAUG






AD-
A-
131
gsgscaag(Uhd)aUfG
A-
575
VPusAfsudCc(Agn)cac
CUGGCAAGUAUGGUG
1019


1565513.1
2894096.1

fGfuguguggasusa
2894097.1

accaUfaCfuugccsasg
UGUGGAUG






AD-
A-
132
gsgscaag(Uhd)aUfG
A-
576
VPusAfsuccdAc(Agn)c
CUGGCAAGUAUGGUG
1020


1565514.1
2894096.1

fGfuguguggasusa
2894098.1

accaUfaCfuugccsasg
UGUGGAUG






AD-
A-
133
csasagu(Ahd)UfgGf
A-
577
VPusGfscadTc(C2p)ac
GGCAAGUAUGGUGU
1021


1565732.1
2894099.1

UfGfuguggaugscsa
2894493.1

acacCfaUfacuugscsc
GUGGAUGCG






AD-
A-
134
csasagu(Ahd)UfgGf
A-
578
VPusGfscaudCc(Agn)c
GGCAAGUAUGGUGU
1022


1565515.1
2894099.1

UfGfuguggaugscsa
2894100.1

acacCfaUfacuugscsc
GUGGAUGCG






AD-
A-
135
usgsagu(Ahd)UfgAf
A-
579
VPusGfsgcdTg(Agn)ug
UUUGAGUAUGACCUC
1023


1565516.1
2894101.1

CfCfucaucagcscsa
2894102.1

agguCfaUfacucasasa
AUCAGCCA






AD-
A-
136
gsasgua(Uhd)GfaCf
A-
580
VPusUfsggdCu(G2p)au
UUGAGUAUGACCUCA
1024


1565733.1
2894494.1

CfUfcaucagccsasa
2894495.1

gaggUfcAfuacucsasa
UCAGCCAG






AD-
A-
137
asgsuaug(Ahd)cCfU
A-
581
VPusCfsugdGc(Tgn)ga
UGAGUAUGACCUCAU
1025


1565517.1
2894103.1

fCfaucagccasgsa
2894104.1

ugagGfuCfauacuscsa
CAGCCAGU






AD-
A-
138
gsusaug(Ahd)CfcUf
A-
582
VPusAfscudGg(C2p)ug
GAGUAUGACCUCAUC
1026


1565734.1
2894105.1

CfAfucagccagsusa
2894496.1

augaGfgUfcauacsusc
AGCCAGUU






AD-
A-
139
gsusaug(Ahd)CfcUf
A-
583
VPusAfscugdGc(Tgn)g
GAGUAUGACCUCAUC
1027


1565518.1
2894105.1

CfAfucagccagsusa
2894106.1

augaGfgUfcauacsusc
AGCCAGUU






AD-
A-
140
usasuga(Chd)CfuCf
A-
584
VPusAfsacdTg(G2p)cu
AGUAUGACCUCAUCA
1028


1565735.1
2894497.1

AfUfcagccagususa
2894498.1

gaugAfgGfucauascsu
GCCAGUUU






AD-
A-
141
asusgac(Chd)UfcAf
A-
585
VPusAfsaadCu(G2p)gc
GUAUGACCUCAUCAG
1029


1565736.1
2894499.1

UfCfagccaguususa
2894500.1

ugauGfaGfgucausasc
CCAGUUUA






AD-
A-
142
usgsacc(Uhd)CfaUf
A-
586
VPusUfsaadAc(Tgn)gg
UAUGACCUCAUCAGC
1030


1565519.1
2894107.1

CfAfgccaguuusasa
2894108.1

cugaUfgAfggucasusa
CAGUUUAU






AD-
A-
143
gsasccu(Chd)auCfAf
A-
587
VPusdAsuadAadCuggc
AUGACCUCAUCAGCC
1031


1565520.1
2894109.1

Gfccaguuuasusa
2894110.1

dTgAfugaggucsasu
AGUUUAUG






AD-
A-
144
ascscuc(Ahd)ucAfGf
A-
588
VPusdCsaudAadAcugg
UGACCUCAUCAGCCA
1032


1565521.1
2894111.1

Cfcaguuuausgsa
2894112.1

dCuGfaugagguscsa
GUUUAUGC






AD-
A-
145
cscsuca(Uhd)CfaGf
A-
589
VPusGfscauAfaAfCfug
GACCUCAUCAGCCAG
1033


1244360.3
2298063.1

CfCfaguuuaugscsa
1803173.1

gcUfgAfugaggsusc
UUUAUGCA






AD-
A-
146
cscsuca(Uhd)caGfCf
A-
590
VPusdGscadTadAacug
GACCUCAUCAGCCAG
1034


1565522.1
2894113.1

Cfaguuuaugscsa
2894114.1

dGcUfgaugaggsusc
UUUAUGCA






AD-
A-
147
cscsuca(Uhd)CfaGf
A-
591
VPusGfscadTa(Agn)ac
GACCUCAUCAGCCAG
1035


1565806.1
2298063.1

CfCfaguuuaugscsa
2894603.1

uggcUfgAfugaggsusc
UUUAUGCA






AD-
A-
148
cscsuca(Uhd)CfaGf
A-
592
VPusGfscadTa(A2p)ac
GACCUCAUCAGCCAG
1036


1565807.1
2298063.1

CfCfaguuuaugscsa
2894604.1

uggcUfgAfugaggsusc
UUUAUGCA






AD-
A-
149
cscsuca(Uhd)caGfCf
A-
593
VPusdGscadTa(Agn)ac
GACCUCAUCAGCCAG
1037


1565808.1
2894113.1

Cfaguuuaugscsa
2894605.1

ugdGcUfgaugaggsusc
UUUAUGCA






AD-
A-
150
cscsuca(Uhd)cagCf
A-
594
VPusGfscadTa(Agn)ac
GACCUCAUCAGCCAG
1038


1565809.1
2894606.1

CfAfguuuaugscsa
2894603.1

uggcUfgAfugaggsusc
UUUAUGCA






AD-
A-
151
cscsuca(Uhd)cagCf
A-
595
VPusdGscadTa(Agn)ac
GACCUCAUCAGCCAG
1039


1565810.1
2894607.1

CfdAguuuaugscsa
2894608.1

ugdGcUfgAfugaggsusc
UUUAUGCA






AD-
A-
152
cscsucu(Uhd)CfaGf
A-
596
VPusGfscadTa(Agn)ac
GACCUCAUCAGCCAG
1040


1565812.1
2894611.1

CfCfaguuuaugscsa
2894612.1

uggcUfgAfagaggsusc
UUUAUGCA






AD-
A-
153
cscsuga(Uhd)CfaGf
A-
597
VPusGfscadTa(Agn)ac
GACCUCAUCAGCCAG
1041


1565813.1
2894613.1

CfCfaguuuaugscsa
2894614.1

uggcUfgAfucaggsusc
UUUAUGCA






AD-
A-
154
cscsaca(Uhd)CfaGf
A-
598
VPusGfscadTa(Agn)ac
GACCUCAUCAGCCAG
1042


1565814.3
2894615.1

CfCfaguuuaugscsa
2894616.1

uggcUfgAfuguggsusc
UUUAUGCA






AD-
A-
155
cscsuca(Uhd)caGfCf
A-
599
VPusdGscadTadAacug
GACCUCAUCAGCCAG
1043


1565522.2
2894113.1

Cfaguuuaugscsa
2894114.1

dGcUfgaugaggsusc
UUUAUGCA






AD-
A-
156
csuscau(Chd)agCfCf
A-
600
VPusdTsgcdAudAaacu
ACCUCAUCAGCCAGU
1044


1565523.1
2894115.1

Afguuuaugcsasa
2894116.1

dGgCfugaugagsgsu
UUAUGCAG






AD-
A-
157
uscsauc(Ahd)GfCfCf
A-
601
VPusGfscadTa(Agn)ac
CCUCAUCAGCCAGUU
1045


1565811.1
2894609.1

aguuuaugscsa
2894610.1

uggcUfgAfugasgsg
UAUGCA






AD-
A-
158
uscsauc(Ahd)GfcCf
A-
602
VPusCfsugdCa(Tgn)aa
CCUCAUCAGCCAGUU
1046


1565524.1
2894117.1

AfGfuuuaugcasgsa
2894118.1

acugGfcUfgaugasgsg
UAUGCAGG






AD-
A-
159
uscsauc(Ahd)gcCfAf
A-
603
VPusdCsugdCadTaaac
CCUCAUCAGCCAGUU
1047


1565525.1
2894119.1

Gfuuuaugcasgsa
2894120.1

dTgGfcugaugasgsg
UAUGCAGG






AD-
A-
160
csasucag(Chd)cAfGf
A-
604
VPusCfscudGc(Agn)ua
CUCAUCAGCCAGUUU
1048


1565526.1
2894121.1

Ufuuaugcagsgsa
2894122.1

aacuGfgCfugaugsasg
AUGCAGGG






AD-
A-
161
asuscag(Chd)CfaGf
A-
605
VPusCfsccdTg(C2p)au
UCAUCAGCCAGUUUA
1049


1565737.1
2894123.1

UfUfuaugcaggsgsa
2894501.1

aaacUfgGfcugausgsa
UGCAGGGC






AD-
A-
162
asuscag(Chd)CfaGf
A-
606
VPusCfsccudGc(Agn)u
UCAUCAGCCAGUUUA
1050


1565527.1
2894123.1

UfUfuaugcaggsgsa
2894124.1

aaacUfgGfcugausgsa
UGCAGGGC






AD-
A-
163
uscsagc(Chd)AfgUf
A-
607
VPusGfsccdCu(G2p)ca
CAUCAGCCAGUUUAU
1051


1565738.1
2894502.1

UfUfaugcagggscsa
2894503.1

uaaaCfuGfgcugasusg
GCAGGGCU






AD-
A-
164
csasgcc(Ahd)GfuUf
A-
608
VPusAfsgcdCc(Tgn)gca
AUCAGCCAGUUUAUG
1052


1565528.1
2894125.1

UfAfugcagggcsusa
2894126.1

uaaAfcUfggcugsasu
CAGGGCUA






AD-
A-
165
asgsccag(Uhd)uUfA
A-
609
VPusUfsagdCc(C2p)ug
UCAGCCAGUUUAUGC
1053


1565739.1
2894127.1

fUfgcagggcusasa
2894504.1

cauaAfaCfuggcusgsa
AGGGCUAC






AD-
A-
166
asgsccag(Uhd)uUfA
A-
610
VPusUfsagcdCc(Tgn)g
UCAGCCAGUUUAUGC
1054


1565529.1
2894127.1

fUfgcagggcusasa
2894128.1

cauaAfaCfuggcusgsa
AGGGCUAC






AD-
A-
167
gscscag(Uhd)UfuAf
A-
611
VPusGfsuadGc(C2p)cu
CAGCCAGUUUAUGCA
1055


1565740.1
2894505.1

UfGfcagggcuascsa
2894506.1

gcauAfaAfcuggcsusg
GGGCUACC






AD-
A-
168
gscscag(Uhd)UfuAf
A-
612
VPusGfsuadGcdAcugc
CAGCCAGUUUAUGCA
1056


1565530.1
2894129.1

UfGfcagugcuascsa
2894130.1

auAfaAfcuggcsusg
GGGCUACC






AD-
A-
169
cscsagu(Uhd)UfaUf
A-
613
VPusGfsgudAg(C2p)cc
AGCCAGUUUAUGCAG
1057


1565741.1
2894507.1

GfCfagggcuacscsa
2894508.1

ugcaUfaAfacuggscsu
GGCUACCC






AD-
A-
170
cscsagu(Uhd)UfaUf
A-
614
VPusGfsgudAgdAccug
AGCCAGUUUAUGCAG
1058


1565531.1
2894131.1

GfCfaggucuacscsa
2894132.1

caUfaAfacuggscsu
GGCUACCC






AD-
A-
171
usgscagggcUfAfCfcc
A-
615
VPusdCsuudAgdAaggg
UAUGCAGGGCUACCC
1059


1565532.1
2894133.1

uu(Chd)uaasgsa
2894134.1

dTaGfcccugcasusa
UUCUAAGG






AD-
A-
172
gsgsgug(Chd)UfgUf
A-
616
VPusCfscgdAg(Tgn)ac
ACGGGUGCUGUGGU
1060


1565533.1
2894135.1

GfGfuguacucgsgsa
2894136.1

accaCfaGfcacccsgsu
GUACUCGGG






AD-
A-
173
gsgsgug(Chd)ugUfG
A-
617
VPusdCscgdAgdTacac
ACGGGUGCUGUGGU
1061


1565534.1
2894137.1

fGfuguacucgsgsa
2894138.1

dCaCfagcacccsgsu
GUACUCGGG






AD-
A-
174
gsasgcc(Uhd)CfuAf
A-
618
VPusCfsgcdCc(Tgn)gga
GGGAGCCUCUAUUUC
1062


1565535.1
2894139.1

UfUfuccagggcsgsa
2894140.1

aauAfgAfggcucscsc
CAGGGCGC






AD-
A-
175
gsasgcc(Uhd)cuAfU
A-
619
VPusdCsgcdCcdTggaa
GGGAGCCUCUAUUUC
1063


1565536.1
2894141.1

fUfuccagggcsgsa
2894142.1

dAuAfgaggcucscsc
CAGGGCGC






AD-
A-
176
cscsucu(Ahd)uuUfC
A-
620
VPusdCsagdCgdCccug
AGCCUCUAUUUCCAG
1064


1565537.1
2894143.1

fCfagggcgcusgsa
2894144.1

dGaAfauagaggscsu
GGCGCUGA






AD-
A-
177
ususcc(Ahd)gGfgCf
A-
621
VPusCfsugdGa(C2p)uc
AUUUCCAGGGCGCUG
1065


1565742.1
2894145.1

GfCfugaguccasgsa
2894509.1

agcgCfcCfuggaasasu
AGUCCAGA






AD-
A-
178
ususcc(Ahd)gGfgCf
A-
622
VPusCfsudGg(Agn)cuc
AUUUCCAGGGCGCUG
1066


1565538.1
2894145.1

GfCfugaguccasgsa
2894146.1

agcgCfcCfuggaasasu
AGUCCAGA






AD-
A-
179
ususcc(Ahd)gGfgCf
A-
623
VPusCfsuggdAc(Tgn)c
AUUUCCAGGGCGCUG
1067


1565539.1
2894145.1

GfCfugaguccasgsa
2894147.1

agcgCfcCfuggaasasu
AGUCCAGA






AD-
A-
180
asgsggcg(Chd)uGfA
A-
624
VPusAfsgudTc(Tgn)gg
CCAGGGCGCUGAGUC
1068


1565540.1
2894148.1

fGfuccagaacsusa
2894149.1

acucAfgCfgcccusgsg
CAGAACUG






AD-
A-
181
gsgsgcg(Chd)UfgAf
A-
625
VPusCfsagdTu(C2p)ug
CAGGGCGCUGAGUCC
1069


1565743.1
2894150.1

GfUfccagaacusgsa
2894510.1

gacuCfaGfcgcccsusg
AGAACUGU






AD-
A-
182
gsgsgcg(Chd)UfgAf
A-
626
VPusCfsadGu(Tgn)cug
CAGGGCGCUGAGUCC
1070


1565541.1
2894150.1

GfUfccagaacusgsa
2894151.1

gacuCfaGfcgcccsusg
AGAACUGU






AD-
A-
183
gsgsgcg(Chd)ugAfG
A-
627
VPusdCsagdTudCugga
CAGGGCGCUGAGUCC
1071


1565542.1
2894152.1

fUfccagaacusgsa
2894153.1

dCuCfagcgcccsusg
AGAACUGU






AD-
A-
184
gsgscgc(Uhd)GfaGf
A-
628
VPusAfscadGu(Tgn)cu
AGGGCGCUGAGUCCA
1072


1565543.1
2894154.1

UfCfcagaacugsusa
2894155.1

ggacUfcAfgcgccscsu
GAACUGUC






AD-
A-
185
gscsgcug(Ahd)gUfCf
A-
629
VPusGfsacdAg(Tgn)uc
GGGCGCUGAGUCCAG
1073


1565544.1
2894156.1

Cfagaacuguscsa
2894157.1

uggaCfuCfagcgcscsc
AACUGUCA






AD-
A-
186
csgscug(Ahd)GfuCf
A-
630
VPusUfsgadCa(G2p)uu
GGCGCUGAGUCCAGA
1074


1565744.1
2894511.1

CfAfgaacugucsasa
2894512.1

cuggAfcUfcagcgscsc
ACUGUCAU






AD-
A-
187
gscsugag(Uhd)cCfAf
A-
631
VPusAfsugdAc(Agn)gu
GCGCUGAGUCCAGAA
1075


1565545.1
2894158.1

Gfaacugucasusa
2894159.1

ucugGfaCfucagcsgsc
CUGUCAUA






AD-
A-
188
csusgag(Uhd)CfcAf
A-
632
VPusUfsaudGa(C2p)ag
CGCUGAGUCCAGAAC
1076


1565745.1
1577552.1

GfAfacugucausasa
2894513.1

uucuGfgAfcucagscsg
UGUCAUAA






AD-
A-
189
csusgag(Uhd)CfcAf
A-
633
VPusUfsadTg(Agn)cag
CGCUGAGUCCAGAAC
1077


1565546.1
1577552.1

GfAfacugucausasa
2894160.1

uucuGfgAfcucagscsg
UGUCAUAA






AD-
A-
190
csusgag(Uhd)CfcAf
A-
634
VPusUfsaugdAc(Agn)g
CGCUGAGUCCAGAAC
1078


1565547.1
1577552.1

GfAfacugucausasa
2894161.1

uucuGfgAfcucagscsg
UGUCAUAA






AD-
A-
191
usgsagu(Chd)CfaGf
A-
635
VPusUfsuadTg(Agn)ca
GCUGAGUCCAGAACU
1079


1565548.1
2894162.1

AfAfcugucauasasa
2894163.1

guucUfgGfacucasgsc
GUCAUAAG






AD-
A-
192
gsasguc(Chd)AfgAf
A-
636
VPusCfsuudAu(G2p)ac
CUGAGUCCAGAACUG
1080


1565746.1
1577518.1

AfCfugucauaasgsa
2894514.1

aguuCfuGfgacucsasg
UCAUAAGA






AD-
A-
193
gsasguc(Chd)agAfAf
A-
637
VPusdCsuudAudGacag
CUGAGUCCAGAACUG
1081


1565549
2894164.1

Cfugucauaasgsa
2894165.1

dTuCfuggacucsasg
UCAUAAGA






AD-
A-
194
asgsucc(Ahd)GfaAf
A-
638
VPusUfscudTa(Tgn)ga
UGAGUCCAGAACUGU
1082


1565550.1
2894166.1

CfUfgucauaagsasa
2894167.1

caguUfcUfggacuscsa
CAUAAGAU






AD-
A-
195
asgsucc(Ahd)gaAfCf
A-
639
VPusdTscudTadTgaca
UGAGUCCAGAACUGU
1083


1565551.1
2894168.1

Ufgucauaagsasa
2894169.1

dGuUfcuggacuscsa
CAUAAGAU






AD-
A-
196
gsuscca(Ghd)AfaCf
A-
640
VPusAfsucuUfaUfGfac
GAGUCCAGAACUGUC
1084


1244365.3
2324726.1

UfGfucauaagasusa
1803353.1

agUfuCfuggacsusc
AUAAGAUA






AD-
A-
197
gsusccag(Ahd)aCfUf
A-
641
VPusAfsucdTu(Agn)ug
GAGUCCAGAACUGUC
1085


1565552.1
2894170.1

Gfucauaagasusa
2894171.1

acagUfuCfuggacsusc
AUAAGAUA






AD-
A-
198
gsusccag(Ahd)aCfUf
A-
642
VPusdAsucdTudAugac
GAGUCCAGAACUGUC
1086


1565553.1
2894170.1

Gfucauaagasusa
2894172.1

dAgUfucuggacsusc
AUAAGAUA






AD-
A-
232
cscsaaa(Chd)UfgAf
A-
676
VPusAfsuucdTc(Tgn)g
CUCCAAACUGAACCCA
1120


1565575.1
2894211.1

AfCfccagagaasusa
2894213.1

gguuCfaGfuuuggsasg
GAGAAUC






AD-
A-
233
csasaac(Uhd)GfaAf
A-
677
VPusGfsaudTc(Tgn)cu
UCCAAACUGAACCCA
1121


1565576.1
2894214.1

CfCfcagagaauscsa
2894215.1

ggguUfcAfguuugsgsa
GAGAAUCU






AD-
A-
234
uscscgu(Ahd)AfgCf
A-
678
VPusGfsgcdGa(C2p)ug
CAUCCGUAAGCAGUC
1122


1565752.1
2894216.1

AfGfucagucgcscsa
2894521.1

acugCfuUfacggasusg
AGUCGCCA






AD-
A-
235
uscscgu(Ahd)AfgCf
A-
679
VPusGfsgdCg(Agn)cug
CAUCCGUAAGCAGUC
1123


1565577.1
2894216.1

AfGfucagucgcscsa
2894217.1

acugCfuUfacggasusg
AGUCGCCA






AD-
A-
236
uscscgu(Ahd)AfgCf
A-
680
VPusGfsgcgdAc(Tgn)g
CAUCCGUAAGCAGUC
1124


1565578.1
2894216.1

AfGfucagucgcscsa
2894218.1

acugCfuUfacggasusg
AGUCGCCA






AD-
A-
237
uscscgu(Ahd)agCfAf
A-
681
VPusdGsgcdGadCugac
CAUCCGUAAGCAGUC
1125


1565579.1
2894219.1

Gfucagucgcscsa
2894220.1

dTgCfuuacggasusg
AGUCGCCA






AD-
A-
238
usasagc(Ahd)GfuCf
A-
682
VPusCfsaudTg(G2p)cg
CGUAAGCAGUCAGUC
1126


1565753.1
2894522.1

AfGfucgccaausgsa
2894523.1

acugAfcUfgcuuascsg
GCCAAUGC






AD-
A-
239
csasguc(Ahd)GfuCf
A-
683
VPusAfsagdGc(Agn)uu
AGCAGUCAGUCGCCA
1127


1565580.1
2894221.1

GfCfcaaugccususa
2894222.1

ggcgAfcUfgacugscsu
AUGCCUUC






AD-
A-
240
uscsagu(Chd)GfcCf
A-
684
VPusAfsugdAa(G2p)gc
AGUCAGUCGCCAAUG
1128


1565754.1
2894524.1

AfAfugccuucasusa
2894525.1

auugGfcGfacugascsu
CCUUCAUC






AD-
A-
241
asgsucg(Chd)CfaAf
A-
685
VPusUfsgadTg(Agn)ag
UCAGUCGCCAAUGCC
1129


1565581.1
2894223.1

UfGfccuucaucsasa
2894224.1

gcauUfgGfcgacusgsa
UUCAUCAU






AD-
A-
242
gscscaa(Uhd)gcCfUf
A-
686
VPusdCsagdAudGauga
UCGCCAAUGCCUUCA
1130


1565582.1
2894225.1

Ufcaucaucusgsa
2894226.1

dAgGfcauuggcsgsa
UCAUCUGU






AD-
A-
243
cscsuuc(Ahd)UfcAf
A-
687
VPusGfsgudGc(C2p)ac
UGCCUUCAUCAUCUG
1131


1565755.1
2894227.1

UfCfuguggcacscsa
2894526.1

agauGfaUfgaaggscsa
UGGCACCU






AD-
A-
244
cscsuuc(Ahd)UfcAf
A-
688
VPusGfsgugdCc(Agn)c
UGCCUUCAUCAUCUG
1132


1565583.1
2894227.1

UfCfuguggcacscsa
2894228.1

agauGfaUfgaaggscsa
UGGCACCU






AD-
A-
245
csusgugg(Chd)aCfCf
2894230.1
689
VPusCfsggdTg(Tgn)aca
AUCUGUGGCACCUUG
1133


1565584.1
2894229.1

Ufuguacaccsgsa


aggUfgCfcacagsasu
UACACCGU






AD-
A-
246
csusaccg(Uhd)cAfAf
A-
690
VPusAfsuadAg(C2p)aa
UGCUACCGUCAACUU
1134


1565756.1
2894231.1

Cfuuugcuuasusa
2894527.1

aguuGfaCfgguagscsa
UGCUUAUG






AD-
A-
247
csusaccg(Uhd)cAfAf
A-
691
VPusAfsuaadGc(Agn)a
UGCUACCGUCAACUU
1135


1565585.1
2894231.1

Cfuuugcuuasusa
2894232.1

aguuGfaCfgguagscsa
UGCUUAUG






AD-
A-
248
usasccg(Uhd)CfaAf
A-
692
VPusCfsauaa(Ggn)caa
GCUACCGUCAACUUU
1136


1073420.3
1991728.1

CfUfuugcuuausgsa
1806295.1

aguUfgAfcgguasgsc
GCUUAUGA






AD-
A-
249
usasccg(Uhd)CfaAf
A-
693
VPusCfsauaAfgCfAfaa
GCUACCGUCAACUUU
1137


1244366.3
1991728.1

CfUfuugcuuausgsa
1803954.1

guUfgAfcgguasgsc
GCUUAUGA






AD-
A-
250
usasccg(Uhd)CfaAf
A-
694
VPusCfsaudAa(G2p)ca
GCUACCGUCAACUUU
1138


1565757.1
1991728.1

CfUfuugcuuausgsa
2894528.1

aaguUfgAfcgguasgsc
GCUUAUGA






AD-
A-
251
usasccg(Uhd)CfaAf
A-
695
VPusCfsadTa(Agn)gca
GCUACCGUCAACUUU
1139


1565821.1
1991728.1

CfUfuugcuuausgsa
2894627.1

aaguUfgAfcgguasgsc
GCUUAUGA






AD-
A-
252
usasccg(Uhd)CfaAf
A-
696
VPusdCsaudAa(G2p)c
GCUACCGUCAACUUU
1140


1565822.1
1991728.1

CfUfuugcuuausgsa
2894628.1

aaadGuUfgAfcgguasgs
GCUUAUGA






AD-
A-
253
usasccc(Uhd)CfaAf
A-
697
VPusCfsaudAa(G2p)ca
GCUACCGUCAACUUU
1141


1565824.1
2894631.1

CfUfuugcuuausgsa
2894632.1

aaguUfgAfggguasgsc
GCUUAUGA






AD-
A-
254
usascgg(Uhd)CfaAf
A-
698
VPusCfsaudAa(G2p)ca
GCUACCGUCAACUUU
1142


1565825.1
2894633.1

CfUfuugcuuausgsa
2894634.1

aaguUfgAfccguasgsc
GCUUAUGA






AD-
A-
255
usasgcg(Uhd)CfaAf
A-
699
VPusCfsaudAa(G2p)ca
GCUACCGUCAACUUU
1143


1565826.1
2894635.1

CfUfuugcuuausgsa
2894636.1

aaguUfgAfcgcuasgsc
GCUUAUGA






AD-
A-
256
usasccg(Uhd)CfaAf
A-
700
VPusCfsaudAa(G2p)ca
GCUACCGUCAACUUU
1144


1565757.2
1991728.1

CfUfuugcuuausgsa
2894528.1

aaguUfgAfcgguasgsc
GCUUAUGA






AD-
A-
257
usasccg(Uhd)caAfCf
A-
701
VPusdCsaudAadGcaaa
GCUACCGUCAACUUU
1145


1565586.1
2894233.1

Ufuugcuuausgsa
2894234.1

dGuUfgacgguasgsc
GCUUAUGA






AD-
A-
258
ascscgu(Chd)AfaCf
A-
702
VPusUfscadTa(Agn)gc
CUACCGUCAACUUUG
1146


1565587.1
2324727.1

UfUfugcuuaugsasa
2894235.1

aaagUfuGfacggusasg
CUUAUGAC






AD-
A-
259
ascscgu(Chd)aaCfUf
A-
703
VPusdTscadTadAgcaa
CUACCGUCAACUUUG
1147


1565588.1
2894236.1

Ufugcuuaugsasa
2894237.1

dAgUfugacggusasg
CUUAUGAC






AD-
A-
260
cscsguc(Ahd)AfCfUf
A-
704
VPusCfsaudAa(G2p)ca
UACCGUCAACUUUGC
1148


1565823.1
2894629.1

uugcuuausgsa
2894630.1

aaguUfgAfcggsusg
UUAUGA






AD-
A-
261
cscsguc(Ahd)acUfU
A-
705
VPusdGsucdAudAagca
UACCGUCAACUUUGC
1149


1565589.1
2894238.1

fUfgcuuaugascsa
2894239.1

dAaGfuugacggsusa
UUAUGACA






AD-
A-
262
csgsuca(Ahd)CfuUf
A-
706
VPusUfsgudCa(Tgn)aa
ACCGUCAACUUUGCU
1150


1565590.1
2894240.1

UfGfcuuaugacsasa
2894241.1

gcaaAfgUfugacgsgsu
UAUGACAC






AD-
A-
263
gsuscaa(Chd)UfuUf
A-
707
VPusGfsugdTc(Agn)ua
CCGUCAACUUUGCUU
1151


1565591.1
2894242.1

GfCfuuaugacascsa
2894243.1

agcaAfaGfuugacsgsg
AUGACACA






AD-
A-
264
uscsaac(Uhd)UfuGf
A-
708
VPusUfsgudGu(C2p)a
CGUCAACUUUGCUUA
1152


1565758.1
2894244.1

CfUfuaugacacsasa
2894529.1

uaagcAfaAfguugascsg
UGACACAG






AD-
A-
265
uscsaac(Uhd)UfuGf
A-
709
VPusUfsgdTg(Tgn)cau
CGUCAACUUUGCUUA
1153


1565592.1
2894244.1

CfUfuaugacacsasa
2894245.1

aagcAfaAfguugascsg
UGACACAG






AD-
A-
266
csasacu(Uhd)UfgCf
A-
710
VPusCfsugdTg(Tgn)ca
GUCAACUUUGCUUAU
1154


1565594.1
2894247.1

UfUfaugacacasgsa
2894248.1

uaagCfaAfaguugsasc
GACACAGG






AD-
A-
267
asascuu(Uhd)GfcUf
A-
711
VPusCfscudGu(G2p)uc
UCAACUUUGCUUAUG
1155


1565759.1
2894530.1

UfAfugacacagsgsa
2894531.1

auaaGfcAfaaguusgsa
ACACAGGC






AD-
A-
268
ascsuuug(Chd)uUfA
A-
712
VPusGfsccdTg(Tgn)gu
CAACUUUGCUUAUGA
1156


1565595.1
2894249.1

fUfgacacaggscsa
2894250.1

cauaAfgCfaaagususg
CACAGGCA






AD-
A-
269
csusuug(Chd)UfuAf
A-
713
VPusUfsgcdCu(G2p)ug
AACUUUGCUUAUGAC
1157


1565760.1
2894532.1

UfGfacacaggcsasa
2894533.1

ucauAfaGfcaaagsusu
ACAGGCAC






AD-
A-
270
ascsagg(Chd)AfcAf
A-
714
VPusUfsugdCu(G2p)a
ACACAGGCACAGGUA
1158


1565761.1
2894534.1

GfGfuaucagcasasa
2894535.1

uaccuGfuGfccugusgsu
UCAGCAAG






AD-
A-
271
asusaag(Uhd)AfcAf
A-
715
VPusAfsaudCa(Tgn)gc
CUAUAAGUACAGCAG
1159


1565596.1
2894251.1

GfCfagcaugaususa
2894252.1

ugcuGfuAfcuuausasg
CAUGAUUG






AD-
A-
272
usasagu(Ahd)CfaGf
A-
716
VPusCfsaadTc(Agn)ug
UAUAAGUACAGCAGC
1160


1565597.1
2894253.1

CfAfgcaugauusgsa
2894254.1

cugcUfgUfacuuasusa
AUGAUUGA






AD-
A-
273
asasgua(Chd)AfgCf
A-
717
VPusUfscadAu(C2p)au
AUAAGUACAGCAGCA
1161


1565762.1
2894255.1

AfGfcaugauugsasa
2894536.1

gcugCfuGfuacuusasu
UGAUUGAC






AD-
A-
274
asasgua(Chd)AfgCf
A-
718
VPusUfscdAa(Tgn)cau
AUAAGUACAGCAGCA
1162


1565598.1
2894255.1

AfGfcaugauugsasa
2894256.1

gcugCfuGfuacuusasu
UGAUUGAC






AD-
A-
275
asasgua(Chd)AfgCf
A-
719
VPusUfscaadTc(Agn)u
AUAAGUACAGCAGCA
1163


1565599.1
2894255.1

AfGfcaugauugsasa
2894257.1

gcugCfuGfuacuusasu
UGAUUGAC






AD-
A-
276
asgsuac(Ahd)GfcAf
A-
720
VPusGfsucdAa(Tgn)ca
UAAGUACAGCAGCAU
1164


1565600.1
2894258.1

GfCfaugauugascsa
2894259.1

ugcuGfcUfguacususa
GAUUGACU






AD-
A-
277
asgsuac(Ahd)gcAfG
A-
721
VPusdGsucdAadTcaug
UAAGUACAGCAGCAU
1165


1565601.1
2894260.1

fCfaugauugascsa
2894261.1

dCuGfcuguacususa
GAUUGACU






AD-
A-
278
gsusacag(Chd)aGfCf
A-
722
VPusAfsgudCa(Agn)uc
AAGUACAGCAGCAUG
1166


1565602.1
2894262.1

Afugauugacsusa
2894263.1

augcUfgCfuguacsusu
AUUGACUA






AD-
A-
279
gsusaca(Ghd)CfaGf
A-
723
VPusAfsgucAfaUfCfau
AAGUACAGCAGCAUG
1167


1565827.1
2894637.1

CfAfugauugacsusa
1804098.1

gcUfgCfuguacsusu
AUUGACUA






AD-
A-
280
gsusacag(Chd)aGfCf
A-
724
VPusAfsgudCa(A2p)uc
AAGUACAGCAGCAUG
1168


1565828.1
2894262.1

Afugauugacsusa
2894638.1

augcUfgCfuguacsusu
AUUGACUA






AD-
A-
281
gsusacag(Chd)aGfCf
A-
725
VPusdAsgudCadAucau
AAGUACAGCAGCAUG
1169


1565829.1
2894262.1

Afugauugacsusa
2894639.1

dGcUfgcuguacsusu
AUUGACUA






AD-
A-
282
gsusacag(Chd)aGfCf
A-
726
VPusdAsgudCa(Agn)uc
AAGUACAGCAGCAUG
1170


1565830.1
2894262.1

Afugauugacsusa
2894640.1

augcUfgCfuguacsusu
AUUGACUA






AD-
A-
283
gsusacag(Chd)aGfCf
A-
727
VPusdAsgudCa(Agn)uc
AAGUACAGCAGCAUG
1171


1565831.1
2894262.1

Afugauugacsusa
2894641.1

audGcUfgcuguacsusu
AUUGACUA






AD-
A-
284
gsusacug(Chd)aGfC
A-
728
VPusdAsgudCa(Agn)uc
AAGUACAGCAGCAUG
1172


1565833.1
2894644.1

fAfugauugacsusa
2894645.1

augcUfgCfaguacsusu
AUUGACUA






AD-
A-
285
gsusagag(Chd)aGfC
A-
729
VPusdAsgudCa(Agn)uc
AAGUACAGCAGCAUG
1173


1565834.1
2894646.1

fAfugauugacsusa
2894647.1

augcUfgCfucuacsusu
AUUGACUA






AD-
A-
286
gsusucag(Chd)aGfC
A-
730
VPusdAsgudCa(Agn)uc
AAGUACAGCAGCAUG
1174


1565835.3
2894648.1

fAfugauugacsusa
2894649.1

augcUfgCfugaacsusu
AUUGACUA






AD-
A-
287
gsusacag(Chd)aGfCf
A-
731
VPusAfsgudCa(Agn)uc
AAGUACAGCAGCAUG
1175


1565602.2
2894262.1

Afugauugacsusa
2894263.1

augcUfgCfuguacsusu
AUUGACUA






AD-
A-
288
usascag(Chd)AfgCf
A-
732
VPusUfsagdTc(Agn)au
AGUACAGCAGCAUGA
1176


1565603.1
2894264.1

AfUfgauugacusasa
2894265.1

caugCfuGfcuguascsu
UUGACUAC






AD-
A-
289
ascsag(Chd)aGfCfAf
A-
733
VPusdAsgudCa(Agn)uc
GUACAGCAGCAUGAU
1177


1565832.1
2894642.1

ugauugacsusa
2894643.1

augcUfgCfugusgsc
UGACUA






AD-
A-
290
ascsagc(Ahd)GfcAf
A-
734
VPusGfsuadGu(C2p)aa
GUACAGCAGCAUGAU
1178


1565763.1
2894266.1

UfGfauugacuascsa
2894537.1

ucauGfcUfgcugusasc
UGACUACA






AD-
A-
291
ascsagc(Ahd)GfcAf
A-
735
VPusGfsudAg(Tgn)caa
GUACAGCAGCAUGAU
1179


1565604.1
2894266.1

UfGfauugacuascsa
2894267.1

ucauGfcUfgcugusasc
UGACUACA






AD-
A-
292
ascsagc(Ahd)GfcAf
A-
736
VPusGfsuagdTc(Agn)a
GUACAGCAGCAUGAU
1180


1565605.1
2894266.1

UfGfauugacuascsa
2894268.1

ucauGfcUfgcugusasc
UGACUACA






AD-
A-
293
csasgcag(Chd)aUfG
A-
737
VPusUfsgudAg(Tgn)ca
UACAGCAGCAUGAUU
1181


1565606.1
2894269.1

fAfuugacuacsasa
2894270.1

aucaUfgCfugcugsusa
GACUACAA






AD-
A-
294
asgscag(Chd)AfuGf
A-
738
VPusUfsugdTa(G2p)uc
ACAGCAGCAUGAUUG
1182


1565764.1
2894538.1

AfUfugacuacasasa
2894539.1

aaucAfuGfcugcusgsu
ACUACAAC






AD-
A-
295
gscsagc(Ahd)UfgAf
A-
739
VPusGfsuudGu(Agn)g
CAGCAGCAUGAUUGA
1183


1565607.1
2894271.1

UfUfgacuacaascsa
2894272.1

ucaauCfaUfgcugcsusg
CUACAACC






AD-
A-
296
csasgca(Uhd)GfaUf
A-
740
VPusGfsgudTg(Tgn)ag
AGCAGCAUGAUUGAC
1184


1565608.1
2894273.1

UfGfacuacaacscsa
2894274.1

ucaaUfcAfugcugscsu
UACAACCC






AD-
A-
297
gscsaug(Ahd)UfuGf
A-
741
VPusGfsggdGu(Tgn)gu
CAGCAUGAUUGACUA
1185


1565609.1
2894275.1

AfCfuacaacccscsa
2894276.1

agucAfaUfcaugcsusg
CAACCCCC






AD-
A-
298
asgscuc(Uhd)UfuGf
A-
742
VPusGfsuudGu(C2p)cc
GAAGCUCUUUGCCUG
1186


1565766.1
2894279.1

CfCfugggacaascsa
2894542.1

aggcAfaAfgagcususc
GGACAACU






AD-
A-
299
asgscuc(Uhd)UfuGf
A-
743
VPusGfsudTg(Tgn)ccc
GAAGCUCUUUGCCUG
1187


1565611.1
2894279.1

CfCfugggacaascsa
2894280.1

aggcAfaAfgagcususc
GGACAACU






AD-
A-
300
gscscuggGfaCfAfAfc
A-
744
VPusAfsugdTu(C2p)aa
UUGCCUGGGACAACU
1188


1565767.1
2894281.1

uug(Ahd)acasusa
2894543.1

guugUfcCfcaggcsasa
UGAACAUG






AD-
A-
301
gscscuggGfaCfAfAfc
A-
745
VPusAfsudGu(Tgn)caa
UUGCCUGGGACAACU
1189


1565612.1
2894281.1

uug(Ahd)acasusa
2894282.1

guugUfcCfcaggcsasa
UGAACAUG






AD-
A-
302
gscscuggGfaCfAfAfc
A-
746
VPusAfsugudTc(Agn)a
UUGCCUGGGACAACU
1190


1565613.1
2894281.1

uug(Ahd)acasusa
2894283.1

guugUfcCfcaggcsasa
UGAACAUG






AD-
A-
303
csusggg(Ahd)CfaAf
A-
747
VPusCfscadTg(Tgn)uca
GCCUGGGACAACUUG
1191


1565614.1
2894284.1

CfUfugaacaugsgsa
2894285.1

aguUfgUfcccagsgsc
AACAUGGU






AD-
A-
304
usgsgga(Chd)AfaCf
A-
748
VPusAfsccdAu(G2p)uu
CCUGGGACAACUUGA
1192


1565768.1
2894544.1

UfUfgaacauggsusa
2894545.1

caagUfuGfucccasgsg
ACAUGGUC






AD-
A-
305
gsgsgac(Ahd)AfcUf
A-
749
VPusGfsacdCa(Tgn)gu
CUGGGACAACUUGAA
1193


1565615.1
2894286.1

UfGfaacaugguscsa
2894287.1

ucaaGfuUfgucccsasg
CAUGGUCA






AD-
A-
306
gsgsgac(Ahd)acUfU
A-
750
VPusdGsacdCadTguuc
CUGGGACAACUUGAA
1194


1565616.1
2894288.1

fGfaacaugguscsa
2894289.1

dAaGfuugucccsasg
CAUGGUCA






AD-
A-
307
gsgsaca(Ahd)CfuUf
A-
751
VPusUfsgadCc(Agn)ug
UGGGACAACUUGAAC
1195


1565617.1
2894290.1

GfAfacauggucsasa
2894291.1

uucaAfgUfuguccscsa
AUGGUCAC






AD-
A-
308
gsascaa(Chd)UfuGf
A-
752
VPusGfsugdAc(C2p)au
GGGACAACUUGAACA
1196


1565769.1
2894292.1

AfAfcauggucascsa
2894546.1

guucAfaGfuugucscsc
UGGUCACU






AD-
A-
309
gsascaa(Chd)UfuGf
A-
753
VPusGfsugadCc(Agn)u
GGGACAACUUGAACA
1197


1565618.1
2894292.1

AfAfcauggucascsa
2894293.1

guucAfaGfuugucscsc
UGGUCACU






AD-
A-
310
ascsaac(Uhd)UfgAf
A-
754
VPusAfsgudGa(C2p)ca
GGACAACUUGAACAU
1198


1565770.1
2894294.1

AfCfauggucacsusa
2894547.1

uguuCfaAfguuguscsc
GGUCACUU






AD-
A-
311
ascsaac(Uhd)UfgAf
A-
755
VPusAfsgdTg(Agn)cca
GGACAACUUGAACAU
1199


1565619.1
2894294.1

AfCfauggucacsusa
2894295.1

uguuCfaAfguuguscsc
GGUCACUU






AD-
A-
312
csasacu(Uhd)GfaAf
A-
756
VPusAfsagdTg(Agn)cc
GACAACUUGAACAUG
1200


1565620.1
2894296.1

CfAfuggucacususa
2894297.1

auguUfcAfaguugsusc
GUCACUUA






AD-
A-
313
asascuug(Ahd)aCfA
A-
757
VPusUfsaadGu(G2p)ac
ACAACUUGAACAUGG
1201


1565771.1
2894548.1

fUfggucacuusasa
2894549.1

caugUfuCfaaguusgsu
UCACUUAU






AD-
A-
314
ascsuug(Ahd)AfcAf
A-
758
VPusAfsuadAg(Tgn)ga
CAACUUGAACAUGGU
1202


1565621.1
1577540.1

UfGfgucacuuasusa
2894298.1

ccauGfuUfcaagususg
CACUUAUG






AD-
A-
315
csusuga(Ahd)CfaUf
A-
759
VPusCfsaudAa(G2p)ug
AACUUGAACAUGGUC
1203


1565772.1
1577508.1

GfGfucacuuausgsa
2894550.1

accaUfgUfucaagsusu
ACUUAUGA






AD-
A-
316
csusuga(Ahd)CfaUf
A-
760
VPusdCsaudAadGugac
AACUUGAACAUGGUC
1204


1565836.1
1577508.1

GfGfucacuuausgsa
2894300.1

dCaUfguucaagsusu
ACUUAUGA






AD-
A-
317
csusuga(Ahd)CfaUf
A-
761
VPusCfsadTa(Agn)gug
AACUUGAACAUGGUC
1205


1565837.1
1577508.1

GfGfucacuuausgsa
2894650.1

accaUfgUfucaagsusu
ACUUAUGA






AD-
A-
318
csusuga(Ahd)CfaUf
A-
762
VPusdCsaudAa(G2p)u
AACUUGAACAUGGUC
1206


1565838.1
1577508.1

GfGfucacuuausgsa
2894651.1

gacdCaUfguucaagsusu
ACUUAUGA






AD-
A-
319
csusugu(Ahd)CfaUf
A-
763
VPusCfsaudAa(G2p)ug
AACUUGAACAUGGUC
1207


1565840.1
2894654.1

GfGfucacuuausgsa
2894655.1

accaUfgUfacaagsusu
ACUUAUGA






AD-
A-
320
csusuca(Ahd)CfaUf
A-
764
VPusCfsaudAa(G2p)ug
AACUUGAACAUGGUC
1208


1565841.1
2894656.1

GfGfucacuuausgsa
2894657.1

accaUfgUfugaagsusu
ACUUAUGA






AD-
A-
321
csusaga(Ahd)CfaUf
A-
765
VPusCfsaudAa(G2p)ug
AACUUGAACAUGGUC
1209


1565842.1
2894658.1

GfGfucacuuausgsa
2894659.1

accaUfgUfucuagsusu
ACUUAUGA






AD-
A-
322
csusuga(Ahd)CfaUf
A-
766
VPusCfsauaAfgUfGfac
AACUUGAACAUGGUC
1210


822899.17
1577508.1

GfGfucacuuausgsa
1577509.1

caUfgUfucaagsusu
ACUUAUGA






AD-
A-
323
csusuga(Ahd)CfaUf
A-
767
VPusCfsaudAa(G2p)ug
AACUUGAACAUGGUC
1211


1565772.2
1577508.1

GfGfucacuuausgsa
2894550.1

accaUfgUfucaagsusu
ACUUAUGA






AD-
A-
324
csusuga(Ahd)caUfG
A-
768
VPusdCsaudAadGugac
AACUUGAACAUGGUC
1212


1565622.1
2894299.1

fGfucacuuausgsa
2894300.1

dCaUfguucaagsusu
ACUUAUGA






AD-
A-
325
ususgaa(Chd)AfuGf
A-
769
VPusUfscadTa(Agn)gu
ACUUGAACAUGGUCA
1213


1565843.1
1577562.1

GfUfcacuuaugsasa
2894660.1

gaccAfuGfuucaasgsu
CUUAUGAC






AD-
A-
326
ususgaa(Chd)AfuGf
A-
770
VPusUfscadTa(A2p)gu
ACUUGAACAUGGUCA
1214


1565844.1
1577562.1

GfUfcacuuaugsasa
2894661.1

gaccAfuGfuucaasgsu
CUUAUGAC






AD-
A-
327
ususgaa(Chd)AfuGf
A-
771
VPusdTscadTa(Agn)gu
ACUUGAACAUGGUCA
1215


1565845.1
1577562.1

GfUfcacuuaugsasa
2894662.1

gadCcAfuguucaasgsu
CUUAUGAC






AD-
A-
328
ususgaa(Chd)audG
A-
772
VPusdTscadTa(Agn)gu
ACUUGAACAUGGUCA
1216


1565846.1
2894663.1

gUfCfacuuaugsasa
2894662.1

gadCcAfuguucaasgsu
CUUAUGAC






AD-
A-
329
ususgau(Chd)AfuGf
A-
773
VPusUfscadTa(Agn)gu
ACUUGAACAUGGUCA
1217


1565848.1
2894666.1

GfUfcacuuaugsasa
2894667.1

gaccAfuGfaucaasgsu
CUUAUGAC






AD-
A-
330
ususgua(Chd)AfuGf
A-
774
VPusUfscadTa(Agn)gu
ACUUGAACAUGGUCA
1218


1565849.1
2894668.1

GfUfcacuuaugsasa
2894669.1

gaccAfuGfuacaasgsu
CUUAUGAC






AD-
A-
331
ususcaa(Chd)AfuGf
A-
775
VPusUfscadTa(Agn)gu
ACUUGAACAUGGUCA
1219


1565850.1
2894670.1

GfUfcacuuaugsasa
2894671.1

gaccAfuGfuugaasgsu
CUUAUGAC






AD-
A-
332
ususgaa(Chd)AfuGf
A-
776
VPusUfscauAfaGfUfga
ACUUGAACAUGGUCA
1220


822926.4
1577562.1

GfUfcacuuaugsasa
1577563.1

ccAfuGfuucaasgsu
CUUAUGAC






AD-
A-
333
ususgaa(Chd)auGfG
A-
777
VPusdTscadTadAguga
ACUUGAACAUGGUCA
1221


1565623.1
2894301.1

fUfcacuuaugsasa
2894302.1

dCcAfuguucaasgsu
CUUAUGAC






AD-
A-
334
usgsa(Ahd)CfaUfGf
A-
778
VPusCfsaudAa(G2p)ug
CUUGAACAUGGUCAC
1222


1565839.1
2894652.1

Gfucacuuausgsa
2894653.1

accaUfgUfucasgsg
UUAUGA






AD-
A-
335
usgsaac(Ahd)ugGfU
A-
779
VPusdGsucdAudAagu
CUUGAACAUGGUCAC
1223


1565624.1
2894303.1

fCfacuuaugascsa
2894304.1

gdAcCfauguucasasg
UUAUGACA






AD-
A-
336
gsasaca(Uhd)GfGfU
A-
780
VPusUfscadTa(Agn)gu
UUGAACAUGGUCACU
1224


1565847.1
2894664.1

fcacuuaugsasa
2894665.1

gaccAfuGfuucsgsg
UAUGAC






AD-
A-
337
gsasaca(Uhd)GfgUf
A-
781
VPusUfsgudCa(Tgn)aa
UUGAACAUGGUCACU
1225


1565625.1
2894305.1

CfAfcuuaugacsasa
2894306.1

gugaCfcAfuguucsasa
UAUGACAU






AD-
A
338
asasca(Uhd)gGfuCf
A-
782
VPusAfsugdTc(Agn)ua
UGAACAUGGUCACUU
1226


1565626.1
2894307.1

AfCfuuaugacasusa
2894308.1

agugAfcCfauguuscsa
AUGACAUC






AD-
A-
339
ascsaugg(Uhd)cAfCf
A-
783
VPusGfsaudGu(C2p)a
GAACAUGGUCACUUA
1227


1565773.1
2894309.1

Ufuaugacauscsa
2894551.1

uaaguGfaCfcaugususc
UGACAUCA






AD-
A-
340
ascsaugg(Uhd)cAfCf
A-
784
VPusGfsadTg(Tgn)cau
GAACAUGGUCACUUA
1228


1565627.1
2894309.1

Ufuaugacauscsa
2894310.1

aaguGfaCfcaugususc
UGACAUCA






AD-
A-
341
ascsaugg(Uhd)cAfCf
A-
785
VPusGfsaugdTc(Agn)u
GAACAUGGUCACUUA
1229


1565628.1
2894309.1

Ufuaugacauscsa
2894311.1

aaguGfaCfcaugususc
UGACAUCA






AD-
A-
342
csasugg(Uhd)CfaCf
A-
786
VPusUfsgadTg(Tgn)ca
AACAUGGUCACUUAU
1230


1565629.1
2894312.1

UfUfaugacaucsasa
2894313.1

uaagUfgAfccaugsusu
GACAUCAA






AD-
A-
343
asusggu(Chd)AfcUf
A-
787
VPusUfsugdAu(G2p)u
ACAUGGUCACUUAUG
1231


1565774.1
2894552.1

UfAfugacaucasasa
2894553.1

cauaaGfuGfaccausgsu
ACAUCAAG






AD-
A-
344
usgsguc(Ahd)CfuUf
A-
788
VPusCfsuudGa(Tgn)gu
CAUGGUCACUUAUGA
1232


1565630.1
2894314.1

AfUfgacaucaasgsa
2894315.1

cauaAfgUfgaccasusg
CAUCAAGC






AD-
A
345
gsgsuca(Chd)UfuAf
A-
789
VPusGfscudTg(Agn)ug
AUGGUCACUUAUGAC
1233


1565631.1
2894316.1

UfGfacaucaagscsa
2894317.1

ucauAfaGfugaccsasu
AUCAAGCU






AD-
A-
346
gsuscac(Uhd)UfaUf
A-
790
VPusAfsgcdTu(G2p)au
UGGUCACUUAUGACA
1234


1565775.1
2894554.1

GfAfcaucaagcsusa
2894555.1

gucaUfaAfgugacscsa
UCAAGCUC






AD-
A-
347
gscsaga(Ahd)GfgAf
A-
791
VPusCfsccdTg(Agn)gca
UGGCAGAAGGAGAUG
1235


1565632.1
2894318.1

GfAfugcucaggsgsa
2894319.1

ucuCfcUfucugcscsa
CUCAGGGC






AD-
A-
348
asasggg(Ahd)GfaGf
A-
792
VPusUfsggdCu(G2p)gc
UGAAGGGAGAGCCAG
1236


1565776.1
2894556.1

CfCfagccagccsasa
2894557.1

uggcUfcUfcccuuscsa
CCAGCCAG






AD-
A
349
gsasuga(Ahd)CfaUf
A-
793
VPusAfsgadTg(G2p)ug
AGGAUGAACAUGGUC
1237


1565777.1
2894558.1

GfGfucaccaucsusa
2894559.1

accaUfgUfucaucscsu
ACCAUCUA






AD-
A-
350
gscsauu(Uhd)auGfG
A-
794
VPusdAsuudAadAcauc
CAGCAUUUAUGGGAU
1238


1565634.1
2894322.1

fGfauguuuaasusa
2894323.1

dCcAfuaaaugcsusg
GUUUAAUG






AD-
A-
351
usgsuuu(Ahd)AfuGf
A-
795
VPusUfsugdAa(C2p)ua
GAUGUUUAAUGACA
1239


1565778.1
2894324.1

AfCfauaguucasasa
2894560.1

ugucAfuUfaaacasusc
UAGUUCAAG






AD-
A-
352
usgsuuu(Ahd)AfuGf
A-
796
VPusUfsudGa(Agn)cua
GAUGUUUAAUGACA
1240


1565635.1
2894324.1

AfCfauaguucasasa
2894325.1

ugucAfuUfaaacasusc
UAGUUCAAG






AD-
A-
353
usgsuuu(Ahd)AfuGf
A-
797
VPusUfsugadAc(Tgn)a
GAUGUUUAAUGACA
1241


1565636.1
2894324.1

AfCfauaguucasasa
2894326.1

ugucAfuUfaaacasusc
UAGUUCAAG






AD-
A-
354
gsusuua(Ahd)UfgAf
A-
798
VPusCfsuudGa(Agn)cu
AUGUUUAAUGACAUA
1242


1565637.1
2894327.1

CfAfuaguucaasgsa
2894328.1

auguCfaUfuaaacsasu
GUUCAAGU






AD-
A-
355
gsusuua(Ahd)ugAfC
A-
799
VPusdCsuudGadAcua
AUGUUUAAUGACAUA
1243


1565638.1
2894329.1

fAfuaguucaasgsa
2894330.1

udGuCfauuaaacsasu
GUUCAAGU






AD-
A-
356
ususuaa(Uhd)GfaCf
A-
800
VPusAfscudTg(Agn)ac
UGUUUAAUGACAUA
1244


1565639.1
2894331.1

AfUfaguucaagsusa
2894332.1

uaugUfcAfuuaaascsa
GUUCAAGUU






AD-
A-
357
ususaaug(Ahd)cAfU
A-
801
VPusAfsacdTu(G2p)aa
GUUUAAUGACAUAG
1245


1565779.1
2894561.1

fAfguucaagususa
2894562.1

cuauGfuCfauuaasasc
UUCAAGUUU






AD-
A-
358
usasaug(Ahd)CfaUf
A-
802
VPusAfsaadCu(Tgn)ga
UUUAAUGACAUAGU
1246


1565640.1
2894333.1

AfGfuucaaguususa
2894334.1

acuaUfgUfcauuasasa
UCAAGUUUU






AD-
A-
359
usasaug(Ahd)caUfA
A-
803
VPusdAsaadCudTgaac
UUUAAUGACAUAGU
1247


1565641.1
2894335.1

fGfuucaaguususa
2894336.1

dTaUfgucauuasasa
UCAAGUUUU






AD-
A-
360
asasuga(Chd)AfuAf
A
804
VPusAfsaadAc(Tgn)ug
UUAAUGACAUAGUUC
1248


1565642.1
2894337.1

GfUfucaaguuususa
2894338.1

aacuAfuGfucauusasa
AAGUUUUC






AD-
A-
361
asasuga(Chd)auAfG
A-
805
VPusdAsaadAcdTugaa
UUAAUGACAUAGUUC
1249


1565643.1
2894339.1

fUfucaaguuususa
2894340.1

dCuAfugucauusasa
AAGUUUUC






AD-
A-
362
asusgac(Ahd)uaGfU
A-
806
VPusdGsaadAadCuuga
UAAUGACAUAGUUCA
1250


1565644.1
2894341.1

fUfcaaguuuuscsa
2894342.1

dAcUfaugucaususa
AGUUUUCU






AD-
A-
363
usgsaca(Uhd)agUfU
A-
807
VPusdAsgadAadAcuug
AAUGACAUAGUUCAA
1251


1565645.1
2894343.1

fCfaaguuuucsusa
2894344.1

dAaCfuaugucasusu
GUUUUCUU






AD-
A-
364
gsascau(Ahd)guUfC
A-
808
VPusdAsagdAadAacuu
AUGACAUAGUUCAAG
1252


1565646.1
2894345.1

fAfaguuuucususa
2894346.1

dGaAfcuaugucsasu
UUUUCUUG






AD
A-
365
ascsaua(Ghd)UfuCf
A-
809
VPusCfsaaga(Agn)aac
UGACAUAGUUCAAGU
1253


1565851.1
2894672.1

AfAfguuuucuusgsa
1806645.1

uugAfaCfuauguscsa
UUUCUUGU






AD-
A-
366
ascsauag(Uhd)uCfA
A-
810
VPusdCsaadGadAaacu
UGACAUAGUUCAAGU
1254


1565852.1
2894347.1

fAfguuuucuusgsa
2894673.1

dTgAfacuauguscsg
UUUCUUGU






AD-
A-
367
ascsauag(Uhd)uCfA
A-
811
VPusCfsaadGa(Agn)aa
UGACAUAGUUCAAGU
1255


1565853.1
2894347.1

fAfguuuucuusgsa
2894674.1

cuugAfaCfuauguscsg
UUUCUUGU






AD-
A-
368
ascsauag(Uhd)uCfA
A-
812
VPusdCsaadGa(Agn)a
UGACAUAGUUCAAGU
1256


1565854.1
2894347.1

fAfguuuucuusgsa
2894675.1

acudTgAfaCfuauguscs
UUUCUUGU






AD-
A-
369
ascsauug(Uhd)uCfA
A-
813
VPusCfsaadGa(Agn)aa
UGACAUAGUUCAAGU
1257


1565857.1
2894679.1

fAfguuuucuusgsa
2894680.1

cuugAfaCfaauguscsg
UUUCUUGU






AD-
A-
370
ascsaaag(Uhd)uCfA
A-
814
VPusCfsaadGa(Agn)aa
UGACAUAGUUCAAGU
1258


1565858.1
2894681.1

fAfguuuucuusgsa
2894682.1

cuugAfaCfuuuguscsg
UUUCUUGU






AD-
A-
371
ascsuuag(Uhd)uCfA
A-
815
VPusCfsaadGa(Agn)aa
UGACAUAGUUCAAGU
1259


1565859.1
2894683.1

fAfguuuucuusgsa
2894684.1

cuugAfaCfuaaguscsg
UUUCUUGU






AD-
A-
372
ascsauag(Uhd)uCfA
A-
816
VPusdCsaadGadAaacu
UGACAUAGUUCAAGU
1260


1565647.1
2894347.1

fAfguuuucuusgsa
2894348.1

dTgAfacuauguscsa
UUUCUUGU






AD-
A-
373
csasuag(Uhd)ucAfA
A-
817
VPusdAscadAgdAaaac
GACAUAGUUCAAGUU
1261


1565648.1
2894349.1

fGfuuuucuugsusa
2894350.1

dTuGfaacuaugsusc
UUCUUGUG






AD-
A-
374
asusagu(Uhd)CfAfA
A-
818
VPusCfsaadGa(Agn)aa
ACAUAGUUCAAGUUU
1262


1565855.1
2894676.1

fguuuucuusgsa
2894677.1

cuugAfaCfuausgsu
UCUUGU






AD-
A-
375
asusagu(Uhd)CfAfA
A-
819
VPusdCsaadGa(Agn)a
ACAUAGUUCAAGUUU
1263


1565856.1
2894676.1

fguuuucuusgsa
2894678.1

acudTgAfaCfuausgsu
UCUUGU






AD-
A-
376
asusagu(Uhd)caAfG
A-
820
VPusdCsacdAadGaaaa
ACAUAGUUCAAGUUU
1264


1565649.1
2894351.1

fUfuuucuugusgsa
2894352.1

dCuUfgaacuausgsu
UCUUGUGA






AD-
A-
377
usasguu(Chd)AfaGf
A-
821
VPusUfscadCa(Agn)ga
CAUAGUUCAAGUUUU
1265


1565650.1
2894353.1

UfUfuucuugugsasa
2894354.1

aaacUfuGfaacuasusg
CUUGUGAU






AD-
A-
378
usasguu(Chd)aaGfU
A-
822
VPusdTscadCadAgaaa
CAUAGUUCAAGUUUU
1266


1565651.1
2894355.1

fUfuucuugugsasa
2894356.1

dAcUfugaacuasusg
CUUGUGAU






AD-
A-
379
asgsuuc(Ahd)AfgUf
A-
823
VPusAfsucdAc(Agn)ag
AUAGUUCAAGUUUUC
1267


1565652.1
2894357.1

UfUfucuugugasusa
2894358.1

aaaaCfuUfgaacusasu
UUGUGAUU






AD-
A-
380
asgsuuc(Ahd)agUfU
A-
824
VPusdAsucdAcdAagaa
AUAGUUCAAGUUUUC
1268


1565653.1
2894359.1

fUfucuugugasusa
2894360.1

dAaCfuugaacusasu
UUGUGAUU






AD-
A-
381
gsusuca(Ahd)GfuUf
A-
825
VPusAfsaudCa(C2p)aa
UAGUUCAAGUUUUC
1269


1565780.1
2894361.1

UfUfcuugugaususa
2894563.1

gaaaAfcUfugaacsusa
UUGUGAUUU






AD-
A-
382
gsusuca(Ahd)GfuUf
A-
826
VPusAfsadTc(Agn)caa
UAGUUCAAGUUUUC
1270


1565654.1
2894361.1

UfUfcuugugaususa
2894362.1

gaaaAfcUfugaacsusa
UUGUGAUUU






AD-
A-
383
ususcaag(Uhd)uUf
A-
827
VPusAfsaadTc(Agn)ca
AGUUCAAGUUUUCU
1271


1565655.1
2894363.1

UfCfuugugauususa
2894364.1

agaaAfaCfuugaascsu
UGUGAUUUG






AD-
A-
384
uscsaag(Uhd)UfuUf
A-
828
VPusCfsaadAu(C2p)ac
GUUCAAGUUUUCUU
1272


1565781.1
2894365.1

CfUfugugauuusgsa
2894564.1

aagaAfaAfcuugasasc
GUGAUUUGG






AD-
A-
385
uscsaag(Uhd)UfuUf
A-
829
VPusCfsadAa(Tgn)cac
GUUCAAGUUUUCUU
1273


1565656.1
2894365.1

CfUfugugauuusgsa
2894366.1

aagaAfaAfcuugasasc
GUGAUUUGG






AD-
A-
386
uscsaag(Uhd)UfuUf
A-
830
VPusCfsaaadTc(Agn)c
GUUCAAGUUUUCUU
1274


1565657.1
2894365.1

CfUfugugauuusgsa
2894367.1

aagaAfaAfcuugasasc
GUGAUUUGG






AD-
A-
387
uscsaag(Uhd)uuUfC
A-
831
VPusdCsaadAudCacaa
GUUCAAGUUUUCUU
1275


1565658.1
2894368.1

fUfugugauuusgsa
2894369.1

dGaAfaacuugasasc
GUGAUUUGG






AD-
A-
388
csasagu(Uhd)uuCfU
A-
832
VPusdCscadAadTcaca
UUCAAGUUUUCUUG
1276


1565659.1
2894370.1

fUfgugauuugsgsa
2894371.1

dAgAfaaacuugsasa
UGAUUUGGG






AD-
A-
389
asasguu(Uhd)ucUf
A-
833
VPusdCsccdAadAucac
UCAAGUUUUCUUGU
1277


1565660.1
2894372.1

UfGfugauuuggsgsa
2894373.1

dAaGfaaaacuusgsa
GAUUUGGGG






AD-
A-
390
usgsaaa(Ahd)CfcAf
A-
834
VPusUfsgcdAa(G2p)ag
CCUGAAAACCAUUGC
1278


1565782.1
2894565.1

UfUfgcucuugcsasa
2894566.1

caauGfgUfuuucasgsg
UCUUGCAU






AD-
A-
391
gsasaaa(Chd)CfaUf
A-
835
VPusAfsugdCa(Agn)ga
CUGAAAACCAUUGCU
1279


1565661.1
2894374.1

UfGfcucuugcasusa
2894375.1

gcaaUfgGfuuuucsasg
CUUGCAUG






AD-
A-
392
gsasaaa(Chd)caUfU
A-
836
VPusdAsugdCadAgagc
CUGAAAACCAUUGCU
1280


1565662.1
2894376.1

fGfcucuugcasusa
2894377.1

dAaUfgguuuucsasg
CUUGCAUG






AD-
A-
393
asasaac(Chd)AfuUf
A-
837
VPusCfsaudGc(Agn)ag
UGAAAACCAUUGCUC
1281


1565663.1
2894378.1

GfCfucuugcausgsa
2894379.1

agcaAfuGfguuuuscsa
UUGCAUGU






AD-
A-
394
asasaac(Chd)auUfG
A-
838
VPusdCsaudGcdAagag
UGAAAACCAUUGCUC
1282


1565664.3
2894380.1

fCfucuugcausgsa
2894381.1

dCaAfugguuuuscsa
UUGCAUGU






AD-
A-
395
asasacc(Ahd)UfuGf
A-
839
VPusAfscadTg(C2p)aa
GAAAACCAUUGCUCU
1283


1565783.1
2894382.1

CfUfcuugcaugsusa
2894567.1

gagcAfaUfgguuususc
UGCAUGUU






AD-
A-
396
asasacc(Ahd)UfuGf
A-
840
VPusAfscaudGc(Agn)a
GAAAACCAUUGCUCU
1284


1565665.1
2894382.1

CfUfcuugcaugsusa
2894383.1

gagcAfaUfgguuususc
UGCAUGUU






AD-
A-
397
asascca(Uhd)UfgCf
A-
841
VPusAfsacdAu(G2p)ca
AAAACCAUUGCUCUU
1285


1565784.1
2894568.1

UfCfuugcaugususa
2894569.1

agagCfaAfugguususu
GCAUGUUA






AD-
A-
398
ascscau(Uhd)GfcUf
A-
842
VPusUfsaadCa(Tgn)gc
AAACCAUUGCUCUUG
1286


1565666.1
2894384.1

CfUfugcauguusasa
2894385.1

aagaGfcAfauggususu
CAUGUUAC






AD-
A-
399
cscsauug(Chd)uCfU
A-
843
VPusGfsuadAc(Agn)ug
AACCAUUGCUCUUGC
1287


1565667.1
2894386.1

fUfgcauguuascsa
2894387.1

caagAfgCfaauggsusu
AUGUUACA






AD-
A-
400
csasuug(Chd)UfcUf
A-
844
VPusUfsgudAa(C2p)au
ACCAUUGCUCUUGCA
1288


1565785.1
2894388.1

UfGfcauguuacsasa
2894570.1

gcaaGfaGfcaaugsgsu
UGUUACAU






AD-
A-
401
csasuug(Chd)UfcUf
A-
845
VPusUfsgdTa(Agn)cau
ACCAUUGCUCUUGCA
1289


1565668.1
2894388.1

UfGfcauguuacsasa
2894389.1

gcaaGfaGfcaaugsgsu
UGUUACAU






AD-
A-
402
asusugc(Uhd)cuUfG
A-
846
VPusdAsugdTadAcaug
CCAUUGCUCUUGCAU
1290


1565669.1
2894390.1

fCfauguuacasusa
2894391.1

dCaAfgagcaausgsg
GUUACAUG






AD-
A-
403
ususgcu(Chd)uuGfC
A-
847
VPusdCsaudGudAacau
CAUUGCUCUUGCAUG
1291


1565670.1
2894392.1

fAfuguuacausgsa
2894393.1

dGcAfagagcaasusg
UUACAUGG






AD-
A-
404
ususgcu(Chd)UfuGf
A-
848
VPusCfsaugUfaAfCfau
CAUUGCUCUUGCAUG
1292


1565860.1
2894685.1

CfAfuguuacausgsa
1804746.1

gcAfaGfagcaasusg
UUACAUGG






AD-
A-
405
ususgca(Chd)uuGfC
A-
849
VPusdCsaudGudAacau
CAUUGCUCUUGCAUG
1293


1565861.1
2894686.1

fAfuguuacausgsa
2894687.1

dGcAfagugcaasusg
UUACAUGG






AD-
A-
406
ususggu(Chd)uuGfC
A-
850
VPusdCsaudGudAacau
CAUUGCUCUUGCAUG
1294


1565862.1
2894688.1

fAfuguuacausgsa
2894689.1

dGcAfagaccaasusg
UUACAUGG






AD-
A-
407
ususccu(Chd)uuGfC
A-
851
VPusdCsaudGudAacau
CAUUGCUCUUGCAUG
1295


1565863.1
2894690.1

fAfuguuacausgsa
2894691.1

dGcAfagaggaasusg
UUACAUGG






AD-
A-
408
ususgcu(Chd)UfudG
A-
852
VPusdCsaudGudAacau
CAUUGCUCUUGCAUG
1296


1565864.1
2894692.1

cdAUfguuacausgsa
2894393.1

dGcAfagagcaasusg
UUACAUGG






AD-
A-
409
ususgcu(Chd)UfuGf
A-
853
VPusCfsaudGu(Agn)ac
CAUUGCUCUUGCAUG
1297


1565866.1
2894685.1

CfAfuguuacausgsa
2894695.1

augcAfaGfagcaasusg
UUACAUGG






AD-
A-
410
ususgcu(Chd)uuGfC
A-
854
VPusdCsaudGudAacau
CAUUGCUCUUGCAUG
1298


1565670.2
2894392.1

fAfuguuacausgsa
2894393.1

dGcAfagagcaasusg
UUACAUGG






AD-
A-
411
usgscuc(Uhd)UfgCf
A-
855
VPusCfscadTg(Tgn)aac
AUUGCUCUUGCAUGU
1299


1565671.1
2894394.1

AfUfguuacaugsgsa
2894395.1

augCfaAfgagcasasu
UACAUGGU






AD-
A-
412
gscsucu(Uhd)GfCfA
A-
856
VPusdCsaudGudAacau
UUGCUCUUGCAUGU
1300


1565865.1
2894693.1

fuguuacausgsa
2894694.1

dGcAfagagcsgsg
UACAUGG






AD-
A-
413
gscsucu(Uhd)GfcAf
A-
857
VPusAfsccdAu(G2p)ua
UUGCUCUUGCAUGU
1301


1565786.1
2894571.1

UfGfuuacauggsusa
2894572.1

acauGfcAfagagcsasa
UACAUGGUU






AD-
A-
414
csuscuug(Chd)aUfG
A-
858
VPusAfsacdCa(Tgn)gu
UGCUCUUGCAUGUUA
1302


1565672.1
2894396.1

fUfuacauggususa
2894397.1

aacaUfgCfaagagscsa
CAUGGUUA






AD-
A-
415
csuscuug(Chd)aUfG
A-
859
VPusdAsacdCadTguaa
UGCUCUUGCAUGUUA
1303


1565673.1
2894396.1

fUfuacauggususa
2894398.1

dCaUfgcaagagscsa
CAUGGUUA






AD-
A-
416
uscsuug(Chd)AfuGf
A-
860
VPusUfsaadCc(Agn)ug
GCUCUUGCAUGUUAC
1304


1565674.1
2894399.1

UfUfacaugguusasa
2894400.1

uaacAfuGfcaagasgsc
AUGGUUAC






AD-
A-
417
csusugc(Ahd)UfgUf
A-
861
VPusGfsuadAc(C2p)au
CUCUUGCAUGUUACA
1305


1565787.1
1577564.1

UfAfcaugguuascsa
2894573.1

guaaCfaUfgcaagsasg
UGGUUACC






AD-
A-
418
csusugc(Ahd)UfgUf
A-
862
VPusGfsuaadCc(Agn)u
CUCUUGCAUGUUACA
1306


1565675.1
1577564.1

UfAfcaugguuascsa
2894401.1

guaaCfaUfgcaagsasg
UGGUUACC






AD-
A-
419
ususgca(Uhd)GfuUf
A-
863
VPusGfsgudAa(C2p)ca
UCUUGCAUGUUACAU
1307


1565788.1
1577566.1

AfCfaugguuacscsa
2894574.1

uguaAfcAfugcaasgsa
GGUUACCA






AD-
A-
420
ususgca(Uhd)GfuUf
A-
864
VPusGfsgdTa(Agn)cca
UCUUGCAUGUUACAU
1308


1565676.1
1577566.1

AfCfaugguuacscsa
2894402.1

uguaAfcAfugcaasgsa
GGUUACCA






AD-
A-
421
ususgca(Uhd)guUfA
A-
865
VPusdGsgudAadCcaug
UCUUGCAUGUUACAU
1309


1565677.1
2894403.1

fCfaugguuacscsa
2894404.1

dTaAfcaugcaasgsa
GGUUACCA






AD-
A-
422
usgscaug(Uhd)uAfC
A-
866
VPusUfsggdTa(Agn)cc
CUUGCAUGUUACAUG
1310


1565678.1
2894405.1

fAfugguuaccsasa
2894406.1

auguAfaCfaugcasasg
GUUACCAC






AD-
A-
423
gscsaug(Uhd)UfaCf
A-
867
VPusGfsugdGu(Agn)ac
UUGCAUGUUACAUG
1311


1565679.1
1577580.1

AfUfgguuaccascsa
2894407.1

caugUfaAfcaugcsasa
GUUACCACA






AD-
A-
424
gscsaug(Uhd)uaCfA
A-
868
VPusdGsugdGudAacca
UUGCAUGUUACAUG
1312


1565680.1
2894408.1

fUfgguuaccascsa
2894409.1

dTgUfaacaugcsasa
GUUACCACA






AD-
A-
425
csasugu(Uhd)AfcAf
A-
869
VPusUfsgudGg(Tgn)aa
UGCAUGUUACAUGG
1313


1565681.1
2894410.1

UfGfguuaccacsasa
2894411.1

ccauGfuAfacaugscsa
UUACCACAA






AD-
A-
426
asusguu(Ahd)CfaUf
A-
870
VPusUfsugdTg(G2p)ua
GCAUGUUACAUGGU
1314


1565789.1
2894575.1

GfGfuuaccacasasa
2894576.1

accaUfgUfaacausgsc
UACCACAAG






AD-
A-
427
usgsuua(Chd)AfuGf
A-
871
VPusCfsuudGu(G2p)g
CAUGUUACAUGGUUA
1315


1565790.1
2894577.1

GfUfuaccacaasgsa
2894578.1

uaaccAfuGfuaacasusg
CCACAAGC






AD-
A-
428
csusccu(Chd)ugGfCf
A-
872
VPusdTsucdGadTgcug
AGCUCCUCUGGCCAG
1316


1565682.1
2894412.1

Cfagcaucgasasa
2894413.1

dGcCfagaggagscsu
CAUCGAAU






AD-
A-
429
asusaua(Ahd)GfuAf
A-
873
VPusUfsaadAu(G2p)ca
GAAUAUAAGUAAGAU
1317


1565791.1
2894579.1

AfGfaugcauuusasa
2894580.1

ucuuAfcUfuauaususc
GCAUUUAC






AD-
A-
430
usasuaag(Uhd)aAfG
A-
874
VPusdGsuadAadTgcau
AAUAUAAGUAAGAUG
1318


1565683.1
2894414.1

fAfugcauuuascsa
2894415.1

dCuUfacuuauasusu
CAUUUACU






AD-
A-
431
asusaag(Uhd)aaGfA
A-
875
VPusdAsgudAadAugca
AUAUAAGUAAGAUGC
1319


1565684.1
2894416.1

fUfgcauuuacsusa
2894417.1

dTcUfuacuuausasu
AUUUACUA






AD-
A-
432
usasagu(Ahd)agAfU
A-
876
VPusdTsagdTadAaugc
UAUAAGUAAGAUGCA
1320


1565685.1
2894418.1

fGfcauuuacusasa
2894419.1

dAuCfuuacuuasusa
UUUACUAC






AD-
A-
433
asasgua(Ahd)GfaUf
A-
877
VPusGfsuagUfaAfAfug
AUAAGUAAGAUGCAU
1321


1565867.1
2894420.1

GfCfauuuacuascsa
1804926.1

caUfcUfuacuusasu
UUACUACA






AD-
A-
434
asasgua(Ahd)GfaUf
A-
878
VPusdGsuadGudAaau
AUAAGUAAGAUGCAU
1322


1565868.1
2894420.1

GfCfauuuacuascsa
2894696.1

gdCaUfcuuacuusgsu
UUACUACA






AD-
A-
435
asasgua(Ahd)GfaUf
A-
879
VPusGfsuadGu(Agn)aa
AUAAGUAAGAUGCAU
1323


1565869.1
2894420.1

GfCfauuuacuascsa
2894697.1

ugcaUfcUfuacuusgsu
UUACUACA






AD-
A-
436
asasgua(Ahd)GfaUf
A-
880
VPusGfsuadGu(A2p)a
AUAAGUAAGAUGCAU
1324


1565870.1
2894420.1

GfCfauuuacuascsa
2894698.1

augcaUfcUfuacuusgsu
UUACUACA






AD-
A-
437
asasgua(Ahd)gaUfG
A-
881
VPusGfsuadGu(Agn)aa
AUAAGUAAGAUGCAU
1325


1565871.1
2894422.1

fCfauuuacuascsa
2894697.1

ugcaUfcUfuacuusgsu
UUACUACA






AD-
A-
438
asasgua(Ahd)gadTg
A-
882
VPusdGsuadGu(Agn)a
AUAAGUAAGAUGCAU
1326


1565872.1
2894699.1

CfdAuuuacuascsa
2894700.1

augdCaUfcUfuacuusgs
UUACUACA






AD-
A-
439
asasgua(Ahd)gaUfG
A-
883
VPusdGsuadGu(Agn)a
AUAAGUAAGAUGCAU
1327


1565873.1
2894422.1

fCfauuuacuascsa
2894700.1

augdCaUfcUfuacuusgs
UUACUACA






AD-
A-
440
asasguu(Ahd)gaUfG
A-
884
VPusGfsuadGu(Agn)aa
AUAAGUAAGAUGCAU
1328


1565874.1
2894701.1

fCfauuuacuascsa
2894702.1

ugcaUfcUfaacuusgsu
UUACUACA






AD-
A-
441
asasgaa(Ahd)gaUfG
A-
885
VPusGfsuadGu(Agn)aa
AUAAGUAAGAUGCAU
1329


1565875.1
2894703.1

fCfauuuacuascsa
2894704.1

ugcaUfcUfuucuusgsu
UUACUACA






AD-
A-
442
asascua(Ahd)gaUfG
A-
886
VPusGfsuadGu(Agn)aa
AUAAGUAAGAUGCAU
1330


1565876.1
2894705.1

fCfauuuacuascsa
2894706.1

ugcaUfcUfuaguusgsu
UUACUACA






AD-
A-
443
asasgua(Ahd)GfaUf
A-
887
VPusGfsuadGu(Agn)aa
AUAAGUAAGAUGCAU
1331


1565686.1
2894420.1

GfCfauuuacuascsa
2894421.1

ugcaUfcUfuacuusasu
UUACUACA






AD-
A-
444
asasgua(Ahd)gaUfG
A-
888
VPusdGsuadGudAaau
AUAAGUAAGAUGCAU
1332


1565687.1
2894422.1

fCfauuuacuascsa
2894423.1

gdCaUfcuuacuusasu
UUACUACA






AD-
A-
445
asgsuaag(Ahd)uGfC
A-
889
VPusUfsgudAg(Tgn)aa
UAAGUAAGAUGCAUU
1333


1565688.1
2894424.1

fAfuuuacuacsasa
2894425.1

augcAfuCfuuacususa
UACUACAG






AD-
A-
446
gsusaag(Ahd)UfGfC
A-
890
VPusGfsuadGu(Agn)aa
AAGUAAGAUGCAUUU
1334


1565877.1
2894707.1

fauuuacuascsa
2894708.1

ugcaUfcUfuacsusu
ACUACA






AD-
A-
447
ususggc(Uhd)UfcUf
A-
891
VPusUfscudGa(Agn)gc
AGUUGGCUUCUAAU
1335


1565689.1
2894426.1

AfAfugcuucagsasa
2894427.1

auuaGfaAfgccaascsu
GCUUCAGAU






AD-
A-
448
csusucu(Ahd)AfuGf
A
892
VPusUfscudAu(C2p)ug
GGCUUCUAAUGCUUC
1336


1565792.1
2894428.1

CfUfucagauagsasa
2894581.1

aagcAfuUfagaagscsc
AGAUAGAA






AD-
A-
449
csusucu(Ahd)AfuGf
A-
893
VPusUfscdTa(Tgn)cug
GGCUUCUAAUGCUUC
1337


1565690.1
2894428.1

CfUfucagauagsasa
2894429.1

aagcAfuUfagaagscsc
AGAUAGAA






AD-
A-
450
csusucu(Ahd)AfuGf
A-
894
VPusUfscuadTc(Tgn)g
GGCUUCUAAUGCUUC
1338


1565691.1
2894428.1

CfUfucagauagsasa
2894430.1

aagcAfuUfagaagscsc
AGAUAGAA
















TABLE 2B







Exemplary Human MYOC siRNA Unmodified Single Strands and Duplex Sequences


Column 1 indicates duplex name; the number following the decimal point in a duplex name merely


refers to a batch production number. Column 2 indicates the sense sequence name. Column 3


indicates the sequence ID for the sequence of column 4. Column 4 provides the unmodified


sequence of a sense strand suitable for use in a duplex described herein. Column 5 provides


the position in the target mRNA (NM_000261.2) of the sense strand of Column 4. Column 6


indicates the antisense sequence name. Column 7 indicates the sequence ID for the sequence of


column 8. Column 8 provides the sequence of an antisense strand suitable for use in a duplex


described herein, without specifying chemical modifications. Column 9 indicates the position


in the target mRNA (NM_000261.2) that is complementary to the antisense strand of Column 8.



















mRNA



mRNA






target

Seq

target




Seq

range

ID

range



Sense
ID

in
Antisense
NO:
antisense
in


Duplex
sequence
NO:
Sense sequence
NM_000
sequence
(anti-
sequence
NM_000


Name
name
(sense)
(5'-3')
261.2
name
sense)
(5'-3')
261.2





AD-
A-
1339
CUCUCAGCACAGCAG
29-49
Å-
1783
UAAGCTCUGCUGUG
27-49


1565444.1
2893965.1

AGCUUA

2893966.1

CUGAGAGGU






AD-
A-
1340
CUCUCAGCACAGCAG
29-49
A-
1784
UAAGCTCTGCUGUG
27-49


1565445.1
2893965.1

AGCUUA

2893967.1

CUGAGAGGU






AD-
A-
1341
CUCUCAGCACAGCAG
29-49
A-
1785
UAAGCUCUGCUGU
27-49


1565692.1
2893965.1

AGCUUA

2894431.1

GCUGAGAGGU






AD-
A-
1342
UCUCAGCACAGCAGA
30-50
A-
1786
UAAAGCTCUGCUGU
28-50


1565446.1
2893968.1

GCUUUA

2893969.1

GCUGAGAGG






AD-
A-
1343
CAGCAGAGCUUUCCA
38-58
Å-
1787
UUCCTCTGGAAAGC
36-58


1565447.1
2893970.1

GAGGAA

2893971.1

UCUGCUGUG






AD-
A-
1344
AAUGAGGUUCUUCU
77-97
A-
1788
UGUGCACAGAAGAA
75-97


1565448.1
2893972.1

GUGCACA

2893973.1

CCUCAUUGC






AD-
A-
1345
AAUGAGGUUCUUCU
77-97
A-
1789
UGUGCACAGAAGAA
75-97


1565693.1
2893972.1

GUGCACA

2894432.1

CCUCAUUGC






AD-
A-
1346
AUGAGGUUCUUCUG
78-98
A-
1790
UCGUGCACAGAAGA
76-98


1565449.1
2893974.1

UGCACGA

2893975.1

ACCUCAUUG






AD-
A-
1347
UGAGGUUCUUCUGU
79-99
A-
1791
UACGUGCACAGAAG
77-99


1565450.1
2893976.1

GCACGUA

2893977.1

AACCUCAUU






AD-
A-
1348
UGAGGUUCUUCUGU
79-99
A-
1792
UACGTGCACAGAAG
77-99


1565694.1
2893976.1

GCACGUA

2894433.1

AACCUCAUU






AD-
A-
1349
GAGGUUCUUCUGUG
 80-100
A-
1793
UAACGUGCACAGAA
 78-100


1565695.1
2894434.1

CACGUUA

2894435.1

GAACCUCAU






AD-
A-
1350
AGGUUCUUCUGUGC
 81-101
A-
1794
UCAACGUGCACAGA
 79-101


1193175.5
2058874.1

ACGUUGA

1801665.1

AGAACCUCA






AD-
A-
1351
AGGUUCUUCUGUGC
 81-101
A-
1795
UCAACGTGCACAGA
 79-101


1565793.1
2893978.1

ACGUUGA

2894582.1

AGAACCUCG






AD-
A-
1352
AGGUUCUUCUGUGC
 81-101
A-
1796
UCAACGTGCACAGA
 79-101


1565795.1
2058874.1

ACGUUGA

2894585.1

AGAACCUCG






AD-
A-
1353
AGGUUCUUCUGUGC
 81-101
A-
1797
UCAACGTGCACAGA
 79-101


1565796.1
2058874.1

ACGUUGA

2894586.1

AGAACCUCG






AD-
A-
1354
AGGUACUUCUGUGC
 81-101
A-
1798
UCAACGTGCACAGA
 79-101


1565797.1
2894587.1

ACGUUGA

2894588.1

AGUACCUCG






AD-
A-
1355
AGGAUCUUCUGUGC
 81-101
A-
1799
UCAACGTGCACAGA
 79-101


1565798.1
2894589.1

ACGUUGA

2894590.1

AGAUCCUCG






AD-
A
1356
AGCUUCUUCUGUGC
 81-101
A-
1800
UCAACGTGCACAGA
 79-101


1565799.1
2894591.1

ACGUUGA

2894592.1

AGAAGCUCG






AD-
A-
1357
AGGUUCUUCUGUGC
 81-101
A-
1801
UCAACGTGCACAGA
 79-101


1565451.1
2893978.1

ACGUUGA

2893979.1

AGAACCUCA






AD-
A-
1358
GGUUCUUCUGUGCA
 82-102
A-
1802
UGCAACGUGCACAG
 80-102


1565452.1
2893980.1

CGUUGCA

2893981.1

AAGAACCUC






AD-
A-
1359
GGUUCUUCUGUGCA
 82-102
A-
1803
UGCAACGUGCACAG
 80-102


1565696.1
2894436.1

CGUUGCA

2894437.1

AAGAACCUC






AD-
A-
1360
GUUCUUCUGUGCAC
 83-101
A-
1804
UCAACGTGCACAGA
 81-101


1565794.1
2894583.1

GUUGA

2894584.1

AGAACCU






AD-
A-
1361
GUUCUUCUGUGCAC
 83-103
A-
1805
UAGCAACGUGCACA
 81-103


1565453.1
2893982.1

GUUGCUA

2893983.1

GAAGAACCU






AD-
A-
1362
UUCUUCUGUGCACG
 84-104
A-
1806
UCAGCAACGUGCAC
 82-104


1565454.1
2893984.1

UUGCUGA

2893985.1

AGAAGAACC






AD-
A-
1363
UCUUCUGUGCACGU
 85-105
A-
1807
UGCAGCAACGUGCA
 83-105


1565455.1
2893986.1

UGCUGCA

2893987.1

CAGAAGAAC






AD-
A-
1364
UCUUCUGUGCACGU
 85-105
A-
1808
UGCAGCAACGUGCA
 83-105


1565456.1
2893988.1

UGCUGCA

2893989.1

CAGAAGAAC






AD-
A-
1365
CUUCUGUGCACGUU
 86-106
A-
1809
UUGCAGCAACGUGC
 84-106


1565457.1
2893990.1

GCUGCAA

2893991.1

ACAGAAGAA






AD-
A-
1366
CUUCUGUGCACGUU
 86-106
A-
1810
UUGCAGCAACGUGC
 84-106


1565697.1
2893990.1

GCUGCAA

2894438.1

ACAGAAGAA






AD-
A-
1367
UUCUGUGCACGUUG
 87-107
A-
1811
UCUGCAGCAACGUG
 85-107


1565698.1
2894439.1

CUGCAGA

2894440.1

CACAGAAGA






AD-
A-
1368
UCUGUGCACGUUGC
 88-108
A-
1812
UGCUGCAGCAACGU
 86-108


1565458.1
2893992.1

UGCAGCA

2893993.1

GCACAGAAG






AD-
A-
1369
CUGUGCACGUUGCU
 89-109
A-
1813
UAGCUGCAGCAACG
 87-109


1565459.1
2893994.1

GCAGCUA

2893995.1

UGCACAGAA






AD-
A-
1370
CUGUGCACGUUGCU
 89-109
A-
1814
UAGCTGCAGCAACG
 87-109


1565699.1
2893994.1

GCAGCUA

2894441.1

UGCACAGAA






AD-
A-
1371
UGUGCACGUUGCUG
 90-110
A-
1815
UAAGCUGCAGCAAC
 88-110


1565700.1
2894442.1

CAGCUUA

2894443.1

GUGCACAGA






AD-
A-
1372
GUGCACGUUGCUGC
 91-111
A-
1816
UAAAGCTGCAGCAA
 89-111


1565460.1
2893996.1

AGCUUUA

2893997.1

CGUGCACAG






AD-
A-
1373
GCACGUUGCUGCAG
 93-113
A-
1817
UCCAAAGCUGCAGC
 91-113


1565461.1
2893998.1

CUUUGGA

2893999.1

AACGUGCAC






AD-
A-
1374
CACGUUGCUGCAGC
 94-114
A-
1818
UCCCAAAGCUGCAG
 92-114


1565462.1
2894000.1

UUUGGGA

2894001.1

CAACGUGCA






AD-
A-
1375
GAGAUGCCAGCUGU
117-137
A-
1819
UAGCTGGACAGCUG
115-137


1565701.1
2894444.1

CCAGCUA

2894445.1

GCAUCUCAG






AD-
A-
1376
GAUGCCAGCUGUCCA
119-139
A-
1820
UGCAGCTGGACAGC
117-139


1565463.1
2894002.1

GCUGCA

2894003.1

UGGCAUCUC






AD-
A-
1377
CUGUCCAGCUGCUGC
127-147
A-
1821
UCAGAAGCAGCAGC
125-147


1565464.1
2894004.1

UUCUGA

2894005.1

UGGACAGCU






AD-
A-
1378
CCAGCUGCUGCUUC
131-151
A-
1822
UAGGCCAGAAGCAG
129-151


1565465.1
2894006.1

UGGCCUA

2894007.1

CAGCUGGAC






AD-
A-
1379
CUGGCCUGCCUGGU
144-164
A-
1823
UUCCCACACCAGGC
142-164


1565466.1
2894008.1

GUGGGAA

2894009.1

AGGCCAGAA






AD-
A-
1380
CUGGCCUGCCUGGU
144-164
A-
1824
UUCCCACACCAGGC
142-164


1565467.1
2894008.1

GUGGGAA

2894010.1

AGGCCAGAA






AD-
A-
1381
CUGGCCUGCCUGGU
144-164
A-
1825
UUCCCACACCAGGC
142-164


1565702.1
2894008.1

GUGGGAA

2894446.1

AGGCCAGAA






AD-
A-
1382
UGGCCUGCCUGGUG
145-165
A-
1826
UAUCCCACACCAGG
143-165


1565468.1
2894011.1

UGGGAUA

2894012.1

CAGGCCAGA






AD-
A-
1383
GCCUGCCUGGUGUG
147-167
A-
1827
UACATCACACACCA
145-167


1565469.1
2894013.1

UGAUGUA

2894014.1

GGCAGGCCA






AD-
A-
1384
GCCUGCCUGGUGUG
147-167
A-
1828
UACATCCCACACCAG
145-167


1565703.1
2894447.1

GGAUGUA

2894448.1

GCAGGCCA






AD-
A-
1385
AGAGUGGCCGAUGC
205-225
A-
1829
UAUACUGGCAUCGG
203-225


1565704.1
2894449.1

CAGUAUA

2894450.1

CCACUCUGG






AD-
A-
1386
AGUGUGGCCAGUCC
231-251
A-
1830
UUCATUGGGACUG
229-251


1565705.1
2894451.1

CAAUGAA

2894452.1

GCCACACUGA






AD-
A-
1387
GUGUGGCCAGUCCC
232-252
A-
1831
UUUCAUTGGGACU
230-252


1565470.1
2894015.1

AAUGAAA

2894016.1

GGCCACACUG






AD-
A-
1388
GUGUGGCCAGUCCC
232-252
A-
1832
UTUCAUTGGGACUG
230-252


1565471.1
2894015.1

AAUGAAA

2894017.1

GCCACACUG






AD-
A-
1389
GCCAGGCCAUGUCAG
271-291
A-
1833
UGATGACUGACAUG
269-291


1565472.1
2894018.1

UCAUCA

2894019.1

GCCUGGCUC






AD-
A-
1390
GCCAGGCCAUGUCAG
271-291
A-
1834
UGAUGACTGACAUG
269-291


1565473.1
2894018.1

UCAUCA

2894020.1

GCCUGGCUC






AD-
A-
1391
GCCAGGCCAUGUCAG
271-291
A-
1835
UGAUGACUGACAU
269-291


1565706.1
2894018.1

UCAUCA

2894453.1

GGCCUGGCUC






AD-
A-
1392
GGCCAUGUCAGUCA
275-295
A-
1836
UUAUGGAUGACUG
273-295


1565474.1
2894021.1

UCCAUAA

2894022.1

ACAUGGCCUG






AD-
A-
1393
GACCUGGAGGCCACC
327-347
A-
1837
UGCUTUGGUGGCC
325-347


1565707.1
2894454.1

AAAGCA

2894455.1

UCCAGGUCUA






AD-
A-
1394
CUGGAGGCCACCAAA
330-350
A-
1838
UCGAGCTUUGGUG
328-350


1565475.1
2894023.1

GCUCGA

2894024.1

GCCUCCAGGU






AD-
A-
1395
GAAACCCAAACCAGA
471-491
A-
1839
UAACTCTCUGGUUU
469-491


1565476.1
2894025.1

GAGUUA

2894026.1

GGGUUUCCA






AD-
A-
1396
CAGCAACCUCCUCCG
503-523
Å-
1840
UUGTCTCGGAGGAG
501-523


1565477.1
2894027.1

AGACAA

2894028.1

GUUGCUGUA






AD-
A-
1397
CAGCAACCUCCUCCG
503-523
A-
1841
UUGUCUCGGAGGA
501-523


1565708.1
2894027.1

AGACAA

2894456.1

GGUUGCUGUA






AD-
A-
1398
GCAACCUCCUCCGAG
505-525
A-
1842
UCUTGTCUCGGAGG
503-525


1565478.1
2894029.1

ACAAGA

2894030.1

AGGUUGCUG






AD-
A-
1399
GCAACCUCCUCCGAG
505-525
A-
1843
UCUUGTCTCGGAGG
503-525


1565479.1
2894029.1

ACAAGA

2894031.1

AGGUUGCUG






AD-
A-
1400
GCAACCUCCUCCGAG
505-525
A-
1844
UCUUGUCUCGGAG
503-525


1565709.1
2894029.1

ACAAGA

2894457.1

GAGGUUGCUG






AD-
A-
1401
AGACAAGUCAGUUC
518-538
A-
1845
UCCUCCAGAACTGA
516-538


1565480.1
2894032.1

UGGAGGA

2894033.1

CUUGUCUCG






AD-
A-
1402
CAAGUCAGUUCUGG
521-541
A-
1846
UCUUCCTCCAGAAC
519-541


1565481.1
2894034.1

AGGAAGA

2894035.1

UGACUUGUC






AD-
A-
1403
GUCAGUUCUGGAGG
524-544
A-
1847
UUCUCUTCCUCCAG
522-544


1565482.1
2894036.1

AAGAGAA

2894037.1

AACUGACUU






AD-
A-
1404
AGAAUCUGGCCAGG
568-588
A-
1848
UCAACCTCCUGGCC
566-588


1565483.1
2894038.1

AGGUUGA

2894039.1

AGAUUCUCA






AD-
A-
1405
UGGCCAGGAGGUUG
574-594
A-
1849
UGCTUTCCAACCUCC
572-594


1565484.1
2894040.1

GAAAGCA

2894041.1

UGGCCAGA






AD-
A-
1406
UGGCCAGGAGGUUG
574-594
A-
1850
UGCUTUCCAACCUC
572-594


1565710.1
2894040.1

GAAAGCA

2894458.1

CUGGCCAGA






AD-
A-
1407
CAGCCAGGAGGUAG
596-616
A-
1851
UGCCTUGCUACCUC
594-616


1565711.1
2894459.1

CAAGGCA

2894460.1

CUGGCUGCU






AD-
A-
1408
UGCCACCAGGCUCCA
661-681
A-
1852
UUUCTCTGGAGCCU
659-681


1565485.1
2894042.1

GAGAAA

2894043.1

GGUGGCACA






AD-
A-
1409
AAUUUGGACACUUU
693-713
A-
1853
UAAGGCCAAAGUGU
691-713


1565486.1
2894044.1

GGCCUUA

2894045.1

CCAAAUUCC






AD-
A-
1410
AAUUUGGACACUUU
693-713
A-
1854
UAAGGCCAAAGUGU
691-713


1565712.1
2894044.1

GGCCUUA

2894461.1

CCAAAUUCC






AD-
A-
1411
ACACUUUGGCCUUCC
700-720
A-
1855
UUUCCUGGAAGGCC
698-720


1565713.1
2894462.1

AGGAAA

2894463.1

AAAGUGUCC






AD-
A-
1412
CUUUGGCCUUCCAG
703-723
A-
1856
UCAGTUCCUGGAAG
701-723


1565487.1
2894046.1

GAACUGA

2894047.1

GCCAAAGUG






AD-
A-
1413
AGGAACUGAAGUCC
715-735
A-
1857
UUAGCUCGGACUUC
713-735


1565714.1
2894048.1

GAGCUAA

2894464.1

AGUUCCUGG






AD-
A-
1414
AGGAACUGAAGUCC
715-735
A-
1858
UUAGCTCGGACUUC
713-735


1565488.1
2894048.1

GAGCUAA

2894049.1

AGUUCCUGG






AD-
A-
1415
GAAGUCCGAGCUAAC
722-742
A-
1859
UCUUCAGUUAGCUC
720-742


1565715.1
2894465.1

UGAAGA

2894466.1

GGACUUCAG






AD-
A-
1416
GCUAACUGAAGUUC
731-751
A-
1860
UAAGCAGGAACUUC
729-751


1565716.1
2894467.1

CUGCUUA

2894468.1

AGUUAGCUC






AD-
A-
1417
CUAACUGAAGUUCC
732-752
A-
1861
UGAAGCAGGAACUU
730-752


1565489.1
2894050.1

UGCUUCA

2894051.1

CAGUUAGCU






AD-
A-
1418
GAAGUUCCUGCUUC
738-758
A-
1862
UAUUCGGGAAGCA
736-758


1565717.1
2894469.1

CCGAAUA

2894470.1

GGAACUUCAG






AD-
A-
1419
GAGAACUAGUUUGG
814-834
A-
1863
UUCCTACCCAAACU
812-834


1565718.1
2894052.1

GUAGGAA

2894471.1

AGUUCUCCA






AD-
A-
1420
GAGAACUAGUUUGG
814-834
A-
1864
UUCCUACCCAAACU
812-834


1565490.1
2894052.1

GUAGGAA

2894053.1

AGUUCUCCA






AD-
A-
1421
ACUAGUUUGGGUAG
818-838
A-
1865
UGCUCUCCUACCCA
816-838


1565719.1
2894054.1

GAGAGCA

2894472.1

AACUAGUUC






AD-
A-
1422
ACUAGUUUGGGUAG
818-838
A-
1866
UGCTCTCUACCCAAA
816-837


1565491.1
2894054.1

GAGAGCA

2894055.1

CUAGUUC






AD-
A-
1423
GGGUAGGAGAGCCU
826-846
A-
1867
UCGUGAGAGGCUC
824-846


1565720.1
2894473.1

CUCACGA

2894474.1

UCCUACCCAA






AD-
A-
1424
GUAGGAGAGCCUCU
828-848
A-
1868
UAGCGUGAGAGGC
826-848


1565721.1
2894475.1

CACGCUA

2894476.1

UCUCCUACCC






AD-
A-
1425
GUAGGAGAGCCUCU
828-848
A-
1869
UAGCGUGAGAGGC
826-848


1565492.1
2894056.1

CACGCUA

2894057.1

UCUCCUACCC






AD-
A-
1426
UAGGAGAGCCUCUC
829-849
A-
1870
UCAGCGTGAGAGGC
827-849


1565493.1
2894058.1

ACGCUGA

2894059.1

UCUCCUACC






AD-
A-
1427
AGGAGAGCCUCUCAC
830-850
A-
1871
UUCAGCGUGAGAG
828-850


1565722.1
2894477.1

GCUGAA

2894478.1

GCUCUCCUAC






AD-
A-
1428
GCCUCUCACGCUGAG
836-856
A-
1872
UCUGTUCUCAGCGU
834-856


1565723.1
2894060.1

AACAGA

2894479.1

GAGAGGCUC






AD-
A-
1429
GCCUCUCACGCUGAG
836-856
A-
1873
UCUGUTCUCAGCGU
834-856


1565494.1
2894060.1

AACAGA

2894061.1

GAGAGGCUC






AD-
A-
1430
GCCUCUCACGCUGAG
836-856
A-
1874
UCUGUTCTCAGCGU
834-856


1565495.1
2894060.1

AACAGA

2894062.1

GAGAGGCUC






AD-
A-
1431
CACGCUGAGAACAGC
842-862
A-
1875
UUUUCUGCUGUUC
840-862


1565724.1
2894480.1

AGAAAA

2894481.1

UCAGCGUGAG






AD-
A-
1432
ACGCUGAGAACAGCA
843-863
A-
1876
UGUUTCTGCUGUUC
841-863


1565496.1
2894063.1

GAAACA

2894064.1

UCAGCGUGA






AD-
A-
1433
CGCUGAGAACAGCAG
844-864
A-
1877
UUGUTUCUGCUGU
842-864


1565725.1
2894065.1

AAACAA

2894482.1

UCUCAGCGUG






AD-
A-
1434
CGCUGAGAACAGCAG
844-864
A-
1878
UUGTUTCUGCUGUU
842-864


1565497.1
2894065.1

AAACAA

2894066.1

CUCAGCGUG






AD-
A-
1435
GCUGAGAACAGCAGA
845-865
Å-
1879
UUUGTUTCUGCUGU
843-865


1565498.1
2894067.1

AACAAA

2894068.1

UCUCAGCGU






AD-
A-
1436
CUGAGAACAGCAGAA
846-866
A-
1880
UAUUGUTUCUGCU
844-866


1565499.1
2894069.1

ACAAUA

2894070.1

GUUCUCAGCG






AD-
A-
1437
UGAGAACAGCAGAAA
847-867
A-
1881
UAAUTGTUUCUGCU
845-867


1565500.1
2894071.1

CAAUUA

2894072.1

GUUCUCAGC






AD-
A-
1438
GAGAACAGCAGAAAC
848-868
A-
1882
UUAATUGUUUCUG
846-868


1565726.1
2894483.1

AAUUAA

2894484.1

CUGUUCUCAG






AD-
A-
1439
AGAACAGCAGAAACA
849-869
A-
1883
UGUAAUTGUUUCU
847-869


1565501.1
2894073.1

AUUACA

2894074.1

GCUGUUCUCA






AD-
A-
1440
AGAACAGCAGAAACA
849-869
A-
1884
UGUAAUTGUUUCU
847-869


1565502.1
2894075.1

AUUACA

2894076.1

GCUGUUCUCA






AD-
A-
1441
GAACAGCAGAAACAA
850-870
A-
1885
UAGUAATUGUUTCU
848-870


1565503.1
2894077.1

UUACUA

2894078.1

GCUGUUCUC






AD-
A-
1442
AACAGCAGAAACAAU
851-871
A-
1886
UCAGTAAUUGUUUC
849-871


1565801.1
1991726.1

UACUGA

2894595.1

UGCUGUUCU






AD-
A-
1443
AACAGCAGAAACAAU
851-871
A-
1887
UCAGTAAUUGUUUC
849-871


1565802.1
1991726.1

UACUGA

2894596.1

UGCUGUUCU






AD-
A-
1444
AACACCAGAAACAAU
851-871
A-
1888
UCAGTAAUUGUTUC
849-871


1565803.1
2894597.1

UACUGA

2894598.1

UGGUGUUCU






AD-
A-
1445
AACUGCAGAAACAAU
851-871
A-
1889
UCAGTAAUUGUTUC
849-871


1565804.1
2894599.1

UACUGA

2894600.1

UGCAGUUCU






AD-
A-
1446
AAGAGCAGAAACAAU
851-871
A-
1890
UCAGTAAUUGUTUC
849-871


1565805.1
2894601.1

UACUGA

2894602.1

UGCUCUUCU






AD-
A-
1447
AACAGCAGAAACAAU
851-871
A-
1891
UCAGUAAUUGUUU
849-871


1073418.5
1991726.1

UACUGA

1802980.1

CUGCUGUUCU






AD-
A-
1448
AACAGCAGAAACAAU
851-871
A-
1892
UCAGTAAUUGUTUC
849-871


1565504.1
2894079.1

UACUGA

2894080.1

UGCUGUUCU






AD-
A-
1449
AACAGCAGAAACAAU
851-871
A-
1893
UCAGTAAUUGUTUC
849-871


1565504.2
2894079.1

UACUGA

2894080.1

UGCUGUUCU






AD-
A-
1450
ACAGCAGAAACAAUU
852-872
A-
1894
UCCAGUAAUUGTUU
850-872


1565505.1
2894081.1

ACUGGA

2894082.1

CUGCUGUUC






AD-
A-
1451
CAGCAGAAACAAUUA
853-871
A-
1895
UCAGTAAUUGUTUC
851-871


1565800.1
2894593.1

CUGA

2894594.1

UGCUGUU






AD-
A-
1452
CAGCAGAAACAAUUA
853-873
A-
1896
UGCCAGTAAUUGUU
851-873


1565506.1
2894083.1

CUGGCA

2894084.1

UCUGCUGUU






AD-
A-
1453
AGCAGAAACAAUUAC
854-874
A-
1897
UUGCCAGUAAUUG
852-874


1565727.1
2894485.1

UGGCAA

2894486.1

UUUCUGCUGU






AD-
A-
1454
GCAGAAACAAUUACU
855-875
A-
1898
UUUGCCAGUAAUU
853-875


1565507.1
2894085.1

GGCAAA

2894086.1

GUUUCUGCUG






AD-
A-
1455
CAGAAACAAUUACUG
856-876
A-
1899
UCUUGCCAGUAAU
854-876


1565728.1
1577510.1

GCAAGA

2894487.1

UGUUUCUGCU






AD-
A-
1456
CAGAAACAAUUACUG
856-876
A-
1900
UCUUGCCAGUAAU
854-876


1565508.1
1577510.1

GCAAGA

2894087.1

UGUUUCUGCU






AD-
A-
1457
AGAAACAAUUACUG
857-877
A-
1901
UACUTGACAGUAAU
855-877


1565509.1
2894088.1

UCAAGUA

2894089.1

UGUUUCUGC






AD-
A-
1458
GAAACAAUUACUGG
858-878
A-
1902
UUACTUGCCAGUAA
856-878


1565730.1
2894490.1

CAAGUAA

2894491.1

UUGUUUCUG






AD-
A-
1459
AAACAAUUACUGGCA
859-879
A-
1903
UAUACUTGCCAGUA
857-879


1565510.1
2894090.1

AGUAUA

2894091.1

AUUGUUUCU






AD-
A-
1460
AACAAUUACUGGCAA
860-880
A-
1904
UCAUACTUGCCAGU
858-880


1565511.1
2894092.1

GUAUGA

2894093.1

AAUUGUUUC






AD-
A-
1461
AACAAUUACUGGCAA
860-880
A-
1905
UCAUACTUGCCAGU
858-880


1565512.1
2894094.1

GUAUGA

2894095.1

AAUUGUUUC






AD-
A-
1462
GGCAAGUAUGGUGU
870-890
A-
1906
UAUCCACACACCAU
868-890


1565731.1
2894096.1

GUGGAUA

2894492.1

ACUUGCCAG






AD-
A-
1463
GGCAAGUAUGGUGU
870-890
A-
1907
UAUCCACACACCAU
868-890


1565513.1
2894096.1

GUGGAUA

2894097.1

ACUUGCCAG






AD-
A-
1464
GGCAAGUAUGGUGU
870-890
A-
1908
UAUCCACACACCAU
868-890


1565514.1
2894096.1

GUGGAUA

2894098.1

ACUUGCCAG






AD-
A-
1465
CAAGUAUGGUGUGU
872-892
A-
1909
UGCATCCACACACCA
870-892


1565732.1
2894099.1

GGAUGCA

2894493.1

UACUUGCC






AD-
A-
1466
CAAGUAUGGUGUGU
872-892
A-
1910
UGCAUCCACACACC
870-892


1565515.1
2894099.1

GGAUGCA

2894100.1

AUACUUGCC






AD-
A-
1467
UGAGUAUGACCUCA
974-994
A-
1911
UGGCTGAUGAGGUC
972-994


1565516.1
2894101.1

UCAGCCA

2894102.1

AUACUCAAA






AD-
A-
1468
GAGUAUGACCUCAU
975-995
A-
1912
UUGGCUGAUGAGG
973-995


1565733.1
2894494.1

CAGCCAA

2894495.1

UCAUACUCAA






AD-
A-
1469
AGUAUGACCUCAUCA
976-996
A-
1913
UCUGGCTGAUGAGG
974-996


1565517.1
2894103.1

GCCAGA

2894104.1

UCAUACUCA






AD-
A-
1470
GUAUGACCUCAUCA
977-997
A-
1914
UACUGGCUGAUGA
975-997


1565734.1
2894105.1

GCCAGUA

2894496.1

GGUCAUACUC






AD-
A-
1471
GUAUGACCUCAUCA
977-997
A-
1915
UACUGGCTGAUGAG
975-997


1565518.1
2894105.1

GCCAGUA

2894106.1

GUCAUACUC






AD-
A-
1472
UAUGACCUCAUCAGC
978-998
A-
1916
UAACTGGCUGAUGA
976-998


1565735.1
2894497.1

CAGUUA

2894498.1

GGUCAUACU






AD-
A-
1473
AUGACCUCAUCAGCC
979-999
A-
1917
UAAACUGGCUGAU
977-999


1565736.1
2894499.1

AGUUUA

2894500.1

GAGGUCAUAC






AD-
A-
1474
UGACCUCAUCAGCCA
 980-1000
A-
1918
UUAAACTGGCUGAU
 978-1000


1565519.1
2894107.1

GUUUAA

2894108.1

GAGGUCAUA






AD-
A-
1475
GACCUCAUCAGCCAG
 981-1001
A-
1919
UAUAAACUGGCTGA
 979-1001


1565520.1
2894109.1

UUUAUA

2894110.1

UGAGGUCAU






AD-
A-
1476
ACCUCAUCAGCCAGU
 982-1002
A-
1920
UCAUAAACUGGCUG
 980-1002


1565521.1
2894111.1

UUAUGA

2894112.1

AUGAGGUCA






AD-
A-
1477
CCUCAUCAGCCAGUU
 983-1003
A-
1921
UGCAUAAACUGGCU
 981-1003


1244360.3
2298063.1

UAUGCA

1803173.1

GAUGAGGUC






AD-
A-
1478
CCUCAUCAGCCAGUU
 983-1003
A-
1922
UGCATAAACUGGCU
 981-1003


1565522.1
2894113.1

UAUGCA

2894114.1

GAUGAGGUC






AD-
A-
1479
CCUCAUCAGCCAGUU
 983-1003
A-
1923
UGCATAAACUGGCU
 981-1003


1565806.1
2298063.1

UAUGCA

2894603.1

GAUGAGGUC






AD-
A-
1480
CCUCAUCAGCCAGUU
 983-1003
A-
1924
UGCATAAACUGGCU
 981-1003


1565807.1
2298063.1

UAUGCA

2894604.1

GAUGAGGUC






AD-
A-
1481
CCUCAUCAGCCAGUU
 983-1003
A-
1925
UGCATAAACUGGCU
 981-1003


1565808.1
2894113.1

UAUGCA

2894605.1

GAUGAGGUC






AD-
A-
1482
CCUCAUCAGCCAGUU
 983-1003
A-
1926
UGCATAAACUGGCU
 981-1003


1565809.1
2894606.1

UAUGCA

2894603.1

GAUGAGGUC






AD-
A-
1483
CCUCAUCAGCCAGUU
 983-1003
A-
1927
UGCATAAACUGGCU
 981-1003


1565810.1
2894607.1

UAUGCA

2894608.1

GAUGAGGUC






AD-
A-
1484
CCUCUUCAGCCAGUU
 983-1003
A-
1928
UGCATAAACUGGCU
 981-1003


1565812.1
2894611.1

UAUGCA

2894612.1

GAAGAGGUC






AD-
A-
1485
CCUGAUCAGCCAGUU
 983-1003
A-
1929
UGCATAAACUGGCU
 981-1003


1565813.1
2894613.1

UAUGCA

2894614.1

GAUCAGGUC






AD-
A-
1486
CCACAUCAGCCAGUU
 983-1003
A-
1930
UGCATAAACUGGCU
 981-1003


1565814.1
2894615.1

UAUGCA

2894616.1

GAUGUGGUC






AD-
A-
1487
CCUCAUCAGCCAGUU
 983-1003
A-
1931
UGCATAAACUGGCU
 981-1003


1565522.2
2894113.1

UAUGCA

2894114.1

GAUGAGGUC






AD-
A-
1488
CUCAUCAGCCAGUUU
 984-1004
A-
1932
UTGCAUAAACUGGC
 982-1004


1565523.1
2894115.1

AUGCAA

2894116.1

UGAUGAGGU






AD-
A-
1489
UCAUCAGCCAGUUU
 985-1003
A-
1933
UGCATAAACUGGCU
 983-1003


1565811.1
2894609.1

AUGCA

2894610.1

GAUGAGG






AD-
A-
1490
UCAUCAGCCAGUUU
 985-1005
A-
1934
UCUGCATAAACUGG
 983-1005


1565524.1
2894117.1

AUGCAGA

2894118.1

CUGAUGAGG






AD-
A-
1491
UCAUCAGCCAGUUU
 985-1005
A-
1935
UCUGCATAAACTGG
 983-1005


1565525.1
2894119.1

AUGCAGA

2894120.1

CUGAUGAGG






AD-
A-
1492
CAUCAGCCAGUUUA
 986-1006
A-
1936
UCCUGCAUAAACUG
 984-1006


1565526.1
2894121.1

UGCAGGA

2894122.1

GCUGAUGAG






AD-
A-
1493
AUCAGCCAGUUUAU
 987-1007
A-
1937
UCCCTGCAUAAACU
 985-1007


1565737.1
2894123.1

GCAGGGA

2894501.1

GGCUGAUGA






AD-
A-
1494
AUCAGCCAGUUUAU
 987-1007
A-
1938
UCCCUGCAUAAACU
 985-1007


1565527.1
2894123.1

GCAGGGA

2894124.1

GGCUGAUGA






AD-
A-
1495
UCAGCCAGUUUAUG
 988-1008
A-
1939
UGCCCUGCAUAAAC
 986-1008


1565738.1
2894502.1

CAGGGCA

2894503.1

UGGCUGAUG






AD-
A-
1496
CAGCCAGUUUAUGC
 989-1009
A-
1940
UAGCCCTGCAUAAA
 987-1009


1565528.1
2894125.1

AGGGCUA

2894126.1

CUGGCUGAU






AD-
A-
1497
AGCCAGUUUAUGCA
 990-1010
A-
1941
UUAGCCCUGCAUAA
 988-1010


1565739.1
2894127.1

GGGCUAA

2894504.1

ACUGGCUGA






AD-
A-
1498
AGCCAGUUUAUGCA
 990-1010
A-
1942
UUAGCCCTGCAUAA
 988-1010


1565529.1
2894127.1

GGGCUAA

2894128.1

ACUGGCUGA






AD-
A-
1499
GCCAGUUUAUGCAG
 991-1011
A-
1943
UGUAGCCCUGCAUA
 989-1011


1565740.1
2894505.1

GGCUACA

2894506.1

AACUGGCUG






AD-
A-
1500
GCCAGUUUAUGCAG
 991-1011
A-
1944
UGUAGCACUGCAUA
 989-1011


1565530.1
2894129.1

UGCUACA

2894130.1

AACUGGCUG






AD-
A-
1501
CCAGUUUAUGCAGG
 992-1012
A-
1945
UGGUAGCCCUGCAU
 990-1012


1565741.1
2894507.1

GCUACCA

2894508.1

AAACUGGCU






AD-
A-
1502
CCAGUUUAUGCAGG
 992-1012
A-
1946
UGGUAGACCUGCAU
 990-1012


1565531.1
2894131.1

UCUACCA

2894132.1

AAACUGGCU






AD-
A-
1503
UGCAGGGCUACCCU
1000-1020
A-
1947
UCUUAGAAGGGTAG
 998-1020


1565532.1
2894133.1

UCUAAGA

2894134.1

CCCUGCAUA






AD-
A-
1504
GGGUGCUGUGGUGU
1052-1072
A-
1948
UCCGAGTACACCAC
1050-1072


1565533.1
2894135.1

ACUCGGA

2894136.1

AGCACCCGU






AD-
A-
1505
GGGUGCUGUGGUGU
1052-1072
A-
1949
UCCGAGTACACCAC
1050-1072


1565534.1
2894137.1

ACUCGGA

2894138.1

AGCACCCGU






AD-
A-
1506
GAGCCUCUAUUUCCA
1073-1093
A-
1950
UCGCCCTGGAAAUA
1071-1093


1565535.1
2894139.1

GGGCGA

2894140.1

GAGGCUCCC






AD-
A-
1507
GAGCCUCUAUUUCCA
1073-1093
A-
1951
UCGCCCTGGAAAUA
1071-1093


1565536.1
2894141.1

GGGCGA

2894142.1

GAGGCUCCC






AD-
A-
1508
CCUCUAUUUCCAGG
1076-1096
A-
1952
UCAGCGCCCUGGAA
1074-1096


1565537.1
2894143.1

GCGCUGA

2894144.1

AUAGAGGCU






AD-
A-
1509
UUCCAGGGCGCUGA
1083-1103
A-
1953
UCUGGACUCAGCGC
1081-1103


1565742.1
2894145.1

GUCCAGA

2894509.1

CCUGGAAAU






AD-
A-
1510
UUCCAGGGCGCUGA
1083-1103
A-
1954
UCUGGACUCAGCGC
1081-1103


1565538.1
2894145.1

GUCCAGA

2894146.1

CCUGGAAAU






AD-
A-
1511
UUCCAGGGCGCUGA
1083-1103
A-
1955
UCUGGACTCAGCGC
1081-1103


1565539.1
2894145.1

GUCCAGA

2894147.1

CCUGGAAAU






AD-
A-
1512
AGGGCGCUGAGUCC
1087-1107
A-
1956
UAGUTCTGGACUCA
1085-1107


1565540.1
2894148.1

AGAACUA

2894149.1

GCGCCCUGG






AD-
A-
1513
GGGCGCUGAGUCCA
1088-1108
A-
1957
UCAGTUCUGGACUC
1086-1108


1565743.1
2894150.1

GAACUGA

2894510.1

AGCGCCCUG






AD-
A-
1514
GGGCGCUGAGUCCA
1088-1108
A-
1958
UCAGUTCUGGACUC
1086-1108


1565541.1
2894150.1

GAACUGA

2894151.1

AGCGCCCUG






AD-
A-
1515
GGGCGCUGAGUCCA
1088-1108
A-
1959
UCAGTUCUGGACUC
1086-1108


1565542.1
2894152.1

GAACUGA

2894153.1

AGCGCCCUG






AD-
A-
1516
GGCGCUGAGUCCAG
1089-1109
A-
1960
UACAGUTCUGGACU
1087-1109


1565543.1
2894154.1

AACUGUA

2894155.1

CAGCGCCCU






AD-
A-
1517
GCGCUGAGUCCAGA
1090-1110
A-
1961
UGACAGTUCUGGAC
1088-1110


1565544.1
2894156.1

ACUGUCA

2894157.1

UCAGCGCCC






AD-
A-
1518
CGCUGAGUCCAGAAC
1091-1111
A-
1962
UUGACAGUUCUGG
1089-1111


1565744.1
2894511.1

UGUCAA

2894512.1

ACUCAGCGCC






AD-
A-
1519
GCUGAGUCCAGAAC
1092-1112
A-
1963
UAUGACAGUUCUG
1090-1112


1565545.1
2894158.1

UGUCAUA

2894159.1

GACUCAGCGC






AD-
A-
1520
CUGAGUCCAGAACU
1093-1113
A-
1964
UUAUGACAGUUCU
1091-1113


1565745.1
1577552.1

GUCAUAA

2894513.1

GGACUCAGCG






AD-
A-
1521
CUGAGUCCAGAACU
1093-1113
A-
1965
UUATGACAGUUCUG
1091-1113


1565546.1
1577552.1

GUCAUAA

2894160.1

GACUCAGCG






AD-
A-
1522
CUGAGUCCAGAACU
1093-1113
A-
1966
UUAUGACAGUUCU
1091-1113


1565547.1
1577552.1

GUCAUAA

2894161.1

GGACUCAGCG






AD-
A-
1523
UGAGUCCAGAACUG
1094-1114
A-
1967
UUUATGACAGUUCU
1092-1114


1565548.1
2894162.1

UCAUAAA

2894163.1

GGACUCAGC






AD-
A-
1524
GAGUCCAGAACUGU
1095-1115
A-
1968
UCUUAUGACAGUU
1093-1115


1565746.1
1577518.1

CAUAAGA

2894514.1

CUGGACUCAG






AD-
A-
1525
GAGUCCAGAACUGU
1095-1115
A-
1969
UCUUAUGACAGTUC
1093-1115


1565549.1
2894164.1

CAUAAGA

2894165.1

UGGACUCAG






AD-
A-
1526
AGUCCAGAACUGUCA
1096-1116
A-
1970
UUCUTATGACAGUU
1094-1116


1565550.1
2894166.1

UAAGAA

2894167.1

CUGGACUCA






AD-
A-
1527
AGUCCAGAACUGUCA
1096-1116
A-
1971
UTCUTATGACAGUU
1094-1116


1565551.1
2894168.1

UAAGAA

2894169.1

CUGGACUCA






AD-
A-
1528
GUCCAGAACUGUCA
1097-1117
A-
1972
UAUCUUAUGACAG
1095-1117


1244365.3
2324726.1

UAAGAUA

1803353.1

UUCUGGACUC






AD-
A-
1529
GUCCAGAACUGUCA
1097-1117
A-
1973
UAUCTUAUGACAGU
1095-1117


1565552.1
2894170.1

UAAGAUA

2894171.1

UCUGGACUC






AD-
A-
1530
GUCCAGAACUGUCA
1097-1117
A-
1974
UAUCTUAUGACAGU
1095-1117


1565553.1
2894170.1

UAAGAUA

2894172.1

UCUGGACUC






AD-
A-
1531
GUCCAGAACUGUCA
1097-1117
A-
1975
UAUCTUAUGACAGU
1095-1117


1565815.1
2894170.1

UAAGAUA

2894617.1

UCUGGACUC






AD-
A-
1532
GUCCAGAACUGUCA
1097-1117
A-
1976
UAUCTUAUGACAGU
1095-1117


1565816.1
2894170.1

UAAGAUA

2894618.1

UCUGGACUC






AD-
A-
1533
GUCCUGAACUGUCA
1097-1117
A-
1977
UAUCTUAUGACAGU
1095-1117


1565818.1
2894621.1

UAAGAUA

2894622.1

UCAGGACUC






AD-
A-
1534
GUCGAGAACUGUCA
1097-1117
A-
1978
UAUCTUAUGACAGU
1095-1117


1565819.1
2894623.1

UAAGAUA

2894624.1

UCUCGACUC






AD-
A-
1535
GUGCAGAACUGUCA
1097-1117
A-
1979
UAUCTUAUGACAGU
1095-1117


1565820.1
2894625.1

UAAGAUA

2894626.1

UCUGGACUC






AD-
A-
1536
GUCCAGAACUGUCA
1097-1117
A-
1980
UAUCTUAUGACAGU
1095-1117


1565552.2
2894170.1

UAAGAUA

2894171.1

UCUGGACUC






AD-
A-
1537
GUCCAGAACUGUCA
1097-1117
A-
1981
UAUCTUAUGACAGU
1095-1117


1565553.2
2894170.1

UAAGAUA

2894172.1

UCUGGACUC






AD-
A-
1538
UCCAGAACUGUCAUA
1098-1118
A-
1982
UUAUCUTAUGACAG
1096-1118


1565554.1
2894173.1

AGAUAA

2894174.1

UUCUGGACU






AD-
A-
1539
CCAGAACUGUCAUAA
1099-1117
A-
1983
UAUCTUAUGACAGU
1097-1117


1565817.1
2894619.1

GAUA

2894620.1

UCUGGGC






AD-
A-
1540
CCAGAACUGUCAUAA
1099-1119
A-
1984
UAUATCTUAUGACA
1097-1119


1565555.1
2894175.1

GAUAUA

2894176.1

GUUCUGGAC






AD-
A-
1541
CAGAACUGUCAUAA
1100-1120
A-
1985
UCAUAUCUUAUGAC
1098-1120


1565747.1
2894177.1

GAUAUGA

2894515.1

AGUUCUGGA






AD-
A-
1542
CAGAACUGUCAUAA
1100-1120
A-
1986
UCATATCUUAUGAC
1098-1120


1565556.1
2894177.1

GAUAUGA

2894178.1

AGUUCUGGA






AD-
A-
1543
CAGAACUGUCAUAA
1100-1120
A-
1987
UCAUAUCUUAUGAC
1098-1120


1565557.1
2894179.1

GAUAUGA

2894180.1

AGUUCUGGA






AD-
A-
1544
AGAACUGUCAUAAG
1101-1121
A-
1988
UUCATATCUUAUGA
1099-1121


1565558.1
2894181.1

AUAUGAA

2894182.1

CAGUUCUGG






AD-
A-
1545
AGAACUGUCAUAAG
1101-1121
A-
1989
UTCATATCUUATGAC
1099-1121


1565559.1
2894183.1

AUAUGAA

2894184.1

AGUUCUGG






AD-
A-
1546
GAACUGUCAUAAGA
1102-1122
A-
1990
UCUCAUAUCUUAU
1100-1122


1565560.1
2894185.1

UAUGAGA

2894186.1

GACAGUUCUG






AD-
A-
1547
AACUGUCAUAAGAU
1103-1123
A-
1991
UGCUCATAUCUUAU
1101-1123


1565561.1
2894187.1

AUGAGCA

2894188.1

GACAGUUCU






AD-
A-
1548
ACUGUCAUAAGAUA
1104-1124
A-
1992
UAGCTCAUAUCUUA
1102-1124


1565562.1
2894189.1

UGAGCUA

2894190.1

UGACAGUUC






AD-
A-
1549
CUGUCAUAAGAUAU
1105-1125
A-
1993
UCAGCUCAUAUCUU
1103-1125


1565748.1
2894191.1

GAGCUGA

2894516.1

AUGACAGUU






AD-
A-
1550
CUGUCAUAAGAUAU
1105-1125
A-
1994
UCAGCTCAUAUCUU
1103-1125


1565563.1
2894191.1

GAGCUGA

2894192.1

AUGACAGUU






AD-
A-
1551
CUGUCAUAAGAUAU
1105-1125
A-
1995
UCAGCTCAUAUCUU
1103-1125


1565564.1
2894191.1

GAGCUGA

2894193.1

AUGACAGUU






AD-
A-
1552
UGUCAUAAGAUAUG
1106-1126
A-
1996
UUCAGCTCAUAUCU
1104-1126


1565565.1
2894194.1

AGCUGAA

2894195.1

UAUGACAGU






AD-
A-
1553
CAUAAGAUAUGAGC
1109-1129
A-
1997
UUAUTCAGCUCAUA
1107-1129


1565566.1
2894196.1

UGAAUAA

2894197.1

UCUUAUGAC






AD-
A-
1554
CUGAAUACCGAGACA
1122-1142
A-
1998
UUUCACTGUCUCGG
1120-1142


1565567.1
2894198.1

GUGAAA

2894199.1

UAUUCAGCU






AD-
A-
1555
UACCGAGACAGUGA
1127-1147
A-
1999
UCAGCCTUCACUGU
1125-1147


1565568.1
2894200.1

AGGCUGA

2894201.1

CUCGGUAUU






AD-
A-
1556
UACCGAGACAGUGA
1127-1147
A-
2000
UCAGCCTUCACTGU
1125-1147


1565569.1
2894202.1

AGGCUGA

2894203.1

CUCGGUAUU






AD-
A-
1557
ACAGUGAAGGCUGA
1134-1154
A-
2001
UUCCTUCUCAGCCU
1132-1154


1565749.1
2894204.1

GAAGGAA

2894517.1

UCACUGUCU






AD-
A-
1558
ACAGUGAAGGCUGA
1134-1154
A-
2002
UUCCUTCUCAGCCU
1132-1154


1565570.1
2894204.1

GAAGGAA

2894205.1

UCACUGUCU






AD-
A-
1559
ACAGUGAAGGCUGA
1134-1154
A-
2003
UUCCUTCTCAGCCU
1132-1154


1565571.1
2894204.1

GAAGGAA

2894206.1

UCACUGUCU






AD-
A-
1560
UGAGAAGGAAAUCC
1145-1165
A-
2004
UCUCCAGGGAUUUC
1143-1165


1565750.1
2894518.1

CUGGAGA

2894519.1

CUUCUCAGC






AD-
A-
1561
GCCAAAGGUGCCAU
1266-1286
A-
2005
UAGGACAAUGGCAC
1264-1286


1565573.1
2894209.1

UGUCCUA

2894210.1

CUUUGGCCU






AD-
A-
1562
CCAAACUGAACCCAG
1288-1308
A-
2006
UAUUCUCUGGGUU
1286-1308


1565751.1
2894211.1

AGAAUA

2894520.1

CAGUUUGGAG






AD-
A-
1563
CCAAACUGAACCCAG
1288-1308
A-
2007
UAUTCTCUGGGUUC
1286-1308


1565574.1
2894211.1

AGAAUA

2894212.1

AGUUUGGAG






AD-
A-
1564
CCAAACUGAACCCAG
1288-1308
A-
2008
UAUUCTCTGGGUUC
1286-1308


1565575.1
2894211.1

AGAAUA

2894213.1

AGUUUGGAG






AD-
A-
1565
CAAACUGAACCCAGA
1289-1309
A-
2009
UGAUTCTCUGGGUU
1287-1309


1565576.1
2894214.1

GAAUCA

2894215.1

CAGUUUGGA






AD-
A-
1566
UCCGUAAGCAGUCA
1339-1359
A-
2010
UGGCGACUGACUGC
1337-1359


1565752.1
2894216.1

GUCGCCA

2894521.1

UUACGGAUG






AD-
A-
1567
UCCGUAAGCAGUCA
1339-1359
A-
2011
UGGCGACUGACUGC
1337-1359


1565577.1
2894216.1

GUCGCCA

2894217.1

UUACGGAUG






AD-
A-
1568
UCCGUAAGCAGUCA
1339-1359
A-
2012
UGGCGACTGACUGC
1337-1359


1565578.1
2894216.1

GUCGCCA

2894218.1

UUACGGAUG






AD-
A-
1569
UCCGUAAGCAGUCA
1339-1359
A-
2013
UGGCGACUGACTGC
1337-1359


1565579.1
2894219.1

GUCGCCA

2894220.1

UUACGGAUG






AD-
A-
1570
UAAGCAGUCAGUCG
1343-1363
A-
2014
UCAUTGGCGACUGA
1341-1363


1565753.1
2894522.1

CCAAUGA

2894523.1

CUGCUUACG






AD-
A-
1571
CAGUCAGUCGCCAAU
1347-1367
A-
2015
UAAGGCAUUGGCG
1345-1367


1565580.1
2894221.1

GCCUUA

2894222.1

ACUGACUGCU






AD-
A-
1572
UCAGUCGCCAAUGCC
1350-1370
A-
2016
UAUGAAGGCAUUG
1348-1370


1565754.1
2894524.1

UUCAUA

2894525.1

GCGACUGACU






AD-
A-
1573
AGUCGCCAAUGCCUU
1352-1372
A-
2017
UUGATGAAGGCAUU
1350-1372


1565581.1
2894223.1

CAUCAA

2894224.1

GGCGACUGA






AD-
A-
1574
GCCAAUGCCUUCAUC
1356-1376
A-
2018
UCAGAUGAUGAAG
1354-1376


1565582.1
2894225.1

AUCUGA

2894226.1

GCAUUGGCGA






AD-
A-
1575
CCUUCAUCAUCUGU
1363-1383
A-
2019
UGGUGCCACAGAUG
1361-1383


1565755.1
2894227.1

GGCACCA

2894526.1

AUGAAGGCA






AD-
A-
1576
CCUUCAUCAUCUGU
1363-1383
A-
2020
UGGUGCCACAGAUG
1361-1383


1565583.1
2894227.1

GGCACCA

2894228.1

AUGAAGGCA






AD-
A-
1577
CUGUGGCACCUUGU
1373-1393
A-
2021
UCGGTGTACAAGGU
1371-1393


1565584.1
2894229.1

ACACCGA

2894230.1

GCCACAGAU






AD-
A-
1578
CUACCGUCAACUUUG
1417-1437
A-
2022
UAUAAGCAAAGUU
1415-1437


1565756.1
2894231.1

CUUAUA

2894527.1

GACGGUAGCA






AD-
A-
1579
CUACCGUCAACUUUG
1417-1437
A-
2023
UAUAAGCAAAGUU
1415-1437


1565585.1
2894231.1

CUUAUA

2894232.1

GACGGUAGCA






AD-
A-
1580
UACCGUCAACUUUGC
1418-1438
A-
2024
UCAUAAGCAAAGUU
1416-1438


1073420.3
1991728.1

UUAUGA

1806295.1

GACGGUAGC






AD-
A-
1581
UACCGUCAACUUUGC
1418-1438
A-
2025
UCAUAAGCAAAGUU
1416-1438


1244366.3
1991728.1

UUAUGA

1803954.1

GACGGUAGC






AD-
A-
1582
UACCGUCAACUUUGC
1418-1438
A-
2026
UCAUAAGCAAAGUU
1416-1438


1565757.1
1991728.1

UUAUGA

2894528.1

GACGGUAGC






AD-
A-
1583
UACCGUCAACUUUGC
1418-1438
A-
2027
UCATAAGCAAAGUU
1416-1438


1565821.1
1991728.1

UUAUGA

2894627.1

GACGGUAGC






AD-
A-
1584
UACCGUCAACUUUGC
1418-1438
A-
2028
UCAUAAGCAAAGUU
1416-1438


1565822.1
1991728.1

UUAUGA

2894628.1

GACGGUAGC






AD-
A-
1585
UACCCUCAACUUUGC
1418-1438
A-
2029
UCAUAAGCAAAGUU
1416-1438


1565824.1
2894631.1

UUAUGA

2894632.1

GAGGGUAGC






AD-
A-
1586
UACGGUCAACUUUG
1418-1438
A-
2030
UCAUAAGCAAAGUU
1416-1438


1565825.1
2894633.1

CUUAUGA

2894634.1

GACCGUAGC






AD-
A-
1587
UAGCGUCAACUUUG
1418-1438
A-
2031
UCAUAAGCAAAGUU
1416-1438


1565826.1
2894635.1

CUUAUGA

2894636.1

GACGCUAGC






AD-
A-
1588
UACCGUCAACUUUGC
1418-1438
A-
2032
UCAUAAGCAAAGUU
1416-1438


1565757.2
1991728.1

UUAUGA

2894528.1

GACGGUAGC






AD-
A-
1589
UACCGUCAACUUUGC
1418-1438
A-
2033
UCAUAAGCAAAGUU
1416-1438


1565586.1
2894233.1

UUAUGA

2894234.1

GACGGUAGC






AD-
A-
1590
ACCGUCAACUUUGCU
1419-1439
A-
2034
UUCATAAGCAAAGU
1417-1439


1565587.1
2324727.1

UAUGAA

2894235.1

UGACGGUAG






AD-
A-
1591
ACCGUCAACUUUGCU
1419-1439
A-
2035
UTCATAAGCAAAGU
1417-1439


1565588.1
2894236.1

UAUGAA

2894237.1

UGACGGUAG






AD-
A-
1592
CCGUCAACUUUGCU
1420-1438
A-
2036
UCAUAAGCAAAGUU
1418-1438


1565823.1
2894629.1

UAUGA

2894630.1

GACGGUG






AD-
A-
1593
CCGUCAACUUUGCU
1420-1440
A-
2037
UGUCAUAAGCAAAG
1418-1440


1565589.1
2894238.1

UAUGACA

2894239.1

UUGACGGUA






AD-
A-
1594
CGUCAACUUUGCUU
1421-1441
A-
2038
UUGUCATAAGCAAA
1419-1441


1565590.1
2894240.1

AUGACAA

2894241.1

GUUGACGGU






AD-
A-
1595
GUCAACUUUGCUUA
1422-1442
A-
2039
UGUGTCAUAAGCAA
1420-1442


1565591.1
2894242.1

UGACACA

2894243.1

AGUUGACGG






AD-
A-
1596
UCAACUUUGCUUAU
1423-1443
A-
2040
UUGUGUCAUAAGC
1421-1443


1565758.1
2894244.1

GACACAA

2894529.1

AAAGUUGACG






AD-
A-
1597
UCAACUUUGCUUAU
1423-1443
A-
2041
UUGTGTCAUAAGCA
1421-1443


1565592.1
2894244.1

GACACAA

2894245.1

AAGUUGACG






AD-
A-
1598
CAACUUUGCUUAUG
1424-1444
A-
2042
UCUGTGTCAUAAGC
1422-1444


1565594.1
2894247.1

ACACAGA

2894248.1

AAAGUUGAC






AD-
A-
1599
AACUUUGCUUAUGA
1425-1445
A-
2043
UCCUGUGUCAUAA
1423-1445


1565759.1
2894530.1

CACAGGA

2894531.1

GCAAAGUUGA






AD-
A-
1600
ACUUUGCUUAUGAC
1426-1446
A-
2044
UGCCTGTGUCAUAA
1424-1446


1565595.1
2894249.1

ACAGGCA

2894250.1

GCAAAGUUG






AD-
A-
1601
CUUUGCUUAUGACA
1427-1447
A-
2045
UUGCCUGUGUCAU
1425-1447


1565760.1
2894532.1

CAGGCAA

2894533.1

AAGCAAAGUU






AD-
A-
1602
ACAGGCACAGGUAUC
1440-1460
A-
2046
UUUGCUGAUACCU
1438-1460


1565761.1
2894534.1

AGCAAA

2894535.1

GUGCCUGUGU






AD-
A-
1603
AUAAGUACAGCAGCA
1489-1509
A-
2047
UAAUCATGCUGCUG
1487-1509


1565596.1
2894251.1

UGAUUA

2894252.1

UACUUAUAG






AD-
A-
1604
UAAGUACAGCAGCA
1490-1510
A-
2048
UCAATCAUGCUGCU
1488-1510


1565597.1
2894253.1

UGAUUGA

2894254.1

GUACUUAUA






AD-
A-
1605
AAGUACAGCAGCAU
1491-1511
A-
2049
UUCAAUCAUGCUGC
1489-1511


1565762.1
2894255.1

GAUUGAA

2894536.1

UGUACUUAU






AD-
A-
1606
AAGUACAGCAGCAU
1491-1511
A-
2050
UUCAATCAUGCUGC
1489-1511


1565598.1
2894255.1

GAUUGAA

2894256.1

UGUACUUAU






AD-
A-
1607
AAGUACAGCAGCAU
1491-1511
A-
2051
UUCAATCAUGCUGC
1489-1511


1565599.1
2894255.1

GAUUGAA

2894257.1

UGUACUUAU






AD-
A-
1608
AGUACAGCAGCAUG
1492-1512
A-
2052
UGUCAATCAUGCUG
1490-1512


1565600.1
2894258.1

AUUGACA

2894259.1

CUGUACUUA






AD-
A-
1609
AGUACAGCAGCAUG
1492-1512
A-
2053
UGUCAATCAUGCUG
1490-1512


1565601.1
2894260.1

AUUGACA

2894261.1

CUGUACUUA






AD-
A-
1610
GUACAGCAGCAUGA
1493-1513
A-
2054
UAGUCAAUCAUGCU
1491-1513


1565602.1
2894262.1

UUGACUA

2894263.1

GCUGUACUU






AD-
A-
1611
GUACAGCAGCAUGA
1493-1513
A-
2055
UAGUCAAUCAUGCU
1491-1513


1565827.1
2894637.1

UUGACUA

1804098.1

GCUGUACUU






AD-
A-
1612
GUACAGCAGCAUGA
1493-1513
A-
2056
UAGUCAAUCAUGCU
1491-1513


1565828.1
2894262.1

UUGACUA

2894638.1

GCUGUACUU






AD-
A-
1613
GUACAGCAGCAUGA
1493-1513
A-
2057
UAGUCAAUCAUGCU
1491-1513


1565829.1
2894262.1

UUGACUA

2894639.1

GCUGUACUU






AD-
A-
1614
GUACAGCAGCAUGA
1493-1513
A-
2058
UAGUCAAUCAUGCU
1491-1513


1565830.1
2894262.1

UUGACUA

2894640.1

GCUGUACUU






AD-
A-
1615
GUACAGCAGCAUGA
1493-1513
A-
2059
UAGUCAAUCAUGCU
1491-1513


1565831.1
2894262.1

UUGACUA

2894641.1

GCUGUACUU






AD-
A-
1616
GUACUGCAGCAUGA
1493-1513
A-
2060
UAGUCAAUCAUGCU
1491-1513


1565833.1
2894644.1

UUGACUA

2894645.1

GCAGUACUU






AD-
A-
1617
GUAGAGCAGCAUGA
1493-1513
A-
2061
UAGUCAAUCAUGCU
1491-1513


1565834.1
2894646.1

UUGACUA

2894647.1

GCUCUACUU






AD-
A-
1618
GUUCAGCAGCAUGA
1493-1513
A-
2062
UAGUCAAUCAUGCU
1491-1513


1565835.1
2894648.1

UUGACUA

2894649.1

GCUGAACUU






AD-
A-
1619
GUACAGCAGCAUGA
1493-1513
A-
2063
UAGUCAAUCAUGCU
1491-1513


1565602.2
2894262.1

UUGACUA

2894263.1

GCUGUACUU






AD-
A-
1620
UACAGCAGCAUGAU
1494-1514
A-
2064
UUAGTCAAUCAUGC
1492-1514


1565603.1
2894264.1

UGACUAA

2894265.1

UGCUGUACU






AD-
A-
1621
ACAGCAGCAUGAUU
1495-1513
A-
2065
UAGUCAAUCAUGCU
1493-1513


1565832.1
2894642.1

GACUA

2894643.1

GCUGUGC






AD-
A-
1622
ACAGCAGCAUGAUU
1495-1515
A-
2066
UGUAGUCAAUCAU
1493-1515


1565763.1
2894266.1

GACUACA

2894537.1

GCUGCUGUAC






AD-
A-
1623
ACAGCAGCAUGAUU
1495-1515
A-
2067
UGUAGTCAAUCAUG
1493-1515


1565604.1
2894266.1

GACUACA

2894267.1

CUGCUGUAC






AD-
A-
1624
ACAGCAGCAUGAUU
1495-1515
A-
2068
UGUAGTCAAUCAUG
1493-1515


1565605.1
2894266.1

GACUACA

2894268.1

CUGCUGUAC






AD-
A-
1625
CAGCAGCAUGAUUG
1496-1516
A-
2069
UUGUAGTCAAUCAU
1494-1516


1565606.1
2894269.1

ACUACAA

2894270.1

GCUGCUGUA






AD-
A-
1626
AGCAGCAUGAUUGA
1497-1517
A-
2070
UUUGTAGUCAAUCA
1495-1517


1565764.1
2894538.1

CUACAAA

2894539.1

UGCUGCUGU






AD-
A-
1627
GCAGCAUGAUUGAC
1498-1518
A-
2071
UGUUGUAGUCAAU
1496-1518


1565607.1
2894271.1

UACAACA

2894272.1

CAUGCUGCUG






AD-
A-
1628
CAGCAUGAUUGACU
1499-1519
A-
2072
UGGUTGTAGUCAAU
1497-1519


1565608.1
2894273.1

ACAACCA

2894274.1

CAUGCUGCU






AD-
A-
1629
GCAUGAUUGACUAC
1501-1521
A-
2073
UGGGGUTGUAGUC
1499-1521


1565609.1
2894275.1

AACCCCA

2894276.1

AAUCAUGCUG






AD-
A-
1630
AGCUCUUUGCCUGG
1531-1551
A-
2074
UGUUGUCCCAGGCA
1529-1551


1565766.1
2894279.1

GACAACA

2894542.1

AAGAGCUUC






AD-
A-
1631
AGCUCUUUGCCUGG
1531-1551
A-
2075
UGUTGTCCCAGGCA
1529-1551


1565611.1
2894279.1

GACAACA

2894280.1

AAGAGCUUC






AD-
A-
1632
GCCUGGGACAACUU
1539-1559
A-
2076
UAUGTUCAAGUUG
1537-1559


1565767.1
2894281.1

GAACAUA

2894543.1

UCCCAGGCAA






AD-
A-
1633
GCCUGGGACAACUU
1539-1559
A-
2077
UAUGUTCAAGUUG
1537-1559


1565612.1
2894281.1

GAACAUA

2894282.1

UCCCAGGCAA






AD-
A-
1634
GCCUGGGACAACUU
1539-1559
A-
2078
UAUGUTCAAGUUG
1537-1559


1565613.1
2894281.1

GAACAUA

2894283.1

UCCCAGGCAA






AD-
A-
1635
CUGGGACAACUUGA
1541-1561
A-
2079
UCCATGTUCAAGUU
1539-1561


1565614.1
2894284.1

ACAUGGA

2894285.1

GUCCCAGGC






AD-
A-
1636
UGGGACAACUUGAA
1542-1562
A-
2080
UACCAUGUUCAAGU
1540-1562


1565768.1
2894544.1

CAUGGUA

2894545.1

UGUCCCAGG






AD-
A-
1637
GGGACAACUUGAAC
1543-1563
A-
2081
UGACCATGUUCAAG
1541-1563


1565615.1
2894286.1

AUGGUCA

2894287.1

UUGUCCCAG






AD-
A-
1638
GGGACAACUUGAAC
1543-1563
A-
2082
UGACCATGUUCAAG
1541-1563


1565616.1
2894288.1

AUGGUCA

2894289.1

UUGUCCCAG






AD-
A-
1639
GGACAACUUGAACA
1544-1564
A-
2083
UUGACCAUGUUCAA
1542-1564


1565617.1
2894290.1

UGGUCAA

2894291.1

GUUGUCCCA






AD-
A-
1640
GACAACUUGAACAU
1545-1565
A-
2084
UGUGACCAUGUUC
1543-1565


1565769.1
2894292.1

GGUCACA

2894546.1

AAGUUGUCCC






AD-
A-
1641
GACAACUUGAACAU
1545-1565
A-
2085
UGUGACCAUGUUC
1543-1565


1565618.1
2894292.1

GGUCACA

2894293.1

AAGUUGUCCC






AD-
A-
1642
ACAACUUGAACAUG
1546-1566
A-
2086
UAGUGACCAUGUU
1544-1566


1565770.1
2894294.1

GUCACUA

2894547.1

CAAGUUGUCC






AD-
A-
1643
ACAACUUGAACAUG
1546-1566
A-
2087
UAGTGACCAUGUUC
1544-1566


1565619.1
2894294.1

GUCACUA

2894295.1

AAGUUGUCC






AD-
A-
1644
CAACUUGAACAUGG
1547-1567
A-
2088
UAAGTGACCAUGUU
1545-1567


1565620.1
2894296.1

UCACUUA

2894297.1

CAAGUUGUC






AD-
A-
1645
AACUUGAACAUGGU
1548-1568
A-
2089
UUAAGUGACCAUG
1546-1568


1565771.1
2894548.1

CACUUAA

2894549.1

UUCAAGUUGU






AD-
A-
1646
ACUUGAACAUGGUC
1549-1569
A-
2090
UAUAAGTGACCAUG
1547-1569


1565621.1
1577540.1

ACUUAUA

2894298.1

UUCAAGUUG






AD-
A-
1647
CUUGAACAUGGUCA
1550-1570
Å-
2091
UCAUAAGUGACCAU
1548-1570


1565772.1
1577508.1

CUUAUGA

2894550.1

GUUCAAGUU






AD-
A-
1648
CUUGAACAUGGUCA
1550-1570
A-
2092
UCAUAAGUGACCAU
1548-1570


1565836.1
1577508.1

CUUAUGA

2894300.1

GUUCAAGUU






AD-
A-
1649
CUUGAACAUGGUCA
1550-1570
A-
2093
UCATAAGUGACCAU
1548-1570


1565837.1
1577508.1

CUUAUGA

2894650.1

GUUCAAGUU






AD-
A-
1650
CUUGAACAUGGUCA
1550-1570
A-
2094
UCAUAAGUGACCAU
1548-1570


1565838.1
1577508.1

CUUAUGA

2894651.1

GUUCAAGUU






AD-
A-
1651
CUUGUACAUGGUCA
1550-1570
A-
2095
UCAUAAGUGACCAU
1548-1570


1565840.1
2894654.1

CUUAUGA

2894655.1

GUACAAGUU






AD-
A-
1652
CUUCAACAUGGUCAC
1550-1570
A-
2096
UCAUAAGUGACCAU
1548-1570


1565841.1
2894656.1

UUAUGA

2894657.1

GUUGAAGUU






AD-
A-
1653
CUAGAACAUGGUCAC
1550-1570
A-
2097
UCAUAAGUGACCAU
1548-1570


1565842.1
2894658.1

UUAUGA

2894659.1

GUUCUAGUU






AD-
A-
1654
CUUGAACAUGGUCA
1550-1570
A-
2098
UCAUAAGUGACCAU
1548-1570


822899.17
1577508.1

CUUAUGA

1577509.1

GUUCAAGUU






AD-
A-
1655
CUUGAACAUGGUCA
1550-1570
A-
2099
UCAUAAGUGACCAU
1548-1570


1565772.2
1577508.1

CUUAUGA

2894550.1

GUUCAAGUU






AD-
A-
1656
CUUGAACAUGGUCA
1550-1570
A-
2100
UCAUAAGUGACCAU
1548-1570


1565622.1
2894299.1

CUUAUGA

2894300.1

GUUCAAGUU






AD-
A-
1657
UUGAACAUGGUCAC
1551-1571
A-
2101
UUCATAAGUGACCA
1549-1571


1565843.1
1577562.1

UUAUGAA

2894660.1

UGUUCAAGU






AD-
A-
1658
UUGAACAUGGUCAC
1551-1571
A-
2102
UUCATAAGUGACCA
1549-1571


1565844.1
1577562.1

UUAUGAA

2894661.1

UGUUCAAGU






AD-
A-
1659
UUGAACAUGGUCAC
1551-1571
A-
2103
UTCATAAGUGACCA
1549-1571


1565845.1
1577562.1

UUAUGAA

2894662.1

UGUUCAAGU






AD-
A-
1660
UUGAACAUGGUCAC
1551-1571
A-
2104
UTCATAAGUGACCA
1549-1571


1565846.1
2894663.1

UUAUGAA

2894662.1

UGUUCAAGU






AD-
A-
1661
UUGAUCAUGGUCAC
1551-1571
A-
2105
UUCATAAGUGACCA
1549-1571


1565848.1
2894666.1

UUAUGAA

2894667.1

UGAUCAAGU






AD-
A-
1662
UUGUACAUGGUCAC
1551-1571
A-
2106
UUCATAAGUGACCA
1549-1571


1565849.1
2894668.1

UUAUGAA

2894669.1

UGUACAAGU






AD-
A-
1663
UUCAACAUGGUCAC
1551-1571
A-
2107
UUCATAAGUGACCA
1549-1571


1565850.1
2894670.1

UUAUGAA

2894671.1

UGUUGAAGU






AD-
A-
1664
UUGAACAUGGUCAC
1551-1571
A-
2108
UUCAUAAGUGACCA
1549-1571


822926.4
1577562.1

UUAUGAA

1577563.1

UGUUCAAGU






AD-
A-
1665
UUGAACAUGGUCAC
1551-1571
A-
2109
UTCATAAGUGACCA
1549-1571


1565623.1
2894301.1

UUAUGAA

2894302.1

UGUUCAAGU






AD-
A-
1666
UGAACAUGGUCACU
1552-1570
A-
2110
UCAUAAGUGACCAU
1550-1570


1565839.1
2894652.1

UAUGA

2894653.1

GUUCAGG






AD-
A-
1667
UGAACAUGGUCACU
1552-1572
A-
2111
UGUCAUAAGUGACC
1550-1572


1565624.1
2894303.1

UAUGACA

2894304.1

AUGUUCAAG






AD-
A-
1668
GAACAUGGUCACUU
1553-1571
A-
2112
UUCATAAGUGACCA
1551-1571


1565847.1
2894664.1

AUGAA

2894665.1

UGUUCGG






AD-
A-
1669
GAACAUGGUCACUU
1553-1573
A-
2113
UUGUCATAAGUGAC
1551-1573


1565625.1
2894305.1

AUGACAA

2894306.1

CAUGUUCAA






AD-
A-
1670
AACAUGGUCACUUA
1554-1574
A-
2114
UAUGTCAUAAGUGA
1552-1574


1565626.1
2894307.1

UGACAUA

2894308.1

CCAUGUUCA






AD-
A-
1671
ACAUGGUCACUUAU
1555-1575
A-
2115
UGAUGUCAUAAGU
1553-1575


1565773.1
2894309.1

GACAUCA

2894551.1

GACCAUGUUC






AD-
A-
1672
ACAUGGUCACUUAU
1555-1575
A-
2116
UGATGTCAUAAGUG
1553-1575


1565627.1
2894309.1

GACAUCA

2894310.1

ACCAUGUUC






AD-
A-
1673
ACAUGGUCACUUAU
1555-1575
A-
2117
UGAUGTCAUAAGUG
1553-1575


1565628.1
2894309.1

GACAUCA

2894311.1

ACCAUGUUC






AD-
A-
1674
CAUGGUCACUUAUG
1556-1576
A-
2118
UUGATGTCAUAAGU
1554-1576


1565629.1
2894312.1

ACAUCAA

2894313.1

GACCAUGUU






AD-
A-
1675
AUGGUCACUUAUGA
1557-1577
A-
2119
UUUGAUGUCAUAA
1555-1577


1565774.1
2894552.1

CAUCAAA

2894553.1

GUGACCAUGU






AD-
A-
1676
UGGUCACUUAUGAC
1558-1578
A-
2120
UCUUGATGUCAUAA
1556-1578


1565630.1
2894314.1

AUCAAGA

2894315.1

GUGACCAUG






AD-
A-
1677
GGUCACUUAUGACA
1559-1579
A-
2121
UGCUTGAUGUCAUA
1557-1579


1565631.1
2894316.1

UCAAGCA

2894317.1

AGUGACCAU






AD-
A-
1678
GUCACUUAUGACAU
1560-1580
A-
2122
UAGCTUGAUGUCAU
1558-1580


1565775.1
2894554.1

CAAGCUA

2894555.1

AAGUGACCA






AD-
A-
1679
GCAGAAGGAGAUGC
1619-1639
A-
2123
UCCCTGAGCAUCUC
1617-1639


1565632.1
2894318.1

UCAGGGA

2894319.1

CUUCUGCCA






AD-
A-
1680
AAGGGAGAGCCAGCC
1659-1679
A-
2124
UUGGCUGGCUGGC
1657-1679


1565776.1
2894556.1

AGCCAA

2894557.1

UCUCCCUUCA






AD-
A-
1681
GAUGAACAUGGUCA
1727-1747
A-
2125
UAGATGGUGACCAU
1725-1747


1565777.1
2894558.1

CCAUCUA

2894559.1

GUUCAUCCU






AD-
A-
1682
GCAUUUAUGGGAUG
1816-1836
A-
2126
UAUUAAACAUCCCA
1814-1836


1565634.1
2894322.1

UUUAAUA

2894323.1

UAAAUGCUG






AD-
A-
1683
UGUUUAAUGACAUA
1828-1848
A-
2127
UUUGAACUAUGUC
1826-1848


1565778.1
2894324.1

GUUCAAA

2894560.1

AUUAAACAUC






AD-
A-
1684
UGUUUAAUGACAUA
1828-1848
A-
2128
UUUGAACUAUGUC
1826-1848


1565635.1
2894324.1

GUUCAAA

2894325.1

AUUAAACAUC






AD-
A-
1685
UGUUUAAUGACAUA
1828-1848
A-
2129
UUUGAACTAUGUCA
1826-1848


1565636.1
2894324.1

GUUCAAA

2894326.1

UUAAACAUC






AD-
A-
1686
GUUUAAUGACAUAG
1829-1849
A-
2130
UCUUGAACUAUGU
1827-1849


1565637.1
2894327.1

UUCAAGA

2894328.1

CAUUAAACAU






AD-
A-
1687
GUUUAAUGACAUAG
1829-1849
A-
2131
UCUUGAACUAUGU
1827-1849


1565638.1
2894329.1

UUCAAGA

2894330.1

CAUUAAACAU






AD-
A-
1688
UUUAAUGACAUAGU
1830-1850
A-
2132
UACUTGAACUAUGU
1828-1850


1565639.1
2894331.1

UCAAGUA

2894332.1

CAUUAAACA






AD-
A-
1689
UUAAUGACAUAGUU
1831-1851
A-
2133
UAACTUGAACUAUG
1829-1851


1565779.1
2894561.1

CAAGUUA

2894562.1

UCAUUAAAC






AD-
A-
1690
UAAUGACAUAGUUC
1832-1852
A-
2134
UAAACUTGAACUAU
1830-1852


1565640.1
2894333.1

AAGUUUA

2894334.1

GUCAUUAAA






AD-
A-
1691
UAAUGACAUAGUUC
1832-1852
A-
2135
UAAACUTGAACTAU
1830-1852


1565641.1
2894335.1

AAGUUUA

2894336.1

GUCAUUAAA






AD-
A-
1692
AAUGACAUAGUUCA
1833-1853
A-
2136
UAAAACTUGAACUA
1831-1853


1565642.1
2894337.1

AGUUUUA

2894338.1

UGUCAUUAA






AD-
A-
1693
AAUGACAUAGUUCA
1833-1853
A-
2137
UAAAACTUGAACUA
1831-1853


1565643.1
2894339.1

AGUUUUA

2894340.1

UGUCAUUAA






AD-
A-
1694
AUGACAUAGUUCAA
1834-1854
A-
2138
UGAAAACUUGAACU
1832-1854


1565644.1
2894341.1

GUUUUCA

2894342.1

AUGUCAUUA






AD-
A-
1695
UGACAUAGUUCAAG
1835-1855
A-
2139
UAGAAAACUUGAAC
1833-1855


1565645.1
2894343.1

UUUUCUA

2894344.1

UAUGUCAUU






AD-
A-
1696
GACAUAGUUCAAGU
1836-1856
A-
2140
UAAGAAAACUUGAA
1834-1856


1565646.1
2894345.1

UUUCUUA

2894346.1

CUAUGUCAU






AD-
A-
1697
ACAUAGUUCAAGUU
1837-1857
A-
2141
UCAAGAAAACUUGA
1835-1857


1565851.1
2894672.1

UUCUUGA

1806645.1

ACUAUGUCA






AD-
A-
1698
ACAUAGUUCAAGUU
1837-1857
A-
2142
UCAAGAAAACUTGA
1835-1857


1565852.1
2894347.1

UUCUUGA

2894673.1

ACUAUGUCG






AD-
A-
1699
ACAUAGUUCAAGUU
1837-1857
A-
2143
UCAAGAAAACUUGA
1835-1857


1565853.1
2894347.1

UUCUUGA

2894674.1

ACUAUGUCG






AD-
A-
1700
ACAUAGUUCAAGUU
1837-1857
A-
2144
UCAAGAAAACUTGA
1835-1857


1565854.1
2894347.1

UUCUUGA

2894675.1

ACUAUGUCG






AD-
A-
1701
ACAUUGUUCAAGUU
1837-1857
A-
2145
UCAAGAAAACUUGA
1835-1857


1565857.1
2894679.1

UUCUUGA

2894680.1

ACAAUGUCG






AD-
A-
1702
ACAAAGUUCAAGUU
1837-1857
A-
2146
UCAAGAAAACUUGA
1835-1857


1565858.1
2894681.1

UUCUUGA

2894682.1

ACUUUGUCG






AD-
A-
1703
ACUUAGUUCAAGUU
1837-1857
A-
2147
UCAAGAAAACUUGA
1835-1857


1565859.1
2894683.1

UUCUUGA

2894684.1

ACUAAGUCG






AD-
A-
1704
ACAUAGUUCAAGUU
1837-1857
A-
2148
UCAAGAAAACUTGA
1835-1857


1565647.1
2894347.1

UUCUUGA

2894348.1

ACUAUGUCA






AD-
A-
1705
CAUAGUUCAAGUUU
1838-1858
A-
2149
UACAAGAAAACTUG
1836-1858


1565648.1
2894349.1

UCUUGUA

2894350.1

AACUAUGUC






AD-
A-
1706
AUAGUUCAAGUUUU
1839-1857
A-
2150
UCAAGAAAACUUGA
1837-1857


1565855.1
2894676.1

CUUGA

2894677.1

ACUAUGU






AD-
A-
1707
AUAGUUCAAGUUUU
1839-1857
A-
2151
UCAAGAAAACUTGA
1837-1857


1565856.1
2894676.1

CUUGA

2894678.1

ACUAUGU






AD-
A-
1708
AUAGUUCAAGUUUU
1839-1859
A-
2152
UCACAAGAAAACUU
1837-1859


1565649.1
2894351.1

CUUGUGA

2894352.1

GAACUAUGU






AD-
A-
1709
UAGUUCAAGUUUUC
1840-1860
A-
2153
UUCACAAGAAAACU
1838-1860


1565650.1
2894353.1

UUGUGAA

2894354.1

UGAACUAUG






AD-
A-
1710
UAGUUCAAGUUUUC
1840-1860
A-
2154
UTCACAAGAAAACU
1838-1860


1565651.1
2894355.1

UUGUGAA

2894356.1

UGAACUAUG






AD-
A-
1711
AGUUCAAGUUUUCU
1841-1861
A-
2155
UAUCACAAGAAAAC
1839-1861


1565652.1
2894357.1

UGUGAUA

2894358.1

UUGAACUAU






AD-
A-
1712
AGUUCAAGUUUUCU
1841-1861
A-
2156
UAUCACAAGAAAAC
1839-1861


1565653.1
2894359.1

UGUGAUA

2894360.1

UUGAACUAU






AD-
A-
1713
GUUCAAGUUUUCUU
1842-1862
A-
2157
UAAUCACAAGAAAA
1840-1862


1565780.1
2894361.1

GUGAUUA

2894563.1

CUUGAACUA






AD-
A-
1714
GUUCAAGUUUUCUU
1842-1862
A-
2158
UAATCACAAGAAAA
1840-1862


1565654.1
2894361.1

GUGAUUA

2894362.1

CUUGAACUA






AD-
A-
1715
UUCAAGUUUUCUUG
1843-1863
A-
2159
UAAATCACAAGAAA
1841-1863


1565655.1
2894363.1

UGAUUUA

2894364.1

ACUUGAACU






AD-
A-
1716
UCAAGUUUUCUUGU
1844-1864
A-
2160
UCAAAUCACAAGAA
1842-1864


1565781.1
2894365.1

GAUUUGA

2894564.1

AACUUGAAC






AD-
A-
1717
UCAAGUUUUCUUGU
1844-1864
A-
2161
UCAAATCACAAGAA
1842-1864


1565656.1
2894365.1

GAUUUGA

2894366.1

AACUUGAAC






AD-
A-
1718
UCAAGUUUUCUUGU
1844-1864
A-
2162
UCAAATCACAAGAA
1842-1864


1565657.1
2894365.1

GAUUUGA

2894367.1

AACUUGAAC






AD-
A-
1719
UCAAGUUUUCUUGU
1844-1864
A-
2163
UCAAAUCACAAGAA
1842-1864


1565658.1
2894368.1

GAUUUGA

2894369.1

AACUUGAAC






AD-
A-
1720
CAAGUUUUCUUGUG
1845-1865
A-
2164
UCCAAATCACAAGA
1843-1865


1565659.1
2894370.1

AUUUGGA

2894371.1

AAACUUGAA






AD-
A-
1721
AAGUUUUCUUGUGA
1846-1866
A-
2165
UCCCAAAUCACAAG
1844-1866


1565660.1
2894372.1

UUUGGGA

2894373.1

AAAACUUGA






AD-
A-
1722
UGAAAACCAUUGCUC
1897-1917
A-
2166
UUGCAAGAGCAAUG
1895-1917


1565782.1
2894565.1

UUGCAA

2894566.1

GUUUUCAGG






AD-
A-
1723
GAAAACCAUUGCUCU
1898-1918
A-
2167
UAUGCAAGAGCAAU
1896-1918


1565661.1
2894374.1

UGCAUA

2894375.1

GGUUUUCAG






AD-
A-
1724
GAAAACCAUUGCUCU
1898-1918
A-
2168
UAUGCAAGAGCAAU
1896-1918


1565662.1
2894376.1

UGCAUA

2894377.1

GGUUUUCAG






AD-
A-
1725
AAAACCAUUGCUCUU
1899-1919
A-
2169
UCAUGCAAGAGCAA
1897-1919


1565663.1
2894378.1

GCAUGA

2894379.1

UGGUUUUCA






AD-
A-
1726
AAAACCAUUGCUCUU
1899-1919
A-
2170
UCAUGCAAGAGCAA
1897-1919


1565664.1
2894380.1

GCAUGA

2894381.1

UGGUUUUCA






AD-
A-
1727
AAACCAUUGCUCUU
1900-1920
A-
2171
UACATGCAAGAGCA
1898-1920


1565783.1
2894382.1

GCAUGUA

2894567.1

AUGGUUUUC






AD-
A-
1728
AAACCAUUGCUCUU
1900-1920
A-
2172
UACAUGCAAGAGCA
1898-1920


1565665.1
2894382.1

GCAUGUA

2894383.1

AUGGUUUUC






AD-
A-
1729
AACCAUUGCUCUUGC
1901-1921
A-
2173
UAACAUGCAAGAGC
1899-1921


1565784.1
2894568.1

AUGUUA

2894569.1

AAUGGUUUU






AD-
A-
1730
ACCAUUGCUCUUGCA
1902-1922
A-
2174
UUAACATGCAAGAG
1900-1922


1565666.1
2894384.1

UGUUAA

2894385.1

CAAUGGUUU






AD-
A-
1731
CCAUUGCUCUUGCA
1903-1923
A-
2175
UGUAACAUGCAAGA
1901-1923


1565667.1
2894386.1

UGUUACA

2894387.1

GCAAUGGUU






AD-
A-
1732
CAUUGCUCUUGCAU
1904-1924
A-
2176
UUGUAACAUGCAAG
1902-1924


1565785.1
2894388.1

GUUACAA

2894570.1

AGCAAUGGU






AD-
A-
1733
CAUUGCUCUUGCAU
1904-1924
A-
2177
UUGTAACAUGCAAG
1902-1924


1565668.1
2894388.1

GUUACAA

2894389.1

AGCAAUGGU






AD-
A-
1734
AUUGCUCUUGCAUG
1905-1925
A-
2178
UAUGTAACAUGCAA
1903-1925


1565669.1
2894390.1

UUACAUA

2894391.1

GAGCAAUGG






AD-
A-
1735
UUGCUCUUGCAUGU
1906-1926
A-
2179
UCAUGUAACAUGCA
1904-1926


1565670.1
2894392.1

UACAUGA

2894393.1

AGAGCAAUG






AD-
A-
1736
UUGCUCUUGCAUGU
1906-1926
A-
2180
UCAUGUAACAUGCA
1904-1926


1565860.1
2894685.1

UACAUGA

1804746.1

AGAGCAAUG






AD-
A-
1737
UUGCACUUGCAUGU
1906-1926
A-
2181
UCAUGUAACAUGCA
1904-1926


1565861.1
2894686.1

UACAUGA

2894687.1

AGUGCAAUG






AD-
A-
1738
UUGGUCUUGCAUGU
1906-1926
A-
2182
UCAUGUAACAUGCA
1904-1926


1565862.1
2894688.1

UACAUGA

2894689.1

AGACCAAUG






AD-
A-
1739
UUCCUCUUGCAUGU
1906-1926
A-
2183
UCAUGUAACAUGCA
1904-1926


1565863.1
2894690.1

UACAUGA

2894691.1

AGAGGAAUG






AD-
A-
1740
UUGCUCUUGCAUGU
1906-1926
A-
2184
UCAUGUAACAUGCA
1904-1926


1565864.1
2894692.1

UACAUGA

2894393.1

AGAGCAAUG






AD-
A-
1741
UUGCUCUUGCAUGU
1906-1926
A-
2185
UCAUGUAACAUGCA
1904-1926


1565866.1
2894685.1

UACAUGA

2894695.1

AGAGCAAUG






AD-
A-
1742
UUGCUCUUGCAUGU
1906-1926
A-
2186
UCAUGUAACAUGCA
1904-1926


1565670.2
2894392.1

UACAUGA

2894393.1

AGAGCAAUG






AD-
A-
1743
UGCUCUUGCAUGUU
1907-1927
A-
2187
UCCATGTAACAUGC
1905-1927


1565671.1
2894394.1

ACAUGGA

2894395.1

AAGAGCAAU






AD-
A-
1744
GCUCUUGCAUGUUA
1908-1926
A-
2188
UCAUGUAACAUGCA
1906-1926


1565865.1
2894693.1

CAUGA

2894694.1

AGAGCGG






AD-
A-
1745
GCUCUUGCAUGUUA
1908-1928
A-
2189
UACCAUGUAACAUG
1906-1928


1565786.1
2894571.1

CAUGGUA

2894572.1

CAAGAGCAA






AD-
A-
1746
CUCUUGCAUGUUAC
1909-1929
A-
2190
UAACCATGUAACAU
1907-1929


1565672.1
2894396.1

AUGGUUA

2894397.1

GCAAGAGCA






AD-
A-
1747
CUCUUGCAUGUUAC
1909-1929
A-
2191
UAACCATGUAACAU
1907-1929


1565673.1
2894396.1

AUGGUUA

2894398.1

GCAAGAGCA






AD-
A-
1748
UCUUGCAUGUUACA
1910-1930
A-
2192
UUAACCAUGUAACA
1908-1930


1565674.1
2894399.1

UGGUUAA

2894400.1

UGCAAGAGC






AD-
A-
1749
CUUGCAUGUUACAU
1911-1931
A-
2193
UGUAACCAUGUAAC
1909-1931


1565787.1
1577564.1

GGUUACA

2894573.1

AUGCAAGAG






AD-
A-
1750
CUUGCAUGUUACAU
1911-1931
A-
2194
UGUAACCAUGUAAC
1909-1931


1565675.1
1577564.1

GGUUACA

2894401.1

AUGCAAGAG






AD-
A-
1751
UUGCAUGUUACAUG
1912-1932
A-
2195
UGGUAACCAUGUAA
1910-1932


1565788.1
1577566.1

GUUACCA

2894574.1

CAUGCAAGA






AD-
A-
1752
UUGCAUGUUACAUG
1912-1932
A-
2196
UGGTAACCAUGUAA
1910-1932


1565676.1
1577566.1

GUUACCA

2894402.1

CAUGCAAGA






AD-
A-
1753
UUGCAUGUUACAUG
1912-1932
A-
2197
UGGUAACCAUGTAA
1910-1932


1565677.1
2894403.1

GUUACCA

2894404.1

CAUGCAAGA






AD-
A-
1754
UGCAUGUUACAUGG
1913-1933
A-
2198
UUGGTAACCAUGUA
1911-1933


1565678.1
2894405.1

UUACCAA

2894406.1

ACAUGCAAG






AD-
A-
1755
GCAUGUUACAUGGU
1914-1934
A-
2199
UGUGGUAACCAUG
1912-1934


1565679.1
1577580.1

UACCACA

2894407.1

UAACAUGCAA






AD-
A-
1756
GCAUGUUACAUGGU
1914-1934
A-
2200
UGUGGUAACCATGU
1912-1934


1565680.1
2894408.1

UACCACA

2894409.1

AACAUGCAA






AD-
A-
1757
CAUGUUACAUGGUU
1915-1935
A-
2201
UUGUGGTAACCAUG
1913-1935


1565681.1
2894410.1

ACCACAA

2894411.1

UAACAUGCA






AD-
A-
1758
AUGUUACAUGGUUA
1916-1936
A-
2.202
UUUGTGGUAACCAU
1914-1936


1565789.1
2894575.1

CCACAAA

2894576.1

GUAACAUGC






AD-
A-
1759
UGUUACAUGGUUAC
1917-1937
A-
2203
UCUUGUGGUAACC
1915-1937


1565790.1
2894577.1

CACAAGA

2894578.1

AUGUAACAUG






AD-
A-
1760
CUCCUCUGGCCAGCA
1976-1996
A-
2204
UTUCGATGCUGGCC
1974-1996


1565682.1
2894412.1

UCGAAA

2894413.1

AGAGGAGCU






AD-
A-
1761
AUAUAAGUAAGAUG
1995-2015
A-
2205
UUAAAUGCAUCUU
1993-2015


1565791.1
2894579.1

CAUUUAA

2894580.1

ACUUAUAUUC






AD-
A-
1762
UAUAAGUAAGAUGC
1996-2016
A-
2206
UGUAAATGCAUCUU
1994-2016


1565683.1
2894414.1

AUUUACA

2894415.1

ACUUAUAUU






AD-
A-
1763
AUAAGUAAGAUGCA
1997-2017
A-
2207
UAGUAAAUGCATCU
1995-2017


1565684.1
2894416.1

UUUACUA

2894417.1

UACUUAUAU






AD-
A-
1764
UAAGUAAGAUGCAU
1998-2018
A-
2208
UTAGTAAAUGCAUC
1996-2018


1565685.1
2894418.1

UUACUAA

2894419.1

UUACUUAUA






AD-
A-
1765
AAGUAAGAUGCAUU
1999-2019
A-
2209
UGUAGUAAAUGCA
1997-2019


1565867.1
2894420.1

UACUACA

1804926.1

UCUUACUUAU






AD-
A-
1766
AAGUAAGAUGCAUU
1999-2019
A-
2.210
UGUAGUAAAUGCA
1997-2019


1565868.1
2894420.1

UACUACA

2894696.1

UCUUACUUGU






AD-
A-
1767
AAGUAAGAUGCAUU
1999-2019
A-
2211
UGUAGUAAAUGCA
1997-2019


1565869.1
2894420.1

UACUACA

2894697.1

UCUUACUUGU






AD-
A-
1768
AAGUAAGAUGCAUU
1999-2019
A-
2212
UGUAGUAAAUGCA
1997-2019


1565870.1
2894420.1

UACUACA

2894698.1

UCUUACUUGU






AD-
A-
1769
AAGUAAGAUGCAUU
1999-2019
A-
2213
UGUAGUAAAUGCA
1997-2019


1565871.1
2894422.1

UACUACA

2894697.1

UCUUACUUGU






AD-
A-
1770
AAGUAAGATGCAUU
1999-2019
A-
2214
UGUAGUAAAUGCA
1997-2019


1565872.1
2894699.1

UACUACA

2894700.1

UCUUACUUGU






AD-
A-
1771
AAGUAAGAUGCAUU
1999-2019
A-
2215
UGUAGUAAAUGCA
1997-2019


1565873.1
2894422.1

UACUACA

2894700.1

UCUUACUUGU






AD-
A-
1772
AAGUUAGAUGCAUU
1999-2019
A-
2216
UGUAGUAAAUGCA
1997-2019


1565874.1
2894701.1

UACUACA

2894702.1

UCUAACUUGU






AD-
A-
1773
AAGAAAGAUGCAUU
1999-2019
A-
2217
UGUAGUAAAUGCA
1997-2019


1565875.1
2894703.1

UACUACA

2894704.1

UCUUUCUUGU






AD-
A-
1774
AACUAAGAUGCAUU
1999-2019
A-
2218
UGUAGUAAAUGCA
1997-2019


1565876.1
2894705.1

UACUACA

2894706.1

UCUUAGUUGU






AD-
A-
1775
AAGUAAGAUGCAUU
1999-2019
A-
2219
UGUAGUAAAUGCA
1997-2019


1565686.1
2894420.1

UACUACA

2894421.1

UCUUACUUAU






AD-
A-
1776
AAGUAAGAUGCAUU
1999-2019
A-
2220
UGUAGUAAAUGCA
1997-2019


1565687.1
2894422.1

UACUACA

2894423.1

UCUUACUUAU






AD-
A-
1777
AGUAAGAUGCAUUU
2000-2020
A-
2221
UUGUAGTAAAUGCA
1998-2020


1565688.1
2894424.1

ACUACAA

2894425.1

UCUUACUUA






AD-
A-
1778
GUAAGAUGCAUUUA
2001-2019
A-
2222
UGUAGUAAAUGCA
1999-2019


1565877.1
2894707.1

CUACA

2894708.1

UCUUACUU






AD-
A-
1779
UUGGCUUCUAAUGC
2021-2041
A-
2.223
UUCUGAAGCAUUA
2019-2041


1565689.1
2894426.1

UUCAGAA

2894427.1

GAAGCCAACU






AD-
A-
1780
CUUCUAAUGCUUCA
2025-2045
A-
2224
UUCUAUCUGAAGCA
2023-2045


1565792.1
2894428.1

GAUAGAA

2894581.1

UUAGAAGCC






AD-
A-
1781
CUUCUAAUGCUUCA
2025-2045
A-
2225
UUCTATCUGAAGCA
2023-2045


1565690.1
2894428.1

GAUAGAA

2894429.1

UUAGAAGCC






AD-
A-
1782
CUUCUAAUGCUUCA
2025-2045
A-
2226
UUCUATCTGAAGCA
2023-2045


1565691.1
2894428.1

GAUAGAA

2894430.1

UUAGAAGCC









Example 2. In Vitro Screening of MYOC siRNA
Experimental Methods
Cell Culture and Transfections:
Human Trabecular Meshwork Cells (HTMC) Cell Transfections

HTMC cells (ATCC) were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl of RNAiMAX per well (Invitrogen, Carlsbad CA. cat #13778-150) to 5 μl of siRNA duplexes per well, with 4 replicates of each siRNA duplex, into a 384-well plate, and incubated at room temperature for 15 minutes. Forty μl of DMEM:F12 Medium (ThermoFisher) containing ˜5×103 cells were then added to the siRNA-transfection mixture. Cells were incubated for 24 hours prior to RNA purification. Experiments were performed at 10 nM, 1 nM, and 0.1 nM.


Total RNA Isolation Using DYNABEADS mRNA Isolation Kit:


RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 70 μl of Lysis/Binding Buffer and 10 μl of lysis buffer containing 3 μl of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 150 μl Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150 μl Elution Buffer, re-captured and supernatant removed.


cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat #4368813):


Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25× dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H2O per reaction was added to RNA isolated above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 h 37° C.


Real Time PCR:

Two μl of cDNA and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) were added to either 0.5 μl of Human GAPDH TaqMan Probe (4326317E) and 0.5 μl MYOC Human probe per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche). Each duplex was tested at least two times and data were normalized to cells transfected with a non-targeting control siRNA. To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with a non-targeting control siRNA.


Results

The results of the multi-dose screen in human trabecular meshwork cells (HTMC) with exemplary human MYOC siRNAs are shown in Table 3 (correspond to siRNAs in Table 2A), The multi-dose experiments were performed at 10 nM, 1 nM, and 0.1 nM final duplex concentrations and the data are expressed as percent message remaining relative to non-targeting control.


Of the exemplary siRNA duplexes evaluated in Table 3 below, 174 achieved a knockdown of MYOC of 2:90%, 347 achieved a knockdown of MYOC of ≥70%, 392 achieved a knockdown of MYOC of ≥50%, 424 achieved a knockdown of MYOC of ≥20%, and 433 achieved a knockdown of MYOC of ≥10% in HTMC cells when administered at the 10 nM concentration.


Of the exemplary siRNA duplexes evaluated in Table 3 below, 142 achieved a knockdown of MYOC of ≥90%, 341 achieved a knockdown of MYOC of ≥70%, 391 achieved a knockdown of MYOC of ≥50%, 424 achieved a knockdown of MYOC of ≥20%, and 431 achieved a knockdown of MYOC of ≥10% in HTMC cells when administered at the 1 nM concentration.


Of the exemplary siRNA duplexes evaluated in Table 3 below, 57 achieved a knockdown of MYOC of ≥90%, 277 achieved a knockdown of MYOC of ≥70%, 361 achieved a knockdown of MYOC of ≥50%, 416 achieved a knockdown of MYOC of ≥20%, and 425 achieved a knockdown of MYOC of ≥10% in HTMC cells when administered at the 0.1 nM concentration.


Of the exemplary siRNA duplexes evaluated in Table 3 below, AD-1565448.1, AD-1193175.5, AD-1565798.1, AD-1565452.1, AD-1565453.1, AD-1565454.1, AD-1565456.1, AD-1565492.1, AD-1565493.1, AD-1565503.1, AD-1073418.5, AD-1565804.1, AD-1565806.1, AD-1244366.3, AD-1565589.1, AD-1565837.1, AD-1565624.1, AD-1565626.1 showed superior knockdown of MYOC in HTMC cells.









TABLE 3







MYOC endogenous in vitro multi-dose screen with one set of exemplary


human MYOC siRNAs (*the number following the decimal point in


a duplex name merely refers to a batch production number)











10 nM
1 nM
0.1 nM














% message

% message

% message



Duplex Name*
remaining
St. Dev.
remaining
St. Dev.
remaining
St. Dev.
















AD-1565444.1
13.58
1.84
14.71
7.84
26.73
7.48


AD-1565445.1
10.87
6.23
13.16
4.98
25.60
5.38


AD-1565692.1
16.41
2.08
18.61
0.75
25.71
4.17


AD-1565446.1
14.37
7.51
6.94
1.71
14.17
3.72


AD-1565447.1
10.38
3.76
10.19
3.32
23.50
6.92


AD-1565448.1
9.24
5.79
4.13
2.66
5.97
2.52


AD-1565693.1
3.99
1.51
6.10
1.34
12.35
2.47


AD-1565449.1
7.20
2.64
3.83
1.28
7.08
3.96


AD-1565450.1
8.78
4.45
6.72
1.51
12.08
2.48


AD-1565694.1
4.18
2.71
9.79
5.26
8.35
1.68


AD-1565695.1
7.35
3.15
6.28
1.55
9.28
3.23


AD-1193175.5
2.63
0.72
2.84
1.11
9.95
4.58


AD-1565793.1
5.77
0.80
2.63
0.70
9.78
3.84


AD-1565795.1
3.59
2.06
6.15
1.52
8.48
3.62


AD-1565796.1
1.04
0.24
3.44
1.14
5.75
1.09


AD-1565797.1
2.72
0.83
3.58
2.60
8.08
3.77


AD-1565798.1
3.95
2.69
2.43
1.56
3.53
1.68


AD-1565799.1
4.64
3.39
3.82
0.78
4.90
2.65


AD-1565451.1
5.31
1.89
4.64
1.66
10.31
5.68


AD-1565452.1
6.08
2.03
7.09
1.62
7.98
5.26


AD-1565696.1
5.58
2.54
9.52
3.27
8.31
3.16


AD-1565794.1
6.22
1.99
4.53
2.58
6.90
3.64


AD-1565453.1
6.30
1.27
8.30
1.28
10.41
6.07


AD-1565454.1
19.20
4.92
6.07
1.17
9.34
1.31


AD-1565455.1
16.24
3.57
6.52
3.62
13.11
3.35


AD-1565456.1
10.95
4.22
4.41
1.79
9.13
3.28


AD-1565457.1
16.55
3.00
5.13
2.63
13.24
6.40


AD-1565697.1
6.35
1.94
9.23
3.77
10.72
0.63


AD-1565698.1
5.42
2.65
12.12
1.42
6.99
1.98


AD-1565458.1
11.01
3.72
4.33
2.88
11.84
3.86


AD-1565459.1
42.85
5.80
36.24
26.02
45.00
15.33


AD-1565699.1
8.28
2.74
10.34
4.26
18.07
7.91


AD-1565700.1
7.73
4.21
11.72
4.13
9.75
1.96


AD-1565460.1
8.30
1.22
8.69
2.89
12.07
3.01


AD-1565461.1
14.00
3.46
5.33
2.92
16.28
3.37


AD-1565462.1
21.75
3.70
20.28
2.83
31.62
4.90


AD-1565701.1
8.93
5.10
9.40
3.76
13.18
1.17


AD-1565463.1
71.43
19.78
76.54
8.03
82.89
9.94


AD-1565464.1
16.39
4.06
10.09
2.80
31.39
3.54


AD-1565465.1
20.94
5.18
12.50
2.79
45.66
3.05


AD-1565466.1
82.31
6.76
75.73
28.49
92.65
18.60


AD-1565467.1
102.55
23.01
84.98
22.44
116.91
27.98


AD-1565702.1
116.28
14.85
85.92
14.00
76.00
18.38


AD-1565468.1
121.69
34.80
86.60
5.71
79.45
23.26


AD-1565469.1
46.82
18.05
34.92
10.42
53.10
9.35


AD-1565703.1
40.06
8.59
41.08
11.27
50.91
16.81


AD-1565704.1
23.10
2.72
32.11
12.42
49.32
8.69


AD-1565705.1
20.82
4.90
14.51
3.79
54.80
11.30


AD-1565470.1
58.45
4.61
77.51
21.21
78.93
12.11


AD-1565471.1
8.36
2.25
14.98
3.23
29.42
6.67


AD-1565472.1
67.13
22.48
107.96
18.35
81.66
15.72


AD-1565473.1
17.14
6.70
12.35
2.42
52.91
13.13


AD-1565706.1
12.69
1.44
17.98
1.45
33.72
10.29


AD-1565474.1
7.35
1.92
11.57
0.74
12.54
0.65


AD-1565707.1
14.28
5.67
12.45
3.33
16.18
4.97


AD-1565475.1
104.91
17.03
92.52
25.04
68.96
7.03


AD-1565476.1
13.25
5.61
10.71
3.39
13.33
2.98


AD-1565477.1
20.62
5.16
21.71
6.04
39.44
2.72


AD-1565708.1
20.35
7.28
34.51
0.60
46.23
16.06


AD-1565478.1
113.51
34.51
84.19
13.48
93.61
8.41


AD-1565479.1
120.66
38.76
113.47
21.10
107.18
23.97


AD-1565709.1
96.03
16.16
82.56
20.98
34.55
10.86


AD-1565480.1
9.45
3.51
15.47
2.78
25.03
4.12


AD-1565481.1
11.63
2.84
8.17
1.49
24.18
8.77


AD-1565482.1
55.07
21.96
62.38
10.34
76.05
18.42


AD-1565483.1
42.47
1.01
32.88
6.07
40.92
10.70


AD-1565484.1
41.71
8.56
35.08
6.28
47.77
12.25


AD-1565710.1
14.95
7.13
25.67
5.90
22.93
2.01


AD-1565711.1
23.82
14.98
23.65
4.48
19.78
3.54


AD-1565485.1
25.24
2.83
19.83
6.64
25.74
2.78


AD-1565486.1
10.05
1.20
14.31
1.72
31.42
7.60


AD-1565712.1
5.20
2.02
19.24
6.00
20.42
4.82


AD-1565713.1
9.27
2.63
19.11
2.21
25.72
3.81


AD-1565487.1
46.68
5.49
28.42
4.77
77.47
6.44


AD-1565714.1
5.67
1.97
5.35
2.39
11.46
2.28


AD-1565488.1
9.36
2.02
11.25
2.36
20.20
1.78


AD-1565715.1
3.35
1.88
5.60
2.17
18.53
6.10


AD-1565716.1
3.87
1.95
8.41
5.17
10.69
3.07


AD-1565489.1
9.86
1.57
14.64
3.55
19.00
5.75


AD-1565717.1
3.41
0.66
2.37
0.59
12.97
3.81


AD-1565718.1
2.99
0.86
3.53
2.40
8.04
3.91


AD-1565490.1
24.04
8.60
11.67
1.80
31.96
10.18


AD-1565719.1
6.39
5.62
2.51
0.45
8.90
2.14


AD-1565491.1
88.01
24.84
75.92
22.03
84.59
11.40


AD-1565720.1
8.86
1.70
4.23
2.27
13.61
2.34


AD-1565721.1
2.67
0.90
4.76
0.47
12.89
4.11


AD-1565492.1
9.24
3.51
4.60
2.16
9.33
1.80


AD-1565493.1
5.01
2.24
6.54
2.52
25.69
11.06


AD-1565722.1
16.33
5.69
19.52
3.24
66.71
10.30


AD-1565723.1
4.09
1.77
10.11
5.93
10.33
2.92


AD-1565494.1
40.03
5.07
32.09
5.50
64.47
10.72


AD-1565495.1
9.19
5.03
12.26
3.30
29.55
9.26


AD-1565724.1
3.97
1.66
3.08
1.67
5.45
1.50


AD-1565496.1
10.35
2.39
20.15
3.54
23.78
8.91


AD-1565725.1
10.58
5.58
4.90
2.19
16.16
7.18


AD-1565497.1
12.46
5.89
10.46
3.87
16.17
5.75


AD-1565498.1
12.18
4.54
8.54
1.66
12.85
5.36


AD-1565499.1
28.70
6.47
25.63
9.41
33.18
6.95


AD-1565500.1
5.40
1.94
4.31
1.08
7.40
2.30


AD-1565726.1
4.06
1.69
2.85
0.29
9.86
5.60


AD-1565501.1
38.18
13.02
39.37
15.62
49.41
13.10


AD-1565502.1
10.38
6.35
10.28
5.46
32.08
11.21


AD-1565503.1
5.08
2.84
7.82
2.01
7.77
0.71


AD-1565801.1
18.47
3.93
10.99
7.48
22.01
4.67


AD-1565802.1
3.76
1.21
4.21
1.61
7.43
1.97


AD-1565803.1
7.26
4.22
7.65
1.94
12.10
3.11


AD-1565804.1
1.84
0.46
1.46
0.81
4.34
0.27


AD-1565805.1
1.81
1.74
1.62
1.12
7.02
3.33


AD-1073418.5
1.66
0.30
2.82
1.23
7.78
4.94


AD-1565504.1
3.08
1.15
3.44
1.47
7.77
2.53


AD-1565504.2
5.57
0.51
6.32
3.44
12.16
4.91


AD-1565505.1
6.00
3.10
9.11
0.70
19.63
5.31


AD-1565800.1
2.31
0.80
3.23
1.74
8.90
5.48


AD-1565506.1
23.85
10.31
28.07
3.84
54.83
10.10


AD-1565727.1
3.70
2.94
4.10
2.15
6.37
2.91


AD-1565507.1
21.31
7.92
27.51
3.72
45.97
12.71


AD-1565728.1
5.43
1.60
3.89
1.30
13.39
1.84


AD-1565508.1
16.45
8.67
8.99
1.68
34.15
11.20


AD-1565509.1
7.04
2.82
6.61
2.08
12.83
2.54


AD-1565730.1
3.38
1.27
6.47
1.28
12.36
3.43


AD-1565510.1
14.18
2.68
24.57
2.78
27.00
1.39


AD-1565511.1
50.88
7.77
59.88
11.33
85.05
19.40


AD-1565512.1
5.77
0.64
16.79
8.26
18.40
4.16


AD-1565731.1
5.79
1.45
4.79
1.98
9.21
1.74


AD-1565513.1
7.85
3.02
8.39
4.73
15.10
2.47


AD-1565514.1
8.18
4.29
8.45
1.99
14.87
2.07


AD-1565732.1
10.04
3.21
20.83
10.19
28.95
5.75


AD-1565515.1
31.33
13.22
25.47
9.82
66.58
12.24


AD-1565516.1
32.42
6.12
22.34
7.88
52.38
10.81


AD-1565733.1
10.20
4.94
7.05
2.67
29.37
6.82


AD-1565517.1
89.04
28.81
134.10
30.29
77.81
27.77


AD-1565734.1
8.69
5.99
8.33
2.02
28.19
7.79


AD-1565518.1
113.31
25.68
99.25
17.54
105.41
19.72


AD-1565735.1
8.25
2.86
7.32
2.99
17.49
2.04


AD-1565736.1
89.87
11.16
82.82
8.36
64.20
3.72


AD-1565519.1
33.76
6.02
27.44
10.42
58.44
19.24


AD-1565520.1
9.40
1.00
20.63
6.01
29.93
7.27


AD-1565521.1
16.54
5.72
10.87
2.71
24.04
8.83


AD-1244360.3
0.93
0.59
2.05
1.46
6.06
1.47


AD-1565522.1
2.11
0.17
2.21
0.63
10.71
6.20


AD-1565806.1
2.01
1.23
1.99
0.78
6.54
4.21


AD-1565807.1
1.14
0.28
2.06
0.28
9.34
3.20


AD-1565808.1
6.46
2.75
13.23
2.10
30.00
3.49


AD-1565809.1
31.44
9.01
31.65
16.95
61.12
8.34


AD-1565810.1
73.75
16.63
55.04
14.78
90.94
25.33


AD-1565812.1
4.64
0.12
8.16
2.01
16.45
9.69


AD-1565813.1
3.88
2.31
5.72
3.22
12.47
5.05


AD-1565814.1
3.74
1.35
2.28
1.09
11.58
10.04


AD-1565522.2
3.48
2.31
5.47
1.39
8.65
3.67


AD-1565523.1
8.60
5.54
16.19
3.18
24.63
10.25


AD-1565811.1
1.86
0.79
3.34
1.00
6.85
1.49


AD-1565524.1
72.95
22.18
71.70
9.91
94.59
16.44


AD-1565525.1
6.68
1.44
29.00
11.53
32.55
7.03


AD-1565526.1
64.82
8.95
81.81
23.93
94.24
1.92


AD-1565737.1
4.73
1.10
5.96
1.36
27.49
7.25


AD-1565527.1
35.16
10.68
57.77
13.50
44.78
5.90


AD-1565738.1
33.81
11.97
37.71
4.91
40.38
9.49


AD-1565528.1
4.57
0.93
13.08
6.54
16.65
3.80


AD-1565739.1
13.69
2.79
7.63
0.94
17.67
1.57


AD-1565529.1
12.48
6.69
12.41
3.01
24.60
4.68


AD-1565740.1
46.59
11.69
24.59
13.81
29.99
8.28


AD-1565530.1
3.85
1.11
16.59
6.50
24.15
14.29


AD-1565741.1
4.79
2.45
5.19
2.76
11.67
1.29


AD-1565531.1
2.88
0.62
8.86
2.91
29.15
16.15


AD-1565532.1
47.20
12.54
54.02
12.23
60.18
10.88


AD-1565533.1
52.58
14.14
52.92
6.95
68.08
20.69


AD-1565534.1
16.47
2.43
36.07
8.10
38.80
1.21


AD-1565535.1
19.47
1.42
18.84
0.59
88.34
12.82


AD-1565536.1
5.75
2.08
26.98
12.23
32.41
10.44


AD-1565537.1
2.45
1.00
14.01
3.91
15.90
7.45


AD-1565742.1
8.77
3.85
14.00
8.58
28.85
4.45


AD-1565538.1
41.11
17.44
17.75
3.59
36.81
13.99


AD-1565539.1
6.54
1.80
23.36
8.41
35.51
12.28


AD-1565540.1
10.90
3.12
33.16
11.52
49.84
15.31


AD-1565743.1
78.79
17.64
56.91
8.13
67.87
7.64


AD-1565541.1
89.10
26.43
118.75
23.82
89.25
13.47


AD-1565542.1
38.04
7.88
75.90
21.39
33.19
9.91


AD-1565543.1
30.20
9.87
29.14
4.70
41.52
4.90


AD-1565544.1
9.42
2.27
28.40
7.23
39.24
12.42


AD-1565744.1
24.73
6.76
22.51
8.57
25.91
5.69


AD-1565545.1
47.11
6.45
68.77
12.27
59.03
19.68


AD-1565745.1
6.33
1.91
4.99
2.25
10.10
3.22


AD-1565546.1
19.20
3.77
28.33
6.33
52.11
19.47


AD-1565547.1
5.54
1.02
15.31
5.28
33.54
9.36


AD-1565548.1
8.24
3.75
14.70
2.67
40.16
5.50


AD-1565746.1
8.80
2.96
7.57
2.68
13.62
1.74


AD-1565549.1
5.83
2.43
10.39
0.49
20.05
4.04


AD-1565550.1
20.30
6.68
20.60
4.03
50.94
7.85


AD-1565551.1
5.55
2.48
14.71
3.54
40.87
9.99


AD-1244365.3
2.81
0.70
6.43
2.73
14.36
7.50


AD-1565552.1
4.23
2.73
4.99
1.74
23.97
7.92


AD-1565553.1
3.13
1.08
4.01
2.83
14.81
3.86


AD-1565815.1
20.58
6.87
17.87
5.67
55.24
13.57


AD-1565816.1
9.35
5.93
6.40
1.94
17.06
5.64


AD-1565818.1
71.18
34.11
91.54
39.75
102.07
7.11


AD-1565819.1
8.53
2.68
16.42
9.17
37.73
10.42


AD-1565820.1
4.30
1.60
16.39
15.44
31.97
6.06


AD-1565552.2
8.71
4.40
17.83
6.52
34.81
10.71


AD-1565553.2
9.88
2.16
15.76
3.45
18.52
1.32


AD-1565554.1
86.10
20.06
92.44
20.16
90.05
11.05


AD-1565817.1
8.82
3.58
9.94
1.98
27.04
8.06


AD-1565555.1
60.03
7.25
79.65
1.93
94.66
18.30


AD-1565747.1
3.71
0.52
4.30
0.82
5.49
1.97


AD-1565556.1
6.54
1.27
18.17
7.56
24.57
9.17


AD-1565557.1
6.55
0.71
11.80
1.61
14.15
4.83


AD-1565558.1
34.90
6.82
45.74
16.31
51.71
5.43


AD-1565559.1
7.56
3.35
16.90
6.13
35.70
6.70


AD-1565560.1
12.01
4.77
20.12
8.08
42.42
14.79


AD-1565561.1
14.87
2.55
41.04
16.82
56.34
19.15


AD-1565562.1
31.27
1.55
39.84
11.11
68.61
16.49


AD-1565748.1
4.73
2.30
7.31
2.75
6.16
3.13


AD-1565563.1
11.56
3.31
14.52
3.50
32.01
6.14


AD-1565564.1
45.30
5.78
31.11
13.84
62.81
24.12


AD-1565565.1
7.88
3.48
17.14
3.33
37.36
12.76


AD-1565566.1
8.76
0.08
25.95
2.71
16.79
4.03


AD-1565567.1
36.72
12.97
31.96
7.23
27.27
12.04


AD-1565568.1
12.21
5.78
15.35
4.76
17.90
8.52


AD-1565569.1
10.87
4.71
14.51
4.01
25.63
7.40


AD-1565749.1
5.31
1.88
6.58
1.58
14.50
3.78


AD-1565570.1
9.58
4.30
9.96
2.16
23.30
4.89


AD-1565571.1
8.78
4.04
10.58
1.49
31.02
14.75


AD-1565750.1
6.27
1.49
5.63
0.86
9.31
2.00


AD-1565573.1
13.43
7.22
17.01
1.73
32.17
10.08


AD-1565751.1
13.71
7.32
10.60
1.99
26.81
4.95


AD-1565574.1
58.13
13.35
39.02
9.73
100.79
15.98


AD-1565575.1
67.71
7.53
62.51
3.58
86.20
24.09


AD-1565576.1
10.63
2.87
18.40
9.43
34.88
5.78


AD-1565752.1
6.45
2.12
7.36
3.77
9.02
2.33


AD-1565577.1
18.86
4.83
15.88
7.49
44.31
13.98


AD-1565578.1
10.01
3.05
15.75
5.12
21.61
4.88


AD-1565579.1
36.46
2.52
13.87
3.07
22.87
6.37


AD-1565753.1
4.80
2.52
4.20
0.60
5.88
2.28


AD-1565580.1
50.99
16.10
54.79
19.39
47.79
8.33


AD-1565754.1
5.40
1.31
5.57
1.86
20.02
4.99


AD-1565581.1
76.36
0.65
50.01
18.97
112.59
14.54


AD-1565582.1
68.84
13.42
59.94
12.89
54.61
10.15


AD-1565755.1
14.04
3.54
17.44
4.58
30.93
9.58


AD-1565583.1
75.79
12.48
58.61
13.48
91.02
22.93


AD-1565584.1
16.03
6.74
31.39
5.39
53.28
7.92


AD-1565756.1
10.12
1.85
13.28
1.55
18.52
5.93


AD-1565585.1
65.26
7.83
76.22
19.35
138.12
8.35


AD-1073420.3
4.68
3.06
8.04
5.93
14.60
5.21


AD-1244366.3
2.80
0.55
2.75
1.99
8.93
3.10


AD-1565757.1
2.58
1.56
3.05
0.76
9.14
2.74


AD-1565821.1
5.45
1.74
4.48
0.95
18.08
6.93


AD-1565822.1
2.64
1.05
3.40
1.03
11.58
3.46


AD-1565824.1
10.07
4.80
5.40
1.41
14.69
3.10


AD-1565825.1
3.43
0.99
4.41
0.69
14.18
3.87


AD-1565826.1
2.60
0.31
5.27
2.04
11.44
3.39


AD-1565757.2
5.70
4.74
3.31
1.11
6.26
1.50


AD-1565586.1
10.54
3.26
9.72
4.39
20.36
3.90


AD-1565587.1
10.26
1.67
23.30
4.07
23.21
6.92


AD-1565588.1
5.50
3.07
8.28
3.79
14.45
5.15


AD-1565823.1
2.90
1.50
5.22
2.56
12.75
5.20


AD-1565589.1
7.82
4.23
10.17
3.33
14.98
7.64


AD-1565590.1
17.34
3.89
25.42
6.05
39.12
6.12


AD-1565591.1
9.81
6.12
16.24
6.75
14.32
3.13


AD-1565758.1
5.05
1.81
6.30
2.45
9.65
2.97


AD-1565592.1
10.85
4.12
20.95
5.38
22.85
8.66


AD-1565594.1
71.45
12.79
106.92
35.47
73.36
15.85


AD-1565759.1
8.02
3.35
10.76
2.33
13.69
2.12


AD-1565595.1
13.55
3.92
40.56
9.53
66.88
17.43


AD-1565760.1
7.84
3.28
13.72
4.96
27.50
7.10


AD-1565761.1
5.55
2.42
6.63
1.87
16.59
1.03


AD-1565596.1
60.69
7.73
48.61
17.04
79.55
24.61


AD-1565597.1
17.28
4.78
27.20
12.46
18.26
5.10


AD-1565762.1
13.16
1.54
10.59
2.12
13.86
3.34


AD-1565598.1
59.38
11.46
56.84
17.50
52.34
9.18


AD-1565599.1
79.70
12.67
150.03
21.77
122.89
36.87


AD-1565600.1
82.87
13.09
57.05
6.94
88.22
21.48


AD-1565601.1
25.18
2.27
22.89
9.55
44.49
5.77


AD-1565602.1
3.97
1.58
6.23
0.60
30.83
8.44


AD-1565827.1
3.03
1.64
4.22
2.36
11.52
3.63


AD-1565828.1
3.65
0.82
2.60
0.33
21.71
9.56


AD-1565829.1
3.60
2.48
2.89
0.44
9.09
3.82


AD-1565830.1
2.98
1.54
5.47
1.73
9.98
5.35


AD-1565831.1
4.47
1.82
7.16
2.87
14.17
5.18


AD-1565833.1
49.82
24.88
57.44
5.52
82.58
21.84


AD-1565834.1
44.76
9.00
21.17
7.13
49.24
18.58


AD-1565835.1
21.43
3.51
28.41
7.50
53.98
13.43


AD-1565602.2
8.60
3.72
16.07
8.09
21.32
2.93


AD-1565603.1
15.64
6.79
20.38
8.40
15.80
3.00


AD-1565832.1
2.84
0.85
4.48
1.91
9.88
4.28


AD-1565763.1
31.78
11.13
55.15
17.04
43.31
1.78


AD-1565604.1
74.65
13.66
64.27
3.82
76.86
24.52


AD-1565605.1
114.63
37.42
90.22
23.73
51.30
6.16


AD-1565606.1
10.93
4.65
19.08
6.65
16.70
4.41


AD-1565764.1
4.24
0.65
4.49
2.04
5.61
2.08


AD-1565607.1
27.14
6.06
36.08
7.73
64.66
4.95


AD-1565608.1
14.35
6.24
31.28
7.21
44.69
9.15


AD-1565609.1
9.04
3.66
20.95
7.58
25.35
5.78


AD-1565766.1
7.96
1.91
9.92
1.72
19.25
1.48


AD-1565611.1
15.58
4.66
25.13
8.99
27.02
3.88


AD-1565767.1
7.79
5.06
13.20
3.78
14.68
3.73


AD-1565612.1
27.72
4.59
35.45
10.64
49.10
6.99


AD-1565613.1
32.54
7.98
63.94
9.99
64.04
15.15


AD-1565614.1
14.27
6.39
25.57
1.52
43.23
30.00


AD-1565768.1
4.44
1.76
5.23
1.23
14.17
5.16


AD-1565615.1
55.18
8.55
79.04
23.74
78.22
6.99


AD-1565616.1
15.40
4.47
35.68
7.48
51.99
10.44


AD-1565617.1
3.84
2.17
11.03
1.79
34.82
1.45


AD-1565769.1
5.91
3.78
3.71
0.60
6.10
2.19


AD-1565618.1
7.81
3.67
7.98
3.83
22.24
8.77


AD-1565770.1
17.14
1.76
14.94
0.31
51.38
16.55


AD-1565619.1
65.85
13.72
60.46
19.23
51.34
12.11


AD-1565620.1
4.02
3.39
4.75
3.11
18.39
8.53


AD-1565771.1
5.16
1.04
6.21
1.84
11.84
4.85


AD-1565621.1
83.75
17.64
64.18
12.51
110.01
8.38


AD-1565772.1
2.83
0.68
4.28
1.68
12.04
3.51


AD-1565836.1
3.31
1.54
6.47
2.88
13.20
3.79


AD-1565837.1
4.13
1.20
4.14
0.94
14.83
4.25


AD-1565838.1
6.38
1.77
5.75
2.18
15.94
4.66


AD-1565840.1
7.19
3.59
5.53
1.80
32.45
6.46


AD-1565841.1
4.72
1.31
8.47
2.00
18.97
8.19


AD-1565842.1
3.04
1.48
4.60
3.10
14.00
3.53


AD-822899.17
3.51
1.21
5.57
2.82
12.90
3.86


AD-1565772.2
2.85
0.87
4.95
1.83
13.34
9.42


AD-1565622.1
9.09
3.35
11.85
1.77
21.74
5.30


AD-1565843.1
9.58
4.09
8.54
1.89
15.97
7.50


AD-1565844.1
2.75
1.37
4.20
1.48
8.94
4.30


AD-1565845.1
27.92
5.48
26.58
6.38
57.51
9.61


AD-1565846.1
48.68
10.51
49.20
29.92
71.91
11.76


AD-1565848.1
94.29
22.52
111.68
12.87
111.38
29.52


AD-1565849.1
37.68
9.68
17.51
5.53
45.10
16.47


AD-1565850.1
57.87
9.01
36.20
10.32
63.68
16.98


AD-822926.4
7.22
4.66
4.35
1.48
17.62
7.83


AD-1565623.1
8.25
3.03
11.68
4.46
14.42
3.89


AD-1565839.1
6.07
2.22
5.99
5.16
11.18
3.84


AD-1565624.1
6.10
1.35
8.89
1.90
9.85
2.62


AD-1565847.1
9.04
3.53
6.91
2.93
38.22
4.27


AD-1565625.1
60.76
1.13
51.43
8.88
33.00
0.71


AD-1565626.1
8.87
3.65
8.36
3.19
9.48
1.76


AD-1565773.1
6.64
1.30
13.77
6.02
38.32
6.58


AD-1565627.1
7.68
2.90
8.60
2.57
9.19
3.34


AD-1565628.1
27.35
5.83
27.90
9.21
42.72
13.86


AD-1565629.1
12.25
4.89
42.80
12.64
37.72
0.21


AD-1565774.1
17.59
5.54
5.12
0.90
18.54
7.94


AD-1565630.1
12.95
1.09
29.20
7.37
36.41
6.87


AD-1565631.1
14.87
4.36
29.86
5.00
30.09
6.35


AD-1565775.1
12.20
0.98
10.60
2.86
11.61
3.39


AD-1565632.1
34.01
6.07
33.10
1.65
43.01
7.59


AD-1565776.1
59.47
10.06
46.53
13.29
76.25
10.64


AD-1565777.1
3.25
3.60
4.24
1.29
7.38
4.40


AD-1565634.1
19.64
6.63
24.71
9.68
12.30
3.61


AD-1565778.1
18.08
3.67
20.05
8.75
19.26
5.00


AD-1565635.1
24.90
4.36
27.27
3.30
17.01
6.09


AD-1565636.1
18.08
5.60
26.04
6.67
19.12
5.80


AD-1565637.1
24.19
7.75
22.69
7.34
22.00
7.85


AD-1565638.1
20.13
4.33
21.73
5.82
23.78
7.73


AD-1565639.1
19.98
6.07
17.46
5.27
17.52
4.96


AD-1565779.1
16.57
5.59
23.11
7.46
18.57
2.30


AD-1565640.1
24.23
7.16
24.86
4.78
22.95
5.65


AD-1565641.1
14.78
4.59
20.27
7.35
15.40
4.17


AD-1565642.1
22.92
7.06
27.36
1.86
22.58
4.00


AD-1565643.1
21.33
5.08
25.45
4.54
21.06
4.94


AD-1565644.1
21.50
4.66
28.68
4.46
21.57
3.60


AD-1565645.1
20.71
1.23
30.21
6.14
24.39
5.16


AD-1565646.1
25.16
2.74
21.62
2.13
26.27
4.91


AD-1565851.1
32.51
12.05
25.85
3.74
28.17
4.26


AD-1565852.1
23.35
2.52
24.85
2.61
25.45
1.40


AD-1565853.1
20.79
5.56
15.58
2.51
23.92
2.07


AD-1565854.1
21.35
2.18
16.74
4.57
37.31
10.98


AD-1565857.1
89.72
24.46
60.67
10.16
72.79
19.16


AD-1565858.1
32.39
5.99
20.22
3.18
34.87
11.29


AD-1565859.1
44.64
4.32
28.84
7.42
41.43
14.25


AD-1565647.1
33.50
6.27
23.93
7.48
20.42
3.95


AD-1565648.1
17.96
3.05
16.97
2.55
15.90
1.48


AD-1565855.1
24.15
5.54
18.07
0.61
37.07
7.91


AD-1565856.1
32.83
3.41
20.30
3.88
46.01
8.44


AD-1565649.1
21.70
5.38
31.01
4.14
14.39
2.35


AD-1565650.1
14.11
2.91
16.32
3.70
13.62
10.74


AD-1565651.1
15.70
3.79
23.56
8.66
14.00
4.41


AD-1565652.1
28.65
5.13
32.11
6.96
41.78
11.65


AD-1565653.1
18.91
3.29
22.20
8.89
16.74
4.17


AD-1565780.1
21.89
7.89
22.48
5.59
25.73
7.52


AD-1565654.1
28.76
5.80
27.06
9.35
36.78
10.64


AD-1565655.1
14.26
1.84
16.84
2.93
22.57
3.51


AD-1565781.1
22.73
5.86
23.64
7.88
26.26
4.73


AD-1565656.1
8.05
3.17
18.34
3.69
20.89
1.60


AD-1565657.1
37.82
7.32
34.46
4.71
46.82
15.41


AD-1565658.1
20.16
5.46
22.60
1.17
33.74
16.69


AD-1565659.1
21.80
5.01
32.48
6.03
27.19
5.57


AD-1565660.1
18.96
4.48
25.75
6.04
33.55
9.37


AD-1565782.1
19.49
5.05
13.48
3.51
23.54
5.97


AD-1565661.1
23.24
4.21
30.64
8.86
21.49
5.88


AD-1565662.1
22.44
1.52
29.91
6.54
23.11
6.46


AD-1565663.1
19.51
3.82
19.55
2.35
22.06
6.15


AD-1565664.1
20.84
2.90
14.97
3.73
16.29
4.16


AD-1565783.1
19.12
7.35
16.94
6.39
19.67
8.30


AD-1565665.1
17.64
0.62
29.45
9.88
29.19
7.74


AD-1565784.1
16.20
5.45
15.66
5.02
13.24
3.05


AD-1565666.1
17.38
4.44
22.81
7.48
24.53
5.40


AD-1565667.1
17.79
4.54
26.44
5.86
16.24
2.00


AD-1565785.1
18.10
7.14
10.86
5.66
17.76
6.07


AD-1565668.1
41.49
8.93
43.99
2.72
37.17
6.96


AD-1565669.1
24.91
3.24
25.11
4.53
14.49
3.34


AD-1565670.1
14.17
6.35
21.96
9.65
29.29
2.34


AD-1565860.1
12.24
3.88
14.80
4.75
36.53
4.17


AD-1565861.1
19.87
5.39
14.91
8.45
25.82
7.66


AD-1565862.1
16.77
5.82
23.11
4.79
28.52
8.22


AD-1565863.1
20.42
7.47
23.10
8.82
22.00
7.25


AD-1565864.1
23.64
12.69
17.75
5.07
21.37
4.31


AD-1565866.1
21.29
7.65
17.97
4.76
25.56
4.55


AD-1565670.2
20.00
4.48
19.21
5.61
15.66
2.61


AD-1565671.1
34.15
10.48
35.84
7.21
26.67
12.46


AD-1565865.1
22.15
7.26
22.19
5.76
29.14
5.04


AD-1565786.1
19.30
7.79
15.01
4.01
23.94
1.39


AD-1565672.1
18.40
4.74
28.01
8.90
16.08
3.92


AD-1565673.1
11.46
5.01
19.74
4.14
18.41
3.39


AD-1565674.1
22.27
7.50
26.45
5.36
22.47
7.98


AD-1565787.1
20.36
2.47
9.49
3.21
18.01
2.96


AD-1565675.1
28.40
5.05
34.29
3.63
29.59
10.48


AD-1565788.1
15.79
4.25
15.72
4.64
16.69
0.95


AD-1565676.1
22.39
5.86
24.17
3.94
28.08
5.73


AD-1565677.1
22.77
5.35
26.27
7.04
22.49
7.99


AD-1565678.1
14.89
2.15
17.14
5.83
19.52
5.04


AD-1565679.1
17.93
3.69
18.38
3.92
21.25
4.78


AD-1565680.1
16.07
3.75
20.49
2.95
19.02
1.58


AD-1565681.1
66.05
9.80
45.76
2.54
76.24
17.73


AD-1565789.1
17.89
6.69
17.09
1.75
15.21
4.11


AD-1565790.1
15.85
0.71
12.83
5.40
24.07
8.61


AD-1565682.1
28.99
9.62
28.53
6.54
34.55
13.61


AD-1565791.1
13.75
1.62
12.50
3.96
34.69
3.47


AD-1565683.1
33.85
9.82
28.85
2.71
27.77
9.17


AD-1565684.1
46.44
16.14
47.20
12.01
92.00
26.93


AD-1565685.1
44.46
12.33
36.23
5.10
58.69
23.40


AD-1565867.1
24.71
5.92
23.04
2.85
31.52
3.79


AD-1565868.1
33.54
8.52
31.27
12.38
37.03
4.99


AD-1565869.1
27.19
9.45
33.17
9.74
30.69
5.68


AD-1565870.1
22.23
4.78
21.55
2.08
29.63
9.89


AD-1565871.1
22.89
4.89
24.65
0.94
21.11
1.91


AD-1565872.1
28.60
9.58
27.43
10.76
23.01
5.72


AD-1565873.1
15.65
4.07
30.34
7.91
29.58
6.70


AD-1565874.1
77.37
12.83
106.84
16.49
58.43
2.43


AD-1565875.1
77.77
23.99
44.29
12.88
55.96
13.56


AD-1565876.1
114.72
42.05
55.55
13.12
52.88
7.61


AD-1565686.1
23.56
4.75
40.73
8.81
43.94
3.80


AD-1565687.1
23.34
1.87
21.53
3.56
22.50
4.26


AD-1565688.1
27.59
6.13
27.27
2.35
24.05
3.29


AD-1565877.1
21.23
2.76
37.13
7.45
45.66
6.89


AD-1565689.1
26.48
4.22
22.79
6.21
24.67
3.21


AD-1565792.1
10.48
2.13
8.23
1.24
21.07
8.93


AD-1565690.1
28.90
2.94
27.49
4.31
22.85
4.91


AD-1565691.1
30.69
8.84
33.68
2.31
34.86
6.66









Example 3. Validation of Human MYOC siRNAs in a Mouse Glaucoma Model

To test human MYOC siRNAs in vivo, a glaucoma mouse model was used: synergistic activation mediator (SAM) mice comprising a humanized MYOC locus comprising a Y437H mutation (SAM-MYOC mice). In these mice, a SAM guide RNA targeting the MYOC promoter (SAM gRNA) can be used to increase expression of the humanized MYOC comprising the Y437H mutation, resulting in increased intraocular pressure (IOP). The SAM-MYOC mice were generated by crossing mice comprising genomically integrated dCas9 synergistic activation mediator (SAM) system components (dCas9-VP64 and MCP-p65-HSF1) as one transcript driven by the endogenous Rosa26 promoter (described in US 2019/0284572 and WO 2019/183123, each of which is herein incorporated by reference) with mice comprising a humanized MYOC locus comprising a Y437H mutation. In these mice, the mouse Myoc sequence from the start codon to the stop codon was replaced with the corresponding human MYOC sequence. The inserted human MYOC sequence comprises a Y437H mutation, which is a mutation associated with elevated IOP and development of glaucoma. Injection of AAV2.Y3F or lentivirus encoding the SAM guide RNA targeting the MYOC promoter resulted in increased humanized MYOC Y437H expression in the limbal ring (trabecular meshwork TM, iris, and ciliary body (CB)), and this correlated with increased IOP, validating the SAM-MYOC mice as a suitable MYOC disease model with increased IOP (data not shown).


siRNAs targeting human MYOC were tested to determine if they could lower the high intraocular pressure (IOP) observed in SAM mice comprising a humanized MYOC locus comprising a Y437H mutation (SAM-MYOC mice) following treatment with the SAM gRNA. The experimental setup is shown in FIG. 1A. The SAM gRNA was administered via intracameral (IC) injection at day 0, and baseline IOP was measured. IOP was then measured over the following five weeks. At week five, MYOC siRNA AD-822899 (1 μL, 15 μg dose) was administered via intravitreal (IVT) injection, and IOP was measured at various timepoints over the subsequent weeks. There were three treatment groups: (1) naïve control mice; (2) SAM-gRNA-treated mice treated with human MYOC siRNA; and (3) SAM-gRNA-treated mice treated with luciferase siRNA. As shown in FIG. 1B, the human MYOC siRNA lowered IOP in the SAM-MYOC mice treated with the SAM gRNA, reversing and returning the IOP to baseline levels starting at D7 after siRNA injection, whereas the luciferase siRNA had no effect on IOP.


Several additional siRNAs targeting human MYOC were then tested at a lower dose to determine if they could lower the IOP observed in the SAM-MYOC mice following treatment with lentiviral SAM gRNA. Mice were bilaterally injected with SAM gRNA and siRNAs. The experimental setup is shown in FIG. 2A. SAM gRNA was administered via intracameral (IC) injection at day 0, and baseline IOP was measured. IOP was then measured over the following five weeks. At week five, MYOC siRNAs AD-822899, AD-1565804, AD-1565837, AD-1193175, or AD-1565503 (1 μL, 7.5 μg dose) were administered via intravitreal (IVT) injection, and IOP was measured at various timepoints over the subsequent weeks. mRNA knockdown in the limbal ring was tested by qPCR, and RNASCOPE® analysis was done. The control groups included naïve control mice, PBS-treated mice, and LV-SAM gRNA-treated mice. As shown in FIG. 2B, each human MYOC siRNA lowered IOP in the SAM-MYOC mice treated with SAM gRNA, reversing and returning the IOP to baseline levels soon after siRNA injection. As shown in FIG. 2C, each human MYOC siRNA decreased human MYOC mRNA expression relative to the LV-SAM-gRNA group as measured by qPCR in a sample from the limbal ring. RNASCOPE® analysis confirmed that the siRNAs mediated knockdown of human MYOC mRNA in the SAM-MYOC mice (FIG. 2D).


Human MYOC siRNAs AD-1565804 or AD-1565837 were then tested at even lower doses to determine if they could lower the IOP observed in the SAM-MYOC mice following treatment with lentiviral SAM-g4. Mice were bilaterally injected with SAM gRNA and siRNA. The experimental setup is shown in FIG. 3A. SAM-g4 was administered via intracameral (IC) injection at day 0, and baseline IOP was measured. IOP was then measured over the following five weeks. At week five, MYOC siRNAs AD-1565804 or AD-1565837 (1 μL, 3.75 μg dose or 1.87 μg dose) were administered via intravitreal (IVT) injection, and IOP was measured at various timepoints over the subsequent weeks. mRNA knockdown in the limbal ring was tested by qPCR, and RNASCOPE® analysis was done. The control groups included naïve control mice, PBS-treated mice, and LV-SAM-gRNA-treated mice. As shown in FIG. 3B, each human MYOC siRNA lowered IOP in the SAM-MYOC mice treated with SAM gRNA at each dose tested, reversing and returning the IOP to baseline levels soon after siRNA injection. As shown in FIG. 3C, each human MYOC siRNA decreased human MYOC mRNA expression relative to the LV-SAM gRNA group as measured by qPCR in a sample from the limbal ring at each dose tested.


Example 4. Validation of Human MYOC siRNAs in a Non-Human Primate (NHP) Glaucoma Model

siRNAs targeting MYOC were tested to determine if they could lower the high intraocular pressure (IOP) observed in a non-human primate (NHP) glaucoma model. The study design is shown in Table 4. The animals received a PBS control or various doses of the duplex AD-1565837 or the duplex AD-1565804.


OE's and intraocular pressure (IOP) were measured pre-study, on days 3, 8, 15, 29, and 57, and once during week 12. Electroretinography (ERG) was performed pre-study, mid-study (week 6), and at termination (week 12). Aqueous humor (AH) was collected pre-dose, week 4, week 8 and at termination. At termination, the following eye samples were collected from the right eye; AH, vitreous humor (VH), cornea, iris, trabecular meshwork TM, ciliary body (CB), sclera (limbal ring without TM), retina, and all remaining tissue in posterior segment of the eye. Blood was collected twice at pre-dose and termination. Hematology, coagulation, and clinical chemistry studies were performed on the blood samples.


Tissues were collected for histopathology at termination including the right eye, optic nerve and extra-ocular and peri-ocular tissues: Brain, right (cerebral hemisphere); Esophagus, proximal; Stomach, esophageal-gastric junction; Eyelid, upper with palpebral conjunctivae, right; Eyelid, lower with palpebral conjunctivae, right; Heart, apex; Jejunum; Kidney, right cortex; Liver, right median lobe; Lacrimal Gl, right; Deep Cervical, lymph node, right; Mandibular, lymph node, right; Sciatic nerve, right; Muscle, rectus femoris; Muscle, diaphragm; Muscle, extraocular (lateral and medial rectus, right); Ovary, right; Mandibular salivary gland, right; Spinal cord, cervical; Spinal cord, thoracic; Spinal cord, lumbar; Thyroid, right; Tongue; Tonsil, right; Trachea; and Uterus.









TABLE 4







NHP DC Selection Study Design













Dose Level




Group
Test Article
(μg/eye)
No. Monkeys
Total














1
PBS
 0
3
31


2
AD-
 10
3


3
1565837
 30
3


4

100
3


5

300
3


6

1000*
2


7
AD-
 10
3


8
1565804
 30
3


9

100
3


10

300
3


11

1000*
2





*Animals treated with highest dose: both eyes will be examined with histopathology read outs.






MYOC protein knock down in the TM was analyzed at day 85. The knock down results are shown in FIG. 4. About a 50% reduction in MYOC protein in the TM was seen with the AD-1565837 duplex, while minimal knockdown was observed with the AD-1565804 duplex. MYOC protein in the aqueous humor was analyzed at days −35, 22, 50, and day 85 (terminal collection). As depicted in FIG. 5, MYOC protein knockdown of about 50% was observed at day 50, but not at day 85. MYOC protein knock down in the vitreous humor, iris, ciliary body, and sclera was analyzed at day 85 (terminal collection). The results are shown in FIG. 6A for vitreous humor and ciliary body, and in FIG. 6B for iris and sclera. At day 85, no knock down was observed in the MYOC mRNA for either duplex evaluated (FIG. 7).

Claims
  • 1. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of myocilin (MYOC), wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from one of the antisense sequences listed in any one of Tables 2A and 2B, and wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from a sense sequence listed in any one of Tables 2A and 2B that corresponds to the antisense sequence.
  • 2. The dsRNA agent of claim 1, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from the antisense strand nucleotide sequence of duplex AD-1565804 and the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from the sense strand nucleotide sequence of duplex AD-1565804.
  • 3. The dsRNA agent of claim 1, wherein the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from the antisense strand nucleotide sequence of duplex AD-1565837 and the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, from the sense strand nucleotide sequence of duplex AD-1565837.
  • 4. The dsRNA agent of any one of claims 1-3, wherein at least one of the sense strand and the antisense strand is conjugated to one or more lipophilic moieties.
  • 5. The dsRNA agent of claim 4, wherein the lipophilic moiety is conjugated via a linker or carrier.
  • 6. The dsRNA agent of claim 4 or 5, wherein one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand.
  • 7. The dsRNA agent of any one of claims 1-3, wherein at least one of the sense strand and the antisense strand is conjugated to one or more of an arginine-glycine-aspartic acid (RGD)-peptide or RGD peptide mimetic.
  • 8. The dsRNA agent of any one of claims 4-6, wherein the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.
  • 9. The dsRNA agent of claim 8, wherein the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.
  • 10. The dsRNA agent of any one of claims 4-6, wherein the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) or the double stranded region.
  • 11. The dsRNA agent of any one of claims 4-6, wherein the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.
  • 12. The double-stranded iRNA agent of any one of claims 4-6, wherein the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.
  • 13. The dsRNA agent of any one of claims 1-12, wherein the dsRNA agent comprises at least one modified nucleotide.
  • 14. The dsRNA agent of claim 13, wherein no more than five of the sense strand nucleotides and not more than five of the nucleotides of the antisense strand are unmodified nucleotides.
  • 15. The dsRNA agent of claim 13, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.
  • 16. The dsRNA agent of any one of claims 13-15, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a 5′-phosphate mimic, a glycol modified nucleotide, and a 2-O—(N-methylacetamide) modified nucleotide; and combinations thereof.
  • 17. The dsRNA agent of any of any one of claims 1-16, wherein at least one strand comprises a 3′ overhang of at least 2 nucleotides.
  • 18. The dsRNA agent of any one of claims 1-17, wherein the double stranded region is 15-30 nucleotide pairs in length.
  • 19. The dsRNA agent of claim 18, wherein the double stranded region is 17-23 nucleotide pairs in length.
  • 20. The dsRNA agent of any one of claims 1-19, wherein each strand has 19-30 nucleotides.
  • 21. The dsRNA agent of any one of claims 1-20, wherein the agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.
  • 22. The dsRNA agent of any one of claims 4-21, further comprising a targeting ligand, e.g., a ligand that targets an ocular tissue.
  • 23. The dsRNA agent of claim 22, wherein the ocular tissue is a trabecular meshwork tissue, a ciliary body, a retinal tissue, a retinal pigment epithelium (RPE) or choroid tissue, e.g., a choroid vessel.
  • 24. The dsRNA agent of any one of claims 1-23, further comprising a phosphate or phosphate mimic at the 5′-end of the antisense strand.
  • 25. The dsRNA agent of claim 24, wherein the phosphate mimic is a 5′-vinyl phosphonate (VP).
  • 26. The dsRNA of any one of claims 1-25, wherein the dsRNA agent targets a hotspot region of an mRNA encoding MYOC.
  • 27. A dsRNA agent that targets a hotspot region of a myocilin (MYOC) mRNA.
  • 28. A cell containing the dsRNA agent of any one of claims 1-27.
  • 29. A pharmaceutical composition for inhibiting expression of a MYOC, comprising the dsRNA agent of any one of claims 1-27.
  • 30. A method of inhibiting expression of MYOC in a cell, the method comprising: (a) contacting the cell with the dsRNA agent of any one of claims 1-27, or a pharmaceutical composition of claim 29; and(b) maintaining the cell produced in step (a) for a time sufficient to reduce levels of MYOC mRNA, MYOC protein, or both of MYOC mRNA and protein, thereby inhibiting expression of MYOC in the cell.
  • 31. The method of claim 30, wherein the cell is within a subject.
  • 32. The method of claim 31, wherein the subject is a human.
  • 33. The method of claim 32, wherein the subject has been diagnosed with a MYOC-associated disorder, e.g., glaucoma (e.g., primary open angle glaucoma (POAG), angle closure glaucoma, congenital glaucoma, and secondary glaucoma).
  • 34. A method of treating a subject diagnosed with a MYOC-associated disorder comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1-27 or a pharmaceutical composition of claim 29, thereby treating the disorder.
  • 35. The method of claim 34, wherein the MYOC-associated disorder is glaucoma.
  • 36. The method of claim 35, wherein glaucoma is primary open angle glaucoma (POAG).
  • 37. The method of any one of claims 34-36, wherein treating comprises amelioration of at least one sign or symptom of the disorder.
  • 38. The method of any one of claims 34-37, wherein the treating comprises (a) inhibiting or reducing the expression or activity of MYOC; (b) reducing the level of misfolded MYOC protein; (c) reducing trabecular meshwork cell death; (d) decreasing intraocular pressure; or (e) increasing visual acuity.
  • 39. The method of any one of claims 31-38, wherein the dsRNA agent is administered to the subject intraocularly, intravenously, or topically.
  • 40. The method of claim 39, wherein the intraocular administration comprises intravitreal administration (e.g., intravitreal injection), transscleral administration (e.g., transscleral injection), subconjunctival administration (e.g., subconjunctival injection), retrobulbar administration (e.g., retrobulbar injection), intracameral administration (e.g., intracameral injection), or subretinal administration (e.g., subretinal injection).
  • 41. The method of any one of claims 31-40, further comprising administering to the subject an additional agent or therapy suitable for treatment or prevention of an MYOC-associated disorder (e.g., laser trabeculoplasty surgery, trabeculectomy surgery, a minimally invasive glaucoma surgery, placement of a drainage tube in the eye, oral medication, or eye drops).
CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application No. 63/215,804, filed on Jun. 28, 2021, claims the benefit of priority to U.S. Provisional Application No. 63/287,404, filed on Dec. 8, 2021, and claims the benefit of priority to U.S. Provisional Application No. 63/351,033, filed on Jun. 10, 2022. The entire contents of the foregoing applications are hereby incorporated herein by reference.

PCT Information
Filing Document Filing Date Country Kind
PCT/US2022/035266 6/28/2022 WO
Provisional Applications (3)
Number Date Country
63351033 Jun 2022 US
63287404 Dec 2021 US
63215804 Jun 2021 US