SMALL RIBOSOMAL PROTEIN SUBUNIT 25 (RPS25) iRNA AGENT COMPOSITIONS AND METHODS OF USE THEREOF

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 Feb. 2, 2022, is named 121301_09803_SL.txt and is 805,258 bytes in size.

BACKGROUND OF THE INVENTION

Nucleotide-repeat expansions underlie a heterogeneous group of primarily neurological diseases that in aggregate impact a large number of patients. Repeats can cause problems through a variety of mechanisms delineated over the past 25 years. For example, expansion of trinucleotide repeats within protein-coding open reading frames (ORFs) cause a gain-of-function toxicity downstream of the production of polyglutamine or (less frequently) polyalanine proteins. This toxicity results from both alterations in the native functions of the protein in which the repeat resides as well as toxicity independent of protein context, related to perturbations in neuronal proteostasis. Repeat expansions located outside of known protein-coding ORFs can elicit changes in the expression of the gene in which they reside, leading to reduced or enhanced expression at the transcript and protein level. Such non-coding repeats can also elicit toxicity as RNA by binding to and sequestering specific RNA-binding proteins via presentation of a repetitive motif.

Repeat-associated non-AUG (RAN)-initiated translation is a non-canonical translational initiation process which enables protein elongation through a repeat strand in the absence of an AUG initiation codon and in multiple reading frames, producing multiple homopolymeric or dipeptide repeat-containing proteins. Originally described in association with CAG-repeat expansions causative for spinocerebellar ataxia type 8 (SCA8), this process also occurs in association with expansions of CAG, CUG, GGGGCC, GGCCCC, and CGG repeats. Repeats can drive RAN translation in a surprising variety of RNA contexts, including within 5′ untranslated regions (UTRs), protein-coding ORFs, or introns and “non-coding” RNAs.

A small ribosomal subunit, RPS25, has recently been identified as a driver of RAN translation of a GGGGCC expansion, a nucleotide repeat expanded in C9 for72 amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), in yeast. Knocking down RPS25 was shown to limit poly-dipeptide production and boost yeast survival without affecting global RNA translation. Knocking down homologs in fruit flies reduced neurodegeneration and in cultured human motor neurons, reduced neurodegeneration. (Yamada, et al. (2019) Nat Neurosci (doi.org/10.1038/s41593-019-0455-7).

There are currently no disease modifying treatments for nucleotide repeat expansion diseases, such as C9orf72 ALS/FTD and Huntington's disease, e.g., Huntington Disease, e.g., Huntington-Like Syndrome Due To C9orf72 Expansions and, therefore, supportive and symptomatic management is the mainstay of treatment. Accordingly, there is a need in the art for compositions and use of such compositions for the treatment of subjects having nucleotide repeat expansion diseases, such as C9orf72 ALS/FTD and Huntington Disease, e.g., Huntington-Like Syndrome Due To C9orf72 Expansions.

BRIEF SUMMARY OF THE INVENTION

The present disclosure provides RNAi agent compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a small ribosomal protein subunit 25 (RPS25) gene. The RPS25 gene may be within a cell, e.g., a cell within a subject, such as a human. The present disclosure also provides methods of using the RNAi agent compositions of the disclosure for inhibiting the expression of an RPS25 gene or for treating a subject who would benefit from inhibiting or reducing the expression of an RPS25 gene, e.g., a subject suffering or prone to suffering from an RPS25-associated disease.

Accordingly, in one aspect, the instant disclosure provides a double stranded ribonucleic acid (RNAi) agent for inhibiting expression of a small ribosomal protein subunit 25 (RPS25) gene, where the RNAi agent includes a sense strand and an antisense strand, and where the antisense strand includes a region of complementarity which includes at least 15 contiguous nucleotides differing by no more than 3 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides) from any one of the antisense sequences listed in any one of Tables 2-14. In certain embodiments, the antisense strand includes a region of complementarity which includes at least 15 contiguous nucleotides of any one of the antisense sequences listed in any one of Tables 2-14. In certain embodiments, the antisense strand includes a region of complementarity which includes at least 19 contiguous nucleotides differing by no more than 3 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides) from any one of the antisense sequences listed in any one of Tables 2-14. In certain embodiments, the antisense strand includes a region of complementarity which includes at least 19 contiguous nucleotides of any one of the antisense sequences listed in any one of Tables 2-14. In certain embodiments, thymine-to-uracil or uracil-to-thymine differences between aligned (compared) sequences are not counted as nucleotides that differ between the aligned (compared) sequences.

Another aspect of the instant disclosure provides a double stranded RNAi agent for inhibiting expression of a small ribosomal protein subunit 25 (RPS25) gene, where the dsRNA agent includes a sense strand and an antisense strand, where the sense strand includes at least 15 contiguous nucleotides differing by no more than 3 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides) from any one of the sense strand sequences presented in Tables 2-14; and where the antisense strand includes at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of antisense strand nucleotide sequences presented in Tables 2-14. In certain embodiments, the sense strand includes at least 15 contiguous nucleotides of any one of the sense strand sequences presented in Tables 2-14; and where the antisense strand includes at least 15 contiguous nucleotides of any one of antisense strand nucleotide sequences presented in Tables 2-14. In certain embodiments, the sense strand includes at least 19 contiguous nucleotides of any one of the sense strand sequences presented in Tables 2-14; and where the antisense strand includes at least 19 contiguous nucleotides of any one of antisense strand nucleotide sequences presented in Tables 2-14 (i.e., differing by 3, 2, 1, or 0 nucleotides) from any one of antisense strand nucleotide sequences presented in Tables 2-14.

An additional aspect of the disclosure provides a double stranded RNAi agent for inhibiting expression of a small ribosomal protein subunit 25 (RPS25) gene, where the dsRNA agent includes a sense strand and an antisense strand, where the sense strand includes at least 15 contiguous nucleotides differing by no more than 3 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, and 9, where a substitution of a uracil for any thymine of SEQ ID NOs: 1, 3, 5, 7, and 9 (when comparing aligned sequences) does not count as a difference that contributes to the differing by no more than 3 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, and 9; and where the antisense strand includes at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, and 10, where a substitution of a uracil for any thymine of SEQ ID NOs: 2, 4, 6, 8, and 10, (when comparing aligned sequences) does not count as a difference that contributes to the differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, and 10, where at least one of the sense strand and the antisense strand includes one or more lipophilic moieties conjugated to one or more internal nucleotide positions, optionally via a linker or carrier.

In one embodiment, the double stranded RNAi agent targeted to RPS25 sense strand includes at least 15 contiguous nucleotides differing by no more than 3 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides) from the nucleotide sequence of the sense strand nucleotide sequence of a duplex in Tables 2-14.

In one embodiment, the double stranded RNAi agent targeted to RPS25 antisense strand includes at least 15 contiguous nucleotides differing by no more than 3 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides) from the antisense nucleotide sequence of duplex in one of Tables 2-14.

Optionally, the double stranded RNAi agent includes at least one modified nucleotide.

In certain embodiments, the lipophilicity of the lipophilic moiety, measured by logK_ow, 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 a related embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

In certain embodiments, all of the nucleotides of the sense strand are modified nucleotides.

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

In certain embodiments, all of the nucleotides of the antisense strand are modified nucleotides. Optionally, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

In one embodiment, at least one of the modified nucleotides is 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, 2′-hydroxly-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 5′-phosphorothioate group, a nucleotide comprising a 5′-methylphosphonate group, a nucleotide comprising a 5′ phosphate or 5′ phosphate mimic, a nucleotide comprising vinyl phosphonate, a nucleotide comprising adenosine-glycol nucleic acid (GNA), a nucleotide comprising thymidine-glycol nucleic acid (GNA) S-Isomer, a nucleotide comprising 2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising 2′-deoxythymidine-3′ phosphate, a nucleotide comprising 2′-deoxyguanosine-3′-phosphate, or a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

In a related embodiment, the modified nucleotide is a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, 3′-terminal deoxy-thymine nucleotides (dT), a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, or a non-natural base comprising nucleotide.

In one embodiment, the modified nucleotide includes a short sequence of 3′-terminal deoxy-thymine nucleotides (dT).

In another embodiment, the modifications on the nucleotides are 2′-O-methyl, 2′ fluoro and GNA modifications.

In an additional embodiment, the double stranded RNAi agent includes at least one phosphorothioate internucleotide linkage. Optionally, the double stranded RNAi agent includes 6-8 phosphorothioate internucleotide linkages.

In certain embodiments, the region of complementarity is at least 17 nucleotides in length. Optionally, the region of complementarity is 19-23 nucleotides in length. Optionally, the region of complementarity is 19 nucleotides in length.

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

In another embodiment, at least one strand includes a 3′ overhang of at least 1 nucleotide. Optionally, at least one strand includes a 3′ overhang of at least 2 nucleotides.

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

In one embodiment, the ligand is

embedded image

where B is a nucleotide base or a nucleotide base analog, optionally where B is adenine, guanine, cytosine, thymine or uracil.

In another embodiment, the region of complementarity to RPS25 includes any one of the antisense sequences in any one of Tables 2-14.

In an additional embodiment, the region of complementarity to RPS25 is that of any one of the antisense sequences in any one of Tables 2-14. In some embodiments, the internal nucleotide positions include all positions except the terminal two positions from each end of the strand.

In a related embodiment, the internal positions include all positions except terminal three positions from each end of the strand. Optionally, the internal positions exclude the cleavage site region of the sense strand.

In some embodiments, the internal positions exclude positions 9-12, counting from the 5′-end of the sense strand. In certain embodiments, the sense strand is 21 nucleotides in length.

In other embodiments, the internal positions exclude positions 11-13, counting from the 3′-end of the sense strand. Optionally, the internal positions exclude the cleavage site region of the antisense strand. In certain embodiments, the sense strand is 21 nucleotides in length.

In some embodiments, the internal positions exclude positions 12-14, counting from the 5′-end of the antisense strand. In certain embodiments, the antisense strand is 23 nucleotides in length.

In another embodiment, the internal positions excluding 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 certain embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In an additional embodiment, one or more lipophilic moieties are conjugated to one or more of the following internal positions: 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. Optionally, one or more lipophilic moieties are conjugated to one or more of the following internal positions: 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 certain embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In certain embodiments, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound. Optionally, the lipophilic moiety is 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-C₃₀hydrocarbon chain, and an optional functional group selected that is hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, or alkyne.

In certain embodiments, the lipophilic moiety contains a saturated or unsaturated C₆-C₁₈hydrocarbon chain. Optionally, the lipophilic moiety contains a saturated or unsaturated C₁₆hydrocarbon chain. In a related embodiment, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s). In certain embodiments, the carrier is a cyclic group that is pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or 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 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, or carbamate.

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

In another embodiment, the double-stranded RNAi agent further includes a phosphate or phosphate mimic at the 5′-end of the antisense strand. Optionally, the phosphate mimic is a 5′-vinyl phosphonate (VP).

In certain embodiments, the double-stranded RNAi agent further includes a targeting ligand that targets a receptor which mediates delivery to a CNS tissue, e.g., a hydrophilic ligand. In certain embodiments, the targeting ligand is a C16 ligand.

In some embodiments, the double-stranded RNAi agent further includes a targeting ligand that targets a brain tissue, e.g., striatum.

In one embodiment, the lipophilic moeity or targeting ligand is conjugated via a bio-cleavable linker that is DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, or a combination thereof.

In a related embodiment, the 3′ end of the sense strand is protected via an end cap which is a cyclic group having an amine, the cyclic group being pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.

In one embodiment, the RNAi agent includes at least one modified nucleotide that is a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a nucleotide that includes a glycol nucleic acid (GNA) or a nucleotide that includes a vinyl phosphonate. Optionally, the RNAi agent includes at least one of each of the following modifications: 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a nucleotide comprising a glycol nucleic acid (GNA) and a nucleotide comprising vinyl phosphonate.

In another embodiment, the RNAi agent includes a pattern of modified nucleotides as provided below in Tables 2-14 where locations of 2′-C16, 2′-O-methyl, GNA, phosphorothioate and 2′-fluoro modifications, irrespective of the individual nucleotide base sequences of the displayed RNAi agents.

Another aspect of the instant disclosure provides a double stranded RNAi agent for inhibiting expression of an RPS25 gene, where the double stranded RNAi agent includes a sense strand complementary to an antisense strand, where the antisense strand includes a region complementary to part of an mRNA encoding RPS25, where each strand is about 14 to about 30 nucleotides in length, where the double stranded RNAi agent is represented by formula (III):

sense: 5′n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q3′

antisense: 3′n_p′-N_a′-(X′X′X′)_k-N_b′-Y′Y′Y′-N_b′-(Z′Z′Z′)_l-N_a′-n_q′5′ (III)

where:

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

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

each N_aand N_a′ independently represents an oligonucleotide sequence including 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence including at least two differently modified nucleotides;

each N_band N_b′ independently represents an oligonucleotide sequence including 0-10 nucleotides which are either modified or unmodified or combinations thereof;

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

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

modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′; and

where the sense strand is conjugated to at least one ligand.

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

- In another embodiment, k is 0; 1 is 0; k is 1; 1 is 1; both k and 1 are 0; or both k andl are 1.
- In certain embodiments, XXX is complementary to X′X′X′, YYY is complementary to Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′.
  
  In another embodiment, the YYY motif occurs at or near the cleavage site of the sense strand.
- In an additional embodiment, the Y′Y′Y′ motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5′-end. Optionally, the Y′ is 2′-O-methyl.

In some embodiments, formula (III) is represented by formula (IIa):

sense: 5′n_p-N_a-Y Y Y-N_a-n_q3′

antisense: 3′n_p′-N_a′-Y′Y′Y′-N_a′-n_q′5′ (IIIa).

In another embodiment, formula (III) is represented by formula (IIIb):

sense: 5′n_p-N_a-Y Y Y-N_b-Z Z Z-N_a-n_q3′

antisense: 3′n_p′-N_a′-Y′Y′Y′-N_b′-Z′Z′Z′-N_a′-n_q′5′ (IIIb)

where each N_band N_b′ independently represents an oligonucleotide sequence including 1-5 modified nucleotides.

In an additional embodiment, formula (III) is represented by formula (IIIc):

sense: 5′n_p-N_a-X X X-N_b-Y Y Y-N_a-n_q3′

antisense: 3′n_p′-N_a′-X′X′X′-N_b′-Y′Y′Y′-N_a′-n_q′5′ (IIIc)

where each N_band N_b′ independently represents an oligonucleotide sequence including 1-5 modified nucleotides.

In certain embodiments, formula (III) is represented by formula (IIId):

sense: 5′n_p-N_a-X X X-N_b-Y Y Y-N_b-Z Z Z-N_a-n_q3′

antisense: 3′n_p′-N_a′-X′X′X′-N_b′-Y′Y′Y′-N_b′-Z′Z′Z′-N_a′-n_q′5′ (IIId)

where each N_band N_b′ independently represents an oligonucleotide sequence including 1-5 modified nucleotides and each N_aand N_a′ independently represents an oligonucleotide sequence including 2-10 modified nucleotides.

In another embodiment, the double stranded region is 15-30 nucleotide pairs in length. Optionally, the double stranded region is 17-23 nucleotide pairs in length.

In certain embodiments, the double stranded region is 17-25 nucleotide pairs in length. Optionally, 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. Optionally, the double stranded region is 21-23 nucleotide pairs in length.

In certain embodiments, each strand has 15-30 nucleotides. Optionally, each strand has 19-30 nucleotides. Optionally, each strand has 19-23 nucleotides.

In certain embodiments, the double stranded region is 19-21 nucleotide pairs in length and each strand has 19-23 nucleotides.

In another embodiment, the modifications on the nucleotides of the RNAi agent are LNA, glycol nucleic acid (GNA), HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C— allyl, 2′-fluoro, 2′-deoxy or 2′-hydroxyl, and combinations thereof. Optionally, the modifications on nucleotides include 2′-O-methyl, 2′-fluoro or GNA, and combinations thereof. In a related embodiment, the modifications on the nucleotides are 2′-O-methyl or 2′-fluoro modifications.

In one embodiment the RNAi agent includes a ligand that is or includes one or more lipophilic, e.g., C16, moieties attached through a bivalent or trivalent branched linker.

In certain embodiments, the ligand is attached to the 3′ end of the sense strand.

In some embodiments, the RNAi agent further includes at least one phosphorothioate or methylphosphonate internucleotide linkage. In a related embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminus of one strand. Optionally, the strand is the antisense strand. In another embodiment, the strand is the sense strand. In a related embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand. Optionally, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the both the 5′- and 3′-terminus of one strand. Optionally, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

In an additional embodiment, the base pair at the 1 position of the 5′-end of the antisense strand of the RNAi agent duplex is an A:U base pair.

In certain embodiments, the Y nucleotides contain a 2′-fluoro modification.

In some embodiments, the Y′ nucleotides contain a 2′-O-methyl modification.

In certain embodiments, p′>0. Optionally, p′=2.

In some embodiments, q′=0, p=0, q=0, and p′ overhang nucleotides are complementary to the target mRNA.

In certain embodiments, q′=0, p=0, q=0, and p′ overhang nucleotides are non-complementary to the target mRNA.

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

In another embodiment, at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage. Optionally, all n_p′ are linked to neighboring nucleotides via phosphorothioate linkages.

In certain embodiments, the RPS25 RNAi agent of the instant disclosure is one of those listed in Tables 2-14. In some embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand include a modification.

Another aspect of the instant disclosure provides a double stranded RNAi agent for inhibiting expression of an RPS25 gene in a cell, where the double stranded RNAi agent includes a sense strand complementary to an antisense strand, where the antisense strand includes a region complementary to part of an mRNA encoding an RPS25 gene, where each strand is about 14 to about 30 nucleotides in length, where the double stranded RNAi agent is represented by formula (III):

sense: 5′n_pN_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q3′

antisense: 3′n_p′-N_a′-(X′X′X′)_k-N_b′-Y′Y′Y′-N_b′-(Z′Z′Z′)_l-N_a′-n_q′5′ (III)

where:

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

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

each N_band N_b′ independently represents an oligonucleotide sequence including 0-10 nucleotides which are either modified or unmodified or combinations thereof;

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

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and where the modifications are 2′-O-methyl or 2′-fluoro modifications;

modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′; and

where the sense strand is conjugated to at least one ligand.

An additional aspect of the instant disclosure provides a double stranded RNAi agent for inhibiting expression of an RPS25 gene in a cell, where the double stranded RNAi agent includes a sense strand complementary to an antisense strand, where the antisense strand includes a region complementary to part of an mRNA encoding RPS25, where each strand is about 14 to about 30 nucleotides in length, where the double stranded RNAi agent is represented by formula (III):

sense: 5′n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q3′

antisense: 3′n_p′-N_a′-(X′X′X′)_k-N_b′-Y′Y′Y′-N_b′-(Z′Z′Z′)_l-N_a′-n_q′5′ (III)

where:

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

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

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

n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage;

each N_band N_b′ independently represents an oligonucleotide sequence including 0-10 nucleotides which are either modified or unmodified or combinations thereof;

modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′; and

where the sense strand is conjugated to at least one ligand.

Another aspect of the instant disclosure provides a double stranded RNAi agent for inhibiting expression of an RPS25 gene in a cell, where the double stranded RNAi agent includes a sense strand complementary to an antisense strand, where the antisense strand includes a region complementary to part of an mRNA encoding RPS25 (SEQ ID NO: 1), where each strand is about 14 to about 30 nucleotides in length, where the double stranded RNAi agent is represented by formula (III):

sense: 5′n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q3′

antisense: 3′n_p′-N_a′-(X′X′X′)_k-N_b′-Y′Y′Y′-N_b′-(Z′Z′Z′)_l-N_a′-n_q′5′ (III)

where:

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

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

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

n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage;

each N_band N_b′ independently represents an oligonucleotide sequence including 0-10 nucleotides which are either modified or unmodified or combinations thereof;

modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′; and

where the sense strand is conjugated to at least one ligand, optioanlly where the ligand is one or more lipophilic, e.g., C16, ligands.

An additional aspect of the instant disclosure provides a double stranded RNAi agent for inhibiting expression of an RPS25 gene in a cell, where the double stranded RNAi agent includes a sense strand complementary to an antisense strand, where the antisense strand includes a region complementary to part of an mRNA encoding RPS25 (SEQ ID NO: 1), where each strand is about 14 to about 30 nucleotides in length, where the double stranded RNAi agent is represented by formula (III):

sense: 5′n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q3′

antisense: 3′n_p′-N_a′-(X′X′X′)_k-N_b′-Y′Y′Y′-N_b′-(Z′Z′Z′)_l-N_a′-n_q′5′ (III)

where:

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

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

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

n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage;

each N_band N_b′ independently represents an oligonucleotide sequence including 0-10 nucleotides which are either modified or unmodified or combinations thereof;

modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′;

where the sense strand includes at least one phosphorothioate linkage; and

where the sense strand is conjugated to at least one ligand, optionally where the ligand is one or more lipophilic, e.g., C16, ligands.

Another aspect of the instant disclosure provides a double stranded RNAi agent for inhibiting expression of an RPS25 gene in a cell, where the double stranded RNAi agent includes a sense strand complementary to an antisense strand, where the antisense strand includes a region complementary to part of an mRNA encoding RPS25 (SEQ ID NO: 1), where each strand is about 14 to about 30 nucleotides in length, where the double stranded RNAi agent is represented by formula (III):

sense: 5′n_p-N_a-Y Y Y-N_a-n_q3′

antisense: 3′n_p′-N_a′-Y′Y′Y′-N_a′-n_q′5′ (IIIa)

where:

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

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

n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage;

YYY and Y′Y′Y′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and where the modifications are 2′-O-methyl or 2′-fluoro modifications;

where the sense strand includes at least one phosphorothioate linkage; and

where the sense strand is conjugated to at least one ligand, optionally where the ligand is one or more lipophilic, e.g., C16, ligands.

An additional aspect of the instant disclosure provides a double stranded RNAi agent for inhibiting expression of an RPS25 gene, where the double stranded RNAi agent targeted to RPS25 includes a sense strand and an antisense strand forming a double stranded region, where the sense strand includes at least 15 contiguous nucleotides differing by no more than 3 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, and 9 and the antisense strand includes at least 15 contiguous nucleotides differing by no more than 3 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, and 10 for RPS25; where a substitution of a uracil for any thymine in the sequences provided in the SEQ ID NOs: 1-10 (when comparing aligned sequences) does not count as a difference that contributes to the differing by no more than 3 nucleotides from any one of the nucleotide sequences provided in SEQ ID NOs: 1-10, where substantially all of the nucleotides of the sense strand include a modification that is a 2′-O-methyl modification, a GNA or a 2′-fluoro modification, where the sense strand includes two phosphorothioate internucleotide linkages at the 5′-terminus, where substantially all of the nucleotides of the antisense strand include a modification selected from the group consisting of a 2′-O-methyl modification and a 2′-fluoro modification, where the antisense strand includes two phosphorothioate internucleotide linkages at the 5′-terminus and two phosphorothioate internucleotide linkages at the 3′-terminus, and where the sense strand is conjugated to one or more lipophilic, e.g., C16, ligands.

Another aspect of the instant disclosure provides a double stranded RNAi agent for inhibiting expression of an RPS25 gene, where the double stranded RNAi agent targeted to RPS25 includes a sense strand and an antisense strand forming a double stranded region, where the sense strand includes at least 15 contiguous nucleotides differing by no more than 3 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, and 9 and the antisense strand includes at least 15 contiguous nucleotides differing by no more than 3 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, and 10 for RPS25, where a substitution of a uracil for any thymine in the sequences provided in the SEQ ID NOs: 1-10 (when comparing aligned sequences) does not count as a difference that contributes to the differing by no more than 3 nucleotides from any one of the nucleotide sequences provided in SEQ ID NOs:1-10; where the sense strand includes at least one 3′-terminal deoxy-thymine nucleotide (dT), and where the antisense strand includes at least one 3′-terminal deoxy-thymine nucleotide (dT).

In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

In another embodiment, each strand has 19-30 nucleotides.

In certain embodiments, the antisense strand of the RNAi agent includes at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5′ region or a precursor thereof. Optionally, the thermally destabilizing modification of the duplex is one or more of

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where B is nucleobase.

Another aspect of the instant disclosure provides a cell containing a double stranded RNAi agent of the instant disclosure.

An additional aspect of the instant disclosure provides a pharmaceutical composition for inhibiting expression of an RPS25 gene that includes a double stranded RNAi agent of the instant disclosure.

In one embodiment, the double stranded RNAi agent is administered in an unbuffered solution. Optionally, the unbuffered solution is saline or water.

In another embodiment, the double stranded RNAi agent is administered with a buffer solution. Optionally, the buffer solution includes acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. In another embodiment, the buffer solution is phosphate buffered saline (PBS).

Another aspect of the disclosure provides a pharmaceutical composition that includes a double stranded RNAi agent of the instant disclosure and a lipid formulation.

In one embodiment, the lipid formulation includes a lipid nanoparticle (LNP).

An additional aspect of the disclosure provides a method of inhibiting expression of an RPS25 gene in a cell, the method involving: (a) contacting the cell with a double stranded RNAi agent of the instant disclosure or a pharmaceutical composition of the instant disclosure; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an RPS25 gene, thereby inhibiting expression of the RPS25 gene in the cell.

In one embodiment, the cell is within a subject. Optionally, the subject is a human.

In certain embodiments, the subject is a rhesus monkey, a cynomolgous monkey, a mouse, or a rat.

In certain embodiments, the human subject suffers from an RPS25-associated disease, e.g., a nucleotide repeat expansion disease, e.g., C9orf72 ALS/FTD, Huntington-Like Syndrome Due To C9orf72 Expansions, Fragile X syndrome (FXS), Myotonic dystrophy (i.e., DM1, and DM2), CAG/polyglutamine disease (e.g., Huntington's disease, Spinal and bulbar muscular atrophy (SBMA), Dentatorubral-pallidoluysian atrophy, Spinocerebellar ataxia type I, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3, Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, Spinocerebellar ataxia type 8, Spinocerebellar ataxia type 12, and Spinocerebellar ataxia type 17), Friedreich ataxia, Unverricht-Lundborg myoclonic epilepsy (EPM1), Oculopharyngeal muscular dystrophy (OPMD), and Fuchs endothelial corneal dystrophy (FECD) In certain embodiments RPS25 expression is inhibited by at least about 50% by the RNAi agent.

Another aspect of the disclosure provides a method of treating a subject having a disorder that would benefit from a reduction in RPS25 expression, e.g., a nucleotide repeat expansion disease, e.g., nucleotide repeat expansion diseases, such as C9orf72 ALS/FTD, Huntington-Like Syndrome Due To C9orf72 Expansions, Fragile X syndrome (FXS), Myotonic dystrophy (i.e., DM1, and DM2), CAG/polyglutamine disease (e.g., Huntington's disease, Spinal and bulbar muscular atrophy (SBMA), Dentatorubral-pallidoluysian atrophy, Spinocerebellar ataxia type I, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3, Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, Spinocerebellar ataxia type 8, Spinocerebellar ataxia type 12, and Spinocerebellar ataxia type 17), Friedreich ataxia, Unverricht-Lundborg myoclonic epilepsy (EPM1), Oculopharyngeal muscular dystrophy (OPMD), and Fuchs endothelial corneal dystrophy (FECD), the method involving administering to the subject a therapeutically effective amount of a double stranded RNAi agent of the disclosure or a pharmaceutical composition of the disclosure, thereby treating the subject.

In certain embodiments, the method further involves administering an additional therapeutic agent to the subject.

In certain embodiments, the double stranded RNAi agent is administered at a dose of about 0.01 mg/kg to about 50 mg/kg.

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

In one embodiment, the method reduces the expression of an RPS25 gene in a brain (e.g., striatum) or spine tissue. Optionally, the brain or spine tissue is striatum, cortex, cerebellum, cervical spine, lumbar spine, or thoracic spine.

Another aspect of the instant disclosure provides a method of inhibiting the expression of RPS25 in a subject, the method involving: administering to the subject a therapeutically effective amount of a double stranded RNAi agent of the disclosure or a pharmaceutical composition of the disclosure, thereby inhibiting the expression of RPS25 in the subject.

An additional aspect of the disclosure provides a method for treating or preventing an disorder or RPS25-associated disease or disorder in a subject, the method involving administering to the subject a therapeutically effective amount of a double stranded RNAi agent of the disclosure or a pharmaceutical composition of the disclosure, thereby treating or preventing an RPS25-associated disease or disorder in the subject.

In certain embodiments, the RPS25-associated disease or disorder is SCA3.

Another aspect of the instant disclosure provides a kit for performing a method of the instant disclosure, the kit including: a) a double stranded RNAi agent of the instant disclosure, and b) instructions for use, and c) optionally, a device for administering the double stranded RNAi agent to the subject.

An additional aspect of the instant disclosure provides a double stranded ribonucleic acid (RNAi) agent for inhibiting expression of an RPS25 gene, where the RNAi agent possesses a sense strand and an antisense strand, and where the antisense strand includes a region of complementarity which includes at least 15 contiguous nucleotides differing by no more than 3 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides), e.g., at least 15 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides), at least 19 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides), from any one of the antisense strand nucleobase sequences of Tables 2-14. In one embodiment, the RNAi agent includes one or more of the following modifications: a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-C-alkyl-modified nucleotide, a nucleotide comprising a glycol nucleic acid (GNA), a phosphorothioate (PS) and a vinyl phosphonate (VP). Optionally, the RNAi agent includes at least one of each of the following modifications: a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-C-alkyl-modified nucleotide, a nucleotide comprising a glycol nucleic acid (GNA), a phosphorothioate and a vinyl phosphonate (VP).

In another embodiment, the RNAi agent includes four or more PS modifications, optionally six to ten PS modifications, optionally eight PS modifications.

In an additional embodiment, each of the sense strand and the antisense strand of the RNAi agent possesses a 5′-terminus and a 3′-terminus, and the RNAi agent includes eight PS modifications positioned at each of the penultimate and ultimate internucleotide linkages from the respective 3′- and 5′-termini of each of the sense and antisense strands of the RNAi agent.

In another embodiment, each of the sense strand and the antisense strand of the RNAi agent includes a 5′-terminus and a 3′-terminus, and the RNAi agent includes only one nucleotide including a GNA. Optionally, the nucleotide including a GNA is positioned on the antisense strand at the seventh nucleobase residue from the 5′-terminus of the antisense strand.

In an additional embodiment, each of the sense strand and the antisense strand of the RNAi agent includes a 5′-terminus and a 3′-terminus, and the RNAi agent includes one to four 2′-C-alkyl-modified nucleotides. Optionally, the 2′-C-alkyl-modified nucleotide is a 2′-C16-modified nucleotide. Optionally, the RNAi agent includes a single 2′-C-alkyl, e.g., C16-modified nucleotide. Optionally, the single 2′-C-alkyl, e.g., C16-modified nucleotide is located on the sense strand at the sixth nucleobase position from the 5′-terminus of the sense strand.

In another embodiment, each of the sense strand and the antisense strand of the RNAi agent includes a 5′-terminus and a 3′-terminus, and the RNAi agent includes two or more 2′-fluoro modified nucleotides. Optionally, each of the sense strand and the antisense strand of the RNAi agent includes two or more 2′-fluoro modified nucleotides. Optionally, the 2′-fluoro modified nucleotides are located on the sense strand at nucleobase positions 7, 9, 10 and 11 from the 5′-terminus of the sense strand and on the antisense strand at nucleobase positions 2, 14 and 16 from the 5′-terminus of the antisense strand.

In an additional embodiment, each of the sense strand and the antisense strand of the RNAi agent includes a 5′-terminus and a 3′-terminus, and the RNAi agent includes one or more VP modifications. Optionally, the RNAi agent includes a single VP modification at the 5′-terminus of the antisense strand.

In another embodiment, each of the sense strand and the antisense strand of the RNAi agent includes a 5′-terminus and a 3′-terminus, and the RNAi agent includes two or more 2′-O-methyl modified nucleotides. Optionally, the RNAi agent includes 2′-O-methyl modified nucleotides at all nucleobase locations not modified by a 2′-fluoro, a 2′-C-alkyl or a glycol nucleic acid (GNA). Optionally, the two or more 2′-O-methyl modified nucleotides are located on the sense strand at positions 1, 2, 3, 4, 5, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 from the 5′-terminus of the sense strand and on the antisense strand at positions 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22 and 23 from the 5′-terminus of the antisense strand.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides RNAi compositions, which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an gene. The RPS25 gene may be within a cell, e.g., a cell within a subject, such as a human. The present disclosure also provides methods of using the RNAi compositions of the disclosure for inhibiting the expression of an RPS25 gene or for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of an RPS25 gene, e.g., an RPS25-associated disease, for example, C9orf72 ALS/FTD.

The RNAi agents of the disclosure include an RNA strand (the antisense strand) having a region which is about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of an RPS25 gene. In certain embodiments, the RNAi agents of the disclosure include an RNA strand (the antisense strand) having a region which is about 21-23 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of an RPS25 gene.

In certain embodiments, the RNAi agents of the disclosure include an RNA strand (the antisense strand) which can include longer lengths, for example up to 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a part of an mRNA transcript of an RPS25 gene. These RNAi agents with the longer length antisense strands preferably include a second RNA strand (the sense strand) of 20-60 nucleotides in length wherein the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

The use of these RNAi agents enables the targeted degradation of mRNAs of an RPS25 gene in mammals Thus, methods and compositions including these RNAi agents are useful for treating a subject who would benefit by a reduction in the levels or activity of an RPS25 protein, such as a subject having an RPS25-associated disease, such as nucleotide repeat expansion diseases, such as C9orf72 ALS/FTD, Huntington-Like Syndrome Due To C9orf72 Expansions, Fragile X syndrome (FXS), Myotonic dystrophy (i.e., DM1, and DM2), CAG/polyglutamine disease (e.g., Huntington's disease, Spinal and bulbar muscular atrophy (SBMA), Dentatorubral-pallidoluysian atrophy, Spinocerebellar ataxia type I, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3, Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, Spinocerebellar ataxia type 8, Spinocerebellar ataxia type 12, and Spinocerebellar ataxia type 17), Friedreich ataxia, Unverricht-Lundborg myoclonic epilepsy (EPM1), Oculopharyngeal muscular dystrophy (OPMD), and Fuchs endothelial corneal dystrophy (FECD).

The following detailed description discloses how to make and use compositions containing RNAi agents to inhibit the expression of an RPS25 gene, as well as compositions and methods for treating subjects having diseases and disorders that would benefit from inhibition or reduction of the expression of the genes.

I. Definitions

In order that the present disclosure may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this disclosure.

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

The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”. The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.

The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means±10%. In certain embodiments, about means±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

The term “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 18 nucleotides of a 21 nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 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, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or intergers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. 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, methods of detection can include determination that the amount of analyte present is below the level of detection of the method.

In the event of a conflict between an indicated target site and the nucleotide sequence for a sense or antisense strand, the indicated sequence takes precedence.

In the event of a conflict between a chemical structure and a chemical name, the chemical structure takes precedence.

The term “rps25” or “RPS25”, also known as “Small Ribosomal Protein S25,” “Ribosomal Protein S25,” “Small Ribosomal Subunit Protein ES25,” “40S Ribosomal Protein S25,” and “S25,” refers to the well-known gene that encodes the protein, RPS25, that is a component of the 40S subunit of the ribosome. RPS25 has been shown to drive “repeat-associated non-AUG (“RAN”)-initiated translation.” RAN-initiated translation,” also referred to as “RAN-translation,” is a non-canonical translational initiation process which enables protein elongation through a repeat strand in the absence of an AUG initiation codon and in multiple reading frames, producing multiple homopolymeric or dipeptide repeat-containing proteins.

Nucleotide and amino acid sequences of RPS25 can be found, for example, at GenBank Accession No. NM_001028.3 (Homo sapiens RPS25, SEQ ID NO: 1, reverse complement, SEQ ID NO: 2); GenBank Accession No. NM_024266.3 (Mus musculus RPS25, SEQ ID NO: 3; reverse complement, SEQ ID NO: 4); GenBank Accession No.: NM_001005528.1 (Rattus norvegicus RPS25, SEQ ID NO: 5, reverse complement, SEQ ID NO: 6); GenBank Accession No.: XM_015115940.1 (Macaca mulatta RPS25, SEQ ID NO: 7, reverse complement, SEQ ID NO: 8); and GenBank Accession No.: NM_001285107.1 (Macaca fascicularis RPS25, SEQ ID NO: 9, reverse complement, SEQ ID NO: 10).

Additional examples of RPS25 sequences can be found in publically available databases, for example, GenBank, OMIM, and UniProt. Additional information on RPS25 can be found, for example, at www.ncbi.nlm.nih.gov/gene/6230.

The term RPS25 as used herein also refers to variations of the RPS25 gene including variants provided in the SNP database, for example, at www.ncbi.nlm.nih.gov/snp/?term=rps25.

The term “C9orf72” gene, also known as “C9orf72-SMCR8 Complex Subunit,” Guanine Nucleotide Exchange C9orf72,” “Chromosome 9 Open Reading Frame 72, “Protein C9orf72,” “DENNL72,” “FTDALS1,” “ALSFTD”, and “FTDALS,” refers to the gene encoding the well-known protein involved in the regulation of endosomal trafficking, C9ORF72. The C9orf72 protein has been shown to interact with Rab proteins that are involved in autophagy and endocytic transport. Expansion of a GGGGCC repeat from 2-22 copies (SEQ ID NO. 14) to 700-1600 copies (SEQ ID NO: 15) in the intronic sequence between alternate 5′ exons in transcripts from this gene is associated with C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia and Huntington's Disease, e.g., Huntington-Like Syndrome Due To C9orf72 Expansions. Alternative splicing results in multiple transcript variants encoding different isoforms.

Nucleotide and amino acid sequences of C9orf72 can be found, for example, at GenBank Accession No. NM_145005.6, transcript variant 1 (SEQ ID NO:11); NM_018325.5, transcript variant 2 (SEQ ID NO:12); and NM_001256054.2, transcript variant 3 (SEQ ID NO:13) (Homo sapiens).

Additional examples of C9orf72 sequences can be found in publically available databases, for example, GenBank, OMIM, and UniProt. Additional information on C9orf72 can be found, for example, at www.ncbi.nlm.nih.gov/gene/?term=c9orf72.

The term C9orf72 as used herein also refers to variations of the C9orf72 gene including variants provided in the SNP database, for example, at www.ncbi.nlm.nih.gov/snp/?term=c9orf72.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an RPS25 gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an RPS25 gene.

The target sequence is about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In certain embodiments, the target sequence is 19-23 nucleotides in length, optionally 21-23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

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.

“G,” “C,” “A,” “T”, and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively in the context of a modified or unmodified nucleotide. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 1). The skilled person is well aware that guanine, cytosine, adenine, thymidine, and uracil can 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 can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can 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.

The terms “iRNA”, “RNAi agent,” “iRNA agent,” “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs the sequence-specific degradation of mRNA. RNAi modulates, e.g., inhibits, the expression of RPS25 in a cell, e.g., a cell within a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the disclosure includes a single stranded RNAi that interacts with a target RNA sequence, e.g., an RPS25 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into double-stranded short interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes these dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). These 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 cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the disclosure relates to a single stranded RNA (ssRNA) (the antisense strand of a siRNA duplex) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., an RPS25 gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded RNA that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs 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 may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

In another embodiment, a “RNAi agent” for use in the compositions and methods of the disclosure is a double stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA” refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., an RPS25 gene. In some embodiments of the disclosure, a double stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, a dsRNA molecule can include ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide, a modified nucleotide. In addition, as used in this specification, an “RNAi agent” may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides. As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the disclosure include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “RNAi agent” for the purposes of this specification and claims.

In certain embodiments of the instant disclosure, inclusion of a deoxy-nucleotide—which is acknowledged as a naturally occurring form of nucleotide—if present within a RNAi agent can be considered to constitute a modified nucleotide.

The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 15-36 base pairs in length, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain embodiments, the duplex region is 19-21 base pairs in length, e.g., 21 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides or nucleotides not directed to the target site of the dsRNA. In some embodiments, the hairpin loop can be 10 or fewer nucleotides. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 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 certain embodiments where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker” (though it is noted that certain other structures defined elsewhere herein can also be referred to as a “linker”). The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs. In one embodiment of the RNAi agent, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

In one embodiment, an RNAi agent of the disclosure is a dsRNA, each strand of which independently comprises 19-23 nucleotides, that interacts with a target RNA sequence, e.g., an RPS25 target mRNA sequence, to direct the cleavage of the target RNA.

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of a RNAi agent, 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, 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) can 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 one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

In certain embodiments, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

In certain embodiments, the overhang on the sense strand or the antisense strand, can include extended lengths longer than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotides in length. In certain embodiments, an extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′ end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′ end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′ end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′ end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. In certain embodiments, the overhang includes a self-complementary portion such that the overhang is capable of forming a hairpin structure that is stable under physiological conditions.

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.

The term “antisense strand” or “guide strand” refers to the strand of a RNAi agent, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., an RPS25 mRNA.

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, e.g., an RPS25 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 molecule. 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 RNAi agent.

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

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

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.

Complementary sequences within a RNAi agent, 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 can 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, can yet be referred to as “fully complementary” for the purposes described herein.

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

The terms “complementary,” “fully complementary” and “substantially complementary” herein can 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 a RNAi 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 RPS25). For example, a polynucleotide is complementary to at least a part of an RPS25 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding RPS25.

Accordingly, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target RPS25 sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target RPS25 sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NOs: 1, 3, 5, 7, or 9 for RPS25, or a fragment of SEQ ID NOs: 1, 3, 5, 7, or 9 for RPS25, such as about 85%, about 90%, about 95%, or about 99% complementary.

In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target RPS25 sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of any one of Tables 2-14 for RPS25, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2-14 for RPS25, such as about 85%, about 90%, about 95%, or about 99% complementary.

In one embodiment, an RNAi agent of the disclosure includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is the same as a target RPS25 sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, or 10, or a fragment of any one of SEQ ID NOs: 2, 4, 6, 8, or 10, such as about 85%, 90%, about 95%, or about 99% complementary.

In one embodiment, at least partial suppression of the expression of an RPS25 gene, is assessed by a reduction of the amount of RPS25 mRNA which can be isolated from or detected in a first cell or group of cells in which an RPS25 gene is transcribed and which has or have been treated such that the expression of an RPS25 gene is inhibited, 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). The degree of inhibition may be expressed in terms of:

$\frac{(mRNA in control cells) - (mRNA in treated cells)}{(mRNA in control cells)} \cdot 100 %$

The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. 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. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, 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., the central nervous system (CNS), optionally via intrathecal, intravitreal or other injection, or to the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. 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, that directs or otherwise stabilizes the RNAi agent at a site of interest, e.g., the CNS. 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.

In one embodiment, contacting a cell with a RNAi agent includes “introducing” or “delivering the RNAi agent into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of a RNAi agent can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing a RNAi agent into a cell may be in vitro or in vivo. For example, for in vivo introduction, a RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or are known in the art.

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, logK_ow, where K_owis 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 logK_owexceeds 0. Typically, the lipophilic moiety possesses a logK_owexceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10. For instance, the logK_owof 6-amino hexanol, for instance, is predicted to be approximately 0.7. Using the same method, the logK_owof 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., logK_ow) 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 one embodiment, 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, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), or a non-primate (such as a a rat, or a mouse). In a preferred embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder, or condition that would benefit from reduction in RPS25 expression; a human at risk for a disease, disorder, or condition that would benefit from reduction in RPS25 expression; a human having a disease, disorder, or condition that would benefit from reduction in RPS25 expression; or human being treated for a disease, disorder, or condition that would benefit from reduction in RPS25 expression as described herein.

As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more signs or symptoms associated with RPS25 gene expression or RPS25 protein production, e.g., RPS25-associated diseases, such as a nucleotide repeat expansion disease, e.g., C9orf72 ALS/FTD and Huntington's disease, e.g., Huntington-Like Syndrome Due To C9orf72 Expansions. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.

The term “lower” in the context of the level of RPS25 in a subject or a disease marker or symptom refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, a decrease is at least 20%. In certain embodiments, the decrease is at least 50% in a disease marker, e.g., protein or gene expression level. “Lower” in the context of the level of RPS25 in a subject is preferably down to a level accepted as within the range of normal for an individual without such disorder. In certain embodiments, “lower” is the decrease in the difference between the level of a marker or symptom for a subject suffering from a disease and a level accepted within the range of normal for an individual, e.g., the level of decrease in bodyweight between an obese individual and an individual having a weight accepted within the range of normal. As used herein, lowering can refer to lowering or predominantly lowering the level of mRNA of an RPS25 and/or C9orf72 or HTT gene having a nucleotide repeat expansion.

As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder, or condition thereof, that would benefit from a reduction in expression of an RPS25 gene or production of an RPS25 protein, refers to a reduction in the likelihood that a subject will develop a symptom associated with such a disease, disorder, or condition, e.g., a symptom of an RPS25-associated disease. The failure to develop a disease, disorder, or condition, or the reduction in the development of a symptom associated with such a disease, disorder, or condition (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention.

As used herein, the term “RPS25-associated disease” or “RPS25-associated disorder” is understood as any disease or disorder that would benefit from reduction in the expression and/or activity of RPS25, e.g., RAN-translation. Exemplary RPS25-associated diseases include nucleotide repeat expansion diseases, such as C9orf72 ALS/FTD, Huntington-Like Syndrome Due To C9orf72 Expansions, Fragile X syndrome (FXS), Myotonic dystrophy (i.e., DM1, and DM2), CAG/polyglutamine disease (e.g., Huntington's disease, Spinal and bulbar muscular atrophy (SBMA), Dentatorubral-pallidoluysian atrophy, Spinocerebellar ataxia type I, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3, Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, Spinocerebellar ataxia type 8, Spinocerebellar ataxia type 12, and Spinocerebellar ataxia type 17), Friedreich ataxia, Unverricht-Lundborg myoclonic epilepsy (EPM1), Oculopharyngeal muscular dystrophy (OPMD), and Fuchs endothelial corneal dystrophy (FECD).

A “nucleotide repeat expansion disease” is any disease or disorder that is the result of expansion of a simple sequence repeat (i.e., a microsatellite). The simple sequence repeat that is expanded may be a tri-, tetra-, penta-, hexa- or dodeca-nucleotide repeat. Exemplary nucleotide repeats include CAG (causing, e.g., Huntington disease, spinal and bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, and Spinocerebellar ataxia type I, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3, Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7 ATXN7, and Spinocerebellar ataxia type 17), CGG (causing, e.g., fragile X syndrome, GCC and CCG (causing, e.g., FRAXE mental retardation), CTG (causing, e.g., myotonic dystrophy type 1, Huntington disease-like 2, spinocerebellar ataxia type 8, Fuchs corneal dystrophy), GAA (causing, e.g., Friedreich ataxia), GCC (causing, e.g., FRAXE mental retardation), GCG (causing, e.g., oculopharyngeal muscular dystrophy), CCTG (causing, e.g., myotonic dystrophy type 1), ATTCT (causing, e.g., spinocerebellar ataxia type 10), TGGAA (causing, e.g., spinocerebellar ataxia type 31), GGCCTG (causing, e.g., spinocerebellar ataxia type 36), GGGGCC (causing, e.g., C9ORF72 frontotemporal dementia/amyotrophic lateral sclerosis), and CCCCGCCCCGCG (SEQ ID NO: 16) (causing, e.g., myoclonic epilepsy).

Subjects having a GGGGCC (or G4C2) hexanucleotide expansion in the C9ORF72 gene can present as amylotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD) even in the same family and, therefore, the neurodegeneration associated with this expansion is referred to herein as “C9orf72 Amyotrophic lateral sclerosis/frontotemporal dementia” or C9orf72 ALS/FTD.” It is an autosomal dominant disease and is the most common form of familial ALS, accounting for about a third of ALS families and 5-10% of sporadic cases in an ALS clinic. It is also a common cause of FTD, explaining about one fourth of familial FTD. Age of symptom onset ranges from 30 to 70 years of age with a mean onset in the late 50s. C9orf72-mediated ALS most often resembles typical ALS, can be bulbar or limb onset, can progress rapidly (though not always) and can be associated with later cognitive symptoms. Thus, C9orf72-mediated ALS is evaluated and treated just as in any ALS patient. The pattern of C9orf72-mediated FTD most commonly is behavioral variant FTD, with the full range of behavioral and cognitive symptoms including disinhibition, apathy and executive dysfunction. Less commonly, C9orf72-mediated FTD presents semantic variant primary progressive aphasia (PPA) or nonfluent variant PPA, and, very rarely, can resemble corticobasal syndrome, progressive supranuclear palsy or an HD-like syndrome. Occasionally parkinsonian features are seen in C9orf72-mediated ALS or FTD.

Normal G4C2 repeats are ˜25 units or less, and high penetrance disease alleles are typically greater than ˜60 repeat units, ranging up to more than 4,000 units; rarely, repeats between 47 and 60 segregate with disease in families A repeat-primed PCR assay is typically used to detect smaller expansions (<80), but accurately sizing larger repeats requires other techniques (e.g. Southern blot hybridization) that provides an estimate of length.

Subjects may exhibit frontotemporal lobar degeneration (FTLD) characterized by progressive changes in behavior, executive dysfunction, and/or language impairment. Of the three FTLD clinical syndromes, behavioral variant FTD (bvFTD) is most often, but not exclusively, present. It is characterized by progressive behavioral impairment and a decline in executive function with predominant frontal lobe atrophy on brain MRI. Motor neuron disease, including upper or lower motor neuron dysfunction (or both) that may or may not fulfill criteria for the full ALS phenotype may also be present. Some degree of parkinsonism, which is present in many individuals with C9orf72-related bvFTD, is typically of the akinetic-rigid type without tremor, and is levodopa unresponsive.

Although the functions of the C9orf72 protein are still being investigated, C9orf72 has been shown to interact with and activate Rab proteins that are involved in regulating the cytoskeleton, autophagy and endocytic transport. In addition, numerous cellular pathways have been demonstrated to be misregulated in neurodegenerative diseases associated with C9orf72 hexanucleotide repeat expansion. For example, altered RNA processing has consistently appeared at the forefront of research into C9orf72 disease. This includes bidirectional transcription of the repeat sequence, accumulation of repeat RNA into nuclear foci sequestering specific RNA-binding proteins (RBPs) and translation of RNA repeats into dipeptide repeat proteins (DPRs) by repeat-associated non-AUG (RAN)-initiated translation. Additionally, disruptions in release of the C9orf72 RNA from RNA polymerase II, translation in the cytoplasm and degradation have been shown to be disrupted by C9orf72 hexanucleotide repeat expansion. Furthermore, several alterations have been identified in the processing of the C9orf72 RNA itself, in terms of its transcription, splicing and localization (see, e.g., Barker, et al., (2017) Frontiers Cell Neurosci 11:1-15).

Irrespective of the mechanism, several groups have identified the presence of sense and antisense C9orf72-containing foci as well as the presence of aberrant dipeptide-repeat (DPR) proteins (poly(GA), poly(GR), poly(GP), poly(PA), and poly(PR)) produced from all reading frames of either sense or antisense repeat-containing C9orf72 RNAs through repeat-associated non-AUG-dependent (RAN) translation in several cell types in the nervous systems of subjects having C9orf72 ALS/FTD (Lagier-Tourenne, et al. (2013) Proc Natl Acad Sci USA doi/10.1073/pnas.1318835110; Jiang, et al. (2016) Neuron 90:535-550). Furthermore, in mice with one allele of C9orf72 inactivated no disease was provoked but, in mice with both C9orf72 alleles inactivated, splenomegaly, enlarged lymph nodes, and mild social interaction deficits, but no motor dysfunction was observed. In addition, in mice expressing human C9orf72 RNAs with up to 450 GGGGCC repeats (SEQ ID NO: 17) it was shown that hexanucleotide expansions caused age-, repeat-length-, and expression-level-dependent accumulation of sense and antisense RNA-containing foci and dipeptide-repeat proteins synthesized by AUG-independent translation, accompanied by loss of hippocampal neurons, increased anxiety, and impaired cognitive function (Jiang, et al. (2016) Neuron 90:535-550).

Huntington's disease-like syndromes (HD-like syndromes, or HDL syndromes) are a family of inherited neurodegenerative diseases that closely resemble Huntington's disease (HD) in that they typically produce a combination of chorea, cognitive decline or dementia and behavioural or psychiatric problems. Subjects having HD-like syndromes do not harbor the characteristic repeats in the huntingtin gene that cause that disorder.

Subjects having Huntington disease-like syndrome due to C9ORF72 expansions are characterized as having movement disorders, including dystonia, chorea, myoclonus, tremor and rigidity. Associated features are also cognitive and memory impairment, early psychiatric disturbances and behavioral problems. The mean age at onset is about 43 years (range 8-60). Early psychiatric and behavioral problems (including depression, apathy, obsessive behaviour, and psychosis) are common. Cognitive symptoms present as executive dysfunction. Movement disorders are prominent: Parkinsonian features and pyramidal features may also be present. A repeat-primed PCR assay is typically used to detect smaller expansions (<80), but accurately sizing larger repeats requires other techniques (e.g. Southern blot hybridization) that provides an estimate of length.

Fragile X syndrome (FXS) is named after the folate-sensitive fragile site at the FRAXA locus on the X chromosome. The most common cause of inherited mental retardation, FXS typically affects males, varies greatly in severity, and is associated with dysmorphic features including enlarged head, ears and testicles. Scientists were puzzled for years that the risk of FXS increased from one generation to the next. Indeed, this particular example of anticipation carried its own name, the Sherman paradox. The discovery in 1991 that FXS and its underlying fragile site are caused by an expanded CGG repeat that changes size over generations explained the paradox. Normal-sized repeats are polymorphic, ranging from 6 to 52, with repeats at the high end of this range being increasingly prone to further expand (“mutable normal”). In FXS families the repeat sizes span a wide range, from “premutations” of ˜60-200 repeats (typically found in maternal grandfathers) to full mutations of several thousand repeats (found in affected FXS males). The mothers of affected FXS males have variably sized expansions and are prone to premature ovarian failure.

The molecular mechanism of FXS is a loss of expression of the developmentally important nervous system protein, FMRP. Full expansions promote hypermethylation of the FMR1 promoter and reduce translation of the transcript, effectively silencing expression of the gene.

Myotonic dystrophy is an autosomal dominant multi-system disease characterized principally by myotonic myopathy. There are two major forms of myotonic dystrophy, both caused by repeat expansions. DM1, also known as Steinert disease, is caused by a CTG expansion in the 3′UTR of the DMPK gene. DM2, which is much less common than DM1 and was previously known as proximal myotonic myopathy, is caused by a CCTG repeat in intron 1 of the CNBP gene (formerly named ZNF9). Despite their similarities, DM1 and DM2 differ in important molecular and clinical respects. Most importantly, DM1 shows robust repeat length/disease severity correlation as well as significant anticipation, whereas DM2 does not.

DM1 is characterized by progressive weakness and myotonia, often associated with cataracts, cardiac arrhythmias, endocrinopathy and cognitive impairment. The range of severity is broad, with differences in repeat length being the key driver of disease severity. “Mild” disease may manifest simply with premature cataracts and baldness, with electromyographically detectable myotonia. “Classic” disease typically manifests in young adulthood and includes distal weakness, symptomatically and often disabling myotonia, as well as significant cardiac conduction defects in addition to cataracts and baldness. Classic disease, when presenting in teen years, is also known as “juvenile” disease. “Congenital” DM1, in which the affected parent is nearly always the mother, is present at birth. The infant is floppy, facial and jaw muscles are weak resulting in failure to thrive, and mental retardation and development delay are common. Rather than displaying myotonia, congenital DM muscles display features of arrested fiber development. Some unaffected individuals have repeats in the “mutable normal” range of 35-49 repeats. Such metastable alleles are prone to expand when transmitted to the next generation; new mutations in families arise through this process. An important, life-threatening feature of DM1 is cardiac involvement which can lead to sudden cardiac death. Repeat length and cardiac abnormalities also are correlated in DM1.

DM2 commonly presents as proximal muscle weakness with variable myotonia, hence its former name proximal myotonic myopathy. Like DM1, it too shows marked clinical heterogeneity ranging from mild forms of disease that may be difficult to detect, to profound and disabling proximal muscle weakness. There is no congenital form of disease nor is there apparent anticipation. Cardiac involvement is less common in DM2 than in DM1, but still requires careful monitoring. Whereas in DM1 cognitive impairment is well described, DM2 shows much less cognitive involvement. The CCTG repeat expansion in DM2 is complex, including repeat elements in addition to the CCTG repeat, and is prone to an extreme range of pathogenic expansions, from 75 units to as many as 11,000 units (mean of roughly 5000 repeats).

The molecular mechanism of disease may be as well worked out for DM1 as it is for any repeat expansion disease. The CTG expansion resides in the 3′UTR of the DMPK transcript, where it does not alter expression of the disease protein, but does form RNA foci and bind to and sequester essential splicing factors. This toxic RNA effect leads to a failure to generate appropriately spliced isoforms of key muscle genes, leading to myotonia and other symptoms of disease. The pathogenic basis of DM2 is less clear, but leading candidates include a toxic RNA effect.

Numerous diseases (e.g., Huntington disease, Spinal and bulbar muscular atrophy (SBMA), Dentatorubral-pallidoluysian atrophy, Spinocerebellar ataxia type I, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3, Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, Spinocerebellar ataxia type 8, Spinocerebellar ataxia type 12, and Spinocerebellar ataxia type 17) belong to the CAG/polyglutamine disease group. All, except SBMA which is an X-linked disorder with dominant toxic features, are dominantly inherited disorders. All are classified as rare diseases. HD, the best known among them, is also the most common, with SCA3 next in line. Six are dominantly inherited ataxias (also known as SCAs) including the four most common SCAs among the 40 discovered thus far (SCAs 1,2,3,6). A seventh disorder, DRPLA, can be thought of as a hybrid between SCA and HD. In all nine, the primary pathogenic mechanism is believed to be proteotoxicity emanating from the encoded disease protein. Other than sharing a common glutamine repeat, the various disease proteins are entirely unrelated and serve very different cellular functions. The distinctive clinical and pathological features of individual CAG/polyglutamine diseases are believed to stem primarily from this differing protein context. At least two other repeat expansion diseases may share elements with the polyglutamine diseases: In SCAB, the antisense transcript can encode a polyglutamine protein through RAN translation and the CAG repeat in SCA12 can encode polyglutamine.

Families with a dominantly inherited disease resembling HD (chorea, cognitive impairment and psychiatric disturbance) may instead have Huntington disease-like 2 (HDL2). This rare phenocopy of HD is caused by a CTG repeat expansion in the Junctophilin-3 (JPH3) gene. Normal repeats are between 6 and 28, whereas expanded repeats are between ˜41 and 58 repeats. Disease typically occurs in midlife and recapitulates many features of HD, with weight loss being a frequent finding. Similar to HD, some individuals with HDL2 can present with juvenile onset disease resembling the Westphal variant of HD (rigidity, parkinsonism, dystonia). The brain MRI often resembles that of HD, showing selective atrophy of the basal ganglia and cortex with relative sparing of the brainstem and cerebellum. The diagnosis of HDL2 cannot be established without molecular genetic testing for the repeat expansion. The pathogenic mechanism remains uncertain and, as with other repeat expansion diseases, may have multiple components. Located in an alternatively spliced exon of the JPH3 gene, the repeat can be transcribed in both directions, leading to CUG (more common) or CAG (less common) repeat-containing transcripts. While a dominant RNA toxic effect may occur, the repeat expansion also reduces levels of the Junctophilin-3 protein, which could prove deleterious to neurons.

Friedreich ataxia is the most common autosomal recessive ataxia, present primarily in Indian and European populations. Before the disease mutation was discovered, Friedreich ataxia was defined as a young onset progressive ataxia with sensory loss, scoliosis, areflexia and cardiomyopathy occurring before age 25. Other disabling features of disease include hearing loss, motor weakness, and diabetes. The discovery of a GAA repeat expansion in the FRDA gene soon led to the recognition that the classic definition of disease, requiring onset before age 25, was incorrect. While most Friedreich ataxia meets this classic definition, roughly a quarter of individuals develop signs of disease after age 25. Moreover, late onset Friedreich ataxia may not show the classically described areflexia and is less likely to have significant cardiac involvement. Most affected individuals are homozygous for the expansion but a small percentage are compound heterozygotes who have an inactivating or deletion mutation in one allele and an expansion in the other allele. There is robust repeat length-disease correlation, with the size of the smaller of the two expansions showing inverse correlation with age of symptom onset and a direct correlation with the probability of significant cardiac dysfunction. Occasionally, however, disease features can vary widely within a family despite similar sized expansions indicating that other factors influence disease severity beyond the mutation.

The basis of disease is impairment in mitochondrial function due to loss of frataxin, a protein required for the assembly of iron-sulfur cluster enzymes in the mitochondria. Frataxin fails to be expressed principally because the GAA expansion directly impedes transcription, although a contributing factor is expansion-induced epigenetic silencing of the upstream promotor.

Unverricht-Lundborg myoclonic epilepsy (EPM1) is the most common cause of myoclonic epilepsy in North America, typically beginning between 6 and 15 years of age and progressing over time. The initial symptom can be either action- or stimulus-induced myoclonus or generalized tonic-clonic seizures, but eventually both are present in affected persons. Ataxia also is a common feature. The EEG shows photosensitive spike and wave abnormalities and the background can be slowed. While cognition is generally normal, mild intellectual deficits may develop over time. The myoclonus is progressive and can be very disabling, leading to wheelchair use for approximately one third of affected individuals.

This autosomal recessive disease is caused by expansion of a dodecamer repeat in the CSTB gene which encodes the lysosomal protein cystatin B. Normal repeats are 2-3 units in length and expansions range from 30 to ˜125 repeats. Most persons with EPM1 will be homozygous for expansions though a small percentage will have an activating mutation on one allele and an expansion on the other.

Oculopharyngeal muscular dystrophy (OPMD) is a dominantly inherited neuromuscular disorder characterized by adult onset progressive weakness, ptosis, ophthalmoparesis and dysphagia. The cause is a small GC(N) expansion in the polyadenylate binding protein 2 (PABP2) gene that modestly enlarges a polyalanine tract in the protein. OPMD is one of at least nine polyalanine diseases, the remainder of which are congenital neurocognitive disorders in which the expansions occur in transcription factors. In contrast, OPMD is an adult onset, progressive, degenerative disease. Reminiscent of the CAG/polyglutamine diseases, OPMD is a proteinopathy: the enlarged alanine tract promotes aggregation of the disease protein, resulting in the formation of intranuclear inclusions in skeletal muscle.

The normal GC(N) repeat length is typically 6 units and expansions are between 8 and 18 in disease. The GC(N) expansions can be either GCG or a mixture of GCG and GCA, both of which encode alanine. A distinctive feature of OPMD is that while most individuals possess a single expanded allele, some affected persons are compound heterozygotes with one allele containing 7 repeats and other 9 repeats. Remarkably, a small percentage of OPMD presents in an autosomal recessive manner wherein affected individuals are homozygous for alleles of 7 repeats. Evidence does not support anticipation in OPMD, but there is some support for a correlation between repeat length and disease severity.

Fuchs endothelial corneal dystrophy (FECD) is included here among the neurological repeat expansion diseases because it affects vision, is relatively common, and is one of the most recently described repeat expansion diseases. FECD is a degenerative condition characterized by progressive loss of corneal endothelium, thickening of the Descemet's membrane and deposition of extracellular matrix in the cornea. This process results in progressive corneal edema and visual loss, typically after age 60. At least five other genes or genetic loci are associated with FECD, but the most common form—a late onset form—is associated with modest expansion of an intronic CTG repeat in the transcription factor four (TCF4) gene. Normal CTG repeats are between 10 and 37, and pathogenic repeats are greater than 50. Little is known about how the expansion contributes to disease, but the current leading hypothesis is a toxic RNA effect.

“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having an RPS25-associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating, or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended to include the amount of a RNAi agent that, when administered to a subject having an RPS25-associated disorder, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the RNAi agent, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effective amount” also includes an amount of a RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. A RNAi agent employed in the methods of the present disclosure may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

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

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the brain (e.g., whole brain or certain segments of brain, e.g., striatum, or certain types of cells in the brain, such as, e.g., neurons and glial cells (astrocytes, oligodendrocytes, microglial cells)). In some embodiments, a “sample derived from a subject” refers to blood drawn from the subject or plasma or serum derived therefrom. In further embodiments, a “sample derived from a subject” refers to brain tissue (or subcomponents thereof) or retinal tissue (or subcomponents thereof) derived from the subject.

II. RNAi Agents of the Disclosure

Described herein are RNAi agents which inhibit the expression of an RPS25 gene. In one embodiment, the RNAi agent includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of an RPS25 gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having an RPS25-associated disease, e.g., a nucleotide repeat expansion disease, e.g., C9orf72 ALS/FTD, Huntington-Like Syndrome Due To C9orf72 Expansions, Fragile X syndrome (FXS), Myotonic dystrophy (i.e., DM1, and DM2), CAG/polyglutamine disease (e.g., Huntington's disease, Spinal and bulbar muscular atrophy (SBMA), Dentatorubral-pallidoluysian atrophy, Spinocerebellar ataxia type I, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3, Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, Spinocerebellar ataxia type 8, Spinocerebellar ataxia type 12, and Spinocerebellar ataxia type 17), Friedreich ataxia, Unverricht-Lundborg myoclonic epilepsy (EPM1), Oculopharyngeal muscular dystrophy (OPMD), and Fuchs endothelial corneal dystrophy (FECD). 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 an RPS25 gene, The region of complementarity is about 15-30 nucleotides or less in length. Upon contact with a cell expressing the RPS25 gene, the RNAi agent inhibits the expression of the RPS25 gene (e.g., a human gene, a primate gene, a non-primate gene) by at least 50% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, western blotting or flowcytometric techniques. In a preferred embodiment, the level of knockdown is assayed at a 10 nM concentration of siRNA in human neuroblastoma BE(2)-C cells using a Dual-Luciferase assay method provided in Example 2 below.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of an RPS25 gene. The other strand (the sense strand) 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. 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.

Generally, the duplex structure is 15 to 30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain preferred embodiments, the duplex structure is 18 to 25 base pairs in length, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24,20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24 or 24-25 base pairs in length, for example, 19-21 basepairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

Similarly, the region of complementarity to the target sequence is 15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, for example 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

In some embodiments, the dsRNA is 15 to 23 nucleotides in length, or 25 to 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the 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 allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).

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 about 15 to 36 base pairs, e.g., 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, for example, 19-21 base pairs. Thus, in one embodiment, 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 one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, a RNAi agent useful to target RPS25 expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can 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.

A dsRNA can be synthesized by standard methods known in the art.

In one aspect, a dsRNA of the disclosure includes at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand sequence for RPS25 may be selected from the group of sequences provided in any one of Tables 2-14, and the corresponding nucleotide sequence of the antisense strand of the sense strand may be selected from the group of sequences of any one of Tables 2-14. In this aspect, 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 in the expression of an RPS25 gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand (passenger strand) in any one of Tables 2-14, and the second oligonucleotide is described as the corresponding antisense strand (guide strand) of the sense strand in any one of Tables 2-14 for RPS25.

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

It will be understood that, although the sequences in Tables 3, 5, 7, 9, 11, 12, and 14 are described as modified or conjugated sequences, the RNA of the RNAi agent of the disclosure e.g., a dsRNA of the disclosure, may comprise any one of the sequences set forth in any one of Tables 2-14 that is un-modified, un-conjugated, or modified or conjugated differently than described therein. For example, the modified sequences provided in Tables 3, 5, 7, 9, 11, and 12 may not require a dT. A lipophilic ligand can be included in any of the positions provided in the instant application.

The skilled person is well aware that dsRNAs having a duplex structure of about 20 to 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided herein, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein, and differing in their ability to inhibit the expression of an RPS25 gene by not more than 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence using the in vitro assay with Cos7 and a 10 nM concentration of the RNA agent and the PCR assay as provided in the examples herein, are contemplated to be within the scope of the present disclosure.

In addition, the RNAs described herein identify a site(s) in an RPS25 transcript that is susceptible to RISC-mediated cleavage. As such, the present disclosure further features RNAi agents that target within this site(s). As used herein, a RNAi agent is said to target within a particular site of an RNA transcript if the RNAi agent promotes cleavage of the transcript anywhere within that particular site. Such a RNAi agent will generally include at least about 15 contiguous nucleotides, preferably at least 19 nucleotides, from one of the sequences provided herein coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in an RPS25 gene.

A RNAi agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, a RNAi agent as described herein contains no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, a RNAi agent as described herein contains no more than 2 mismatches. In one embodiment, a RNAi agent as described herein contains no more than 1 mismatch. In one embodiment, a RNAi agent as described herein contains 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches to the target sequence, the mismatch can optionally be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand which is complementary to a region of an RPS25 gene, 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 a RNAi agent containing a mismatch to a target sequence is effective in inhibiting the expression of an RPS25 gene. Consideration of the efficacy of RNAi agents with mismatches in inhibiting expression of an RPS25 gene is important, especially if the particular region of complementarity in an RPS25 gene is known to have polymorphic sequence variation within the population.

III. Modified RNAi Agents of the Disclosure

In one embodiment, the RNA of the RNAi agent of the disclosure e.g., a dsRNA, is un-modified, and does not comprise, e.g., chemical modifications or conjugations known in the art and described herein. In preferred embodiments, the RNA of a RNAi agent of the disclosure, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the disclosure, substantially all of the nucleotides of a RNAi agent of the disclosure are modified. In other embodiments of the disclosure, all of the nucleotides of a RNAi agent of the disclosure are modified. RNAi agents of the disclosure in which “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides. In still other embodiments of the disclosure, RNAi agents of the disclosure can include not more than 5, 4, 3, 2 or 1 modified nucleotides.

The nucleic acids featured in the disclosure can be synthesized 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, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); 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; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNAi agents useful in the embodiments described herein 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 some embodiments, a modified RNAi agent 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, the entire contents of each of which are hereby incorporated herein 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 CH₂component 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, the entire contents of each of which are hereby incorporated herein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use in RNAi agents, in which 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, a 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 a 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, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the RNAi agents of the disclosure are described in, 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 can also contain one or more substituted sugar moieties. The RNAi agents, 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 can be substituted or unsubstituted C₁to C₁₀alkyl or C₂to C₁₀alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_nO]_mCH₃, O(CH₂)._nOCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_n[(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: C₁to C₁₀lower 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 a RNAi agent, or a group for improving the pharmacodynamic properties of a RNAi agent, 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)₂group, also known as 2′-DMAOE, as described in examples herein below, 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. Further exemplary modifications include: 5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides, 5′-Me-2′-deoxynucleotides, (both R and S isomers in these three families); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-O-hexadecyl, and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of a RNAi agent, 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. RNAi agents can 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. The entire contents of each of the foregoing are hereby incorporated herein by reference.

A RNAi agent of the disclosure can 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 et al., (1991) Angewandte Chemie, International Edition, 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. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

A RNAi agent of the disclosure can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting 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, 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).

A RNAi agent of the disclosure can also be modified to include one or more bicyclic sugar moities. 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 (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4′-CH2-O-2′ bridge. 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, 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 entire contents of each of the foregoing are hereby incorporated herein by reference.

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

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

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)-O-2′ bridge. In one embodiment, 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 entire contents of each of which are hereby incorporated herein by reference.

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 hereby incorporated by reference).

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 entire contents of each of which are hereby incorporated herein by reference.

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 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 entire contents of which are incorporated herein by reference.

A. Modified RNAi Agents Comprising Motifs of the Disclosure

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 entire contents of which are incorporated herein by reference. 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 ligand, e.g., a C16 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.

Accordingly, the disclosure provides double stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., an RPS25 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be 15-30 nucleotides in length. For example, each strand may be 16-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length. In certain embodiments, each strand is 19-23 nucleotides in length.

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

In one embodiment, the RNAi agent may contain one or more overhang regions or capping groups at the 3′-end, 5′-end, or both ends of one or both strands. The overhang can be 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. In preferred embodiments, the nucleotide overhang region is 2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In one embodiment, the nucleotides in the overhang region of the RNAi agent can each independently be a modified or unmodified nucleotide including, but no limited to 2′-sugar modified, such as, 2-F, 2′-O-methyl, thymidine (T), and any combinations thereof.

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

The 5′- or 3′-overhangs at the sense strand, antisense strand or both strands of the RNAi agent may be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3′-end of the sense strand, antisense strand, or both strands. In one embodiment, this 3′-overhang is present in the antisense strand. In one embodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthen the interference activity of the RNAi, without affecting its overall stability. For example, the single-stranded overhang may be located at the 3′-terminal end of the sense strand or, alternatively, at the 3′-terminal end of the antisense strand. The RNAi may also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa. Generally, the antisense strand of the RNAi has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While not wishing to be bound by theory, the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process.

In one embodiment, the RNAi agent is a double ended bluntmer of 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 7, 8, 9 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′ end.

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

In yet another embodiment, the RNAi agent is a double ended bluntmer of 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′ end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′ end; the antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′ end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2 nucleotide overhang. Preferably, the 2 nucleotide overhang is at the 3′-end of the antisense strand. When the 2 nucleotide overhang is at the 3′-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand. In one embodiment, every nucleotide in the sense strand and the antisense strand of the RNAi agent, including the nucleotides that are part of the motifs are modified nucleotides. In one embodiment each residue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g., in an alternating motif. Optionally, the RNAi agent further comprises a ligand (e.g., a lipophilic ligand, optionally a C16 ligand).

In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3 ‘ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3’ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises sense and antisense strands, wherein the RNAi agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at position 11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand and the 5′ end of the second strand form a blunt end and the second strand is 1˜4 nucleotides longer at its 3′ end than the first strand, wherein the duplex region region which is at least 25 nucleotides in length, and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotide of the second strand length to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and wherein dicer cleavage of the RNAi agent preferentially results in an siRNA comprising the 3′ end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.

In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand.

For an RNAi agent having a duplex region of 17-23 nucleotide in length, the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5′-end. Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, the count starting from the 1^stnucleotide from the 5′-end of the antisense strand, or, the count starting from the 1^stpaired nucleotide within the duplex region from the 5′-end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand and the other motifs may be a wing modification. The term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other then the chemistry of the motifs are distinct from each other and when the motifs are separated by one or more nucleotide than the chemistries can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, each wing modification may occur at one end relative to the first motif which is at or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the RNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that may be present on the sense strand.

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

In another embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two paired nucleotides within the duplex region at the 3′-end, 5′-end or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two or three nucleotides.

When the sense strand and the antisense strand of the RNAi agent each contain at least two wing modifications, the sense strand and the antisense strand can be so aligned that two modifications each from one strand fall on one end of the duplex region, having an overlap of one, two or three nucleotides; two modifications each from one strand fall on the other end of the duplex region, having an overlap of one, two or three nucleotides; two modifications one strand fall on each side of the lead motif, having an overlap of one, two, or three nucleotides in the duplex region.

In one embodiment, the RNAi agent comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mismatch may occur in the overhang region or the duplex region. The base pair may 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 one embodiment, the RNAi agent 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 independently selected 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 one embodiment, 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.

In another embodiment, the nucleotide at the 3′-end of the sense strand is deoxy-thymine (dT). In another embodiment, the nucleotide at the 3′-end of the antisense strand is deoxy-thymine (dT). In one embodiment, there is a short sequence of deoxy-thymine nucleotides, for example, two dT nucleotides on the 3′-end of the sense or antisense strand.

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

5′n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q3′ (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

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

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

each n_pand n_qindependently represent an overhang nucleotide;

wherein N_band 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. Preferably YYY is all 2′-F modified nucleotides.

In one embodiment, the N_aor N_bcomprise modifications of alternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif 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^stnucleotide, from the 5′-end; or optionally, the count starting at the 1^stpaired nucleotide within the duplex region, from the 5′-end.

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

5′n_p-N_a-YYY-N_b-ZZZ-N_a-n_q3′ (Ib);

5′n_p-N_a-XXX-N_b-YYY-N_a-n_q3′ (Ic); or

5′n_p-N_a-XXX-N_b-YYY-N_b-ZZZ-N_a-n_q3′ (Id).

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

Each N_aindependently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), N_brepresents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_acan 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 N_bindependently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_bis 0, 1, 2, 3, 4, 5 or 6. Each N_acan 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′n_p-N_a-YYY-N_a-n_q3′ (Ia).

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

In one embodiment, the antisense strand sequence of the RNAi may be represented by formula (II):

5′n_q′-N_a′-(Z′Z′Z′)_k-N_b′-Y′Y′Y′-N_b′-(X′X′X′)_l-N′_a-n_p′3′ (II)

wherein:

k and 1 are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_a′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides,

each sequence comprising at least two differently modified nucleotides;

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

each n_p′ and n_q′ independently represent an overhang nucleotide;

wherein N_b′ and Y′ do not have the same modification;

and

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

In one embodiment, the N_a′ or N_b′ comprise modifications 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 nucleotide 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 1^stnucleotide, from the 5′-end; or optionally, the count starting at the 1^stpaired nucleotide within the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

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

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

5′n_q′-N_a′-Z′Z′Z′-N_b′-Y′Y′Y′-N_a′-n_p′3′ (IIb);

5′n_q′-N_a′-Y′Y′Y′-N_b′-X′X′X′-n_p′3′ (IIc); or

5′n_q′-N_a′-Z′Z′Z′-N_b′-Y′Y′Y′-N_b′-X′X′X′-N_a′-n_p′3′ (IId).

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

When the antisense strand is represented as formula (IIC), N_b′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IId), each N_b′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N_bis 0, 1, 2, 3, 4, 5 or 6.

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

5′n_p′-N_a′-Y′Y′Y′-N_a-n_q′3′ (Ia).

When the antisense strand is represented as formula (IIa), each N_a′ 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, 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 one embodiment, 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^stnucleotide from the 5′-end, or optionally, the count starting at the 1^stpaired 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 one embodiment the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 1^stnucleotide from the 5′-end, or optionally, the count starting at the 1^stpaired 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 a antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.

Accordingly, the 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 (III):

sense: 5′n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q3′

antisense: 3′n_p′-N_a′-(X′X′X′)_k-N_b′-Y′Y′Y′-N_b′-(Z′Z′Z′)_l-N_a′-n_q′5′ (III)

wherein:

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

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

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

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

wherein

each n_p′, n_p, n_q′, and n_q, 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 modifications on three consecutive nucleotides.

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

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

5′n_p-N_a-Y Y Y-N_a-n_q3′3′n_p′-N_a′-Y′Y′Y′-N_a′n_q′5′ (IIa)

5′n_p-N_a-Y Y Y-N_b-Z Z Z-N_a-n_q3′3′n_p′-N_a′-Y′Y′Y′-N_b′-Z′Z′Z′-N_a′n_q′5′ (IIIb)

5′n_p-N_a-X X X-N_b-Y Y Y-N_an_q3′3′n_p′-N_a′-X′X′X′-N_b′-Y′Y′Y′-N_a′-n_q′5′ (IIIc)

5′n_p-N_a-X X X-N_b-Y Y Y-N_b-Z Z Z-N_a-n_q3′3′n_p′-N_a′-X′X′X′-N_b′-Y′Y′Y′-N_b′-Z′Z′Z′-N_a-n_q′5′ (IIId)

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

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

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

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

In one embodiment, when the RNAi agent is represented by formula (IIId), the N_amodifications are 2′-O-methyl or 2′-fluoro modifications. In another embodiment, when the RNAi agent is represented by formula (IIId), the N_amodifications are 2′-O-methyl or 2′-fluoro modifications and n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In yet another embodiment, when the RNAi agent is represented by formula (IIId), the N_amodifications are 2′-O-methyl or 2′-fluoro modifications, n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more C16 (or related) moieties attached through a bivalent or trivalent branched linker (described below). In another embodiment, when the RNAi agent is represented by formula (IIId), the N_amodifications are 2′-O-methyl or 2′-fluoro modifications, n_p′>0 and at least one n_p′ 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 lipophilic, e.g., C16 (or related) moieties, optionally attached through a bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIa), the N_amodifications are 2′-O-methyl or 2′-fluoro modifications, n_p′>0 and at least one n_p′ 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 lipophilic, e.g., C16 (or related) moieties attached through a bivalent or trivalent branched linker.

In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIa), (IIIb), (IIIc), and (IIId), 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 one embodiment, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIa), (IIIb), (IIIc), and (IIId), 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 one embodiment, two RNAi agents represented by formula (III), (IIa), (IIIb), (IIIc), and (IIId) 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 entire contents of each of which are hereby 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.

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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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 antisense 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 R, 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., C C2′, C3′, C4′ or 04′) 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, R¹and R²independently are H, halogen, OR₃, or alkyl; and R₃is 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 target mRNA, such as:

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wherein R is H, OH, OCH₃, F, NH₂, NHMe, NMe₂or O-alkyl.

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

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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 comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5′-end. In some other embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 14, and 16 from the 5′-end. In still some other embodiments, the antisense 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, the dsRNA molecule of the disclosure comprising a sense and antisense strands, wherein: the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1), positions 1 to 23 of said sense strand comprise at least 8 ribonucleotides; antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3 ‘ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3’ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell; and wherein the antisense strand contains at least one thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is in the seed region of the antisense strand (i.e. at position 2-9 of the 5′-end of the antisense strand). For example, the thermally destabilizing nucleotide occurs between positions opposite or complimentary to positions 14-17 of the 5′-end of the sense strand, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following 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; and (vi) the dsRNA comprises at least four 2′-fluoro modifications; and (vii) the dsRNA comprises a duplex region of 12-30 nucleotide pairs in length.

In some embodiments, the dsRNA molecule of the disclosure comprises a sense and antisense strands, wherein said dsRNA molecule comprises a sense strand having a length which is at least 25 and at most 29 nucleotides and an antisense strand having a length which is at most 30 nucleotides with the sense strand comprises a modified nucleotide that is susceptible to enzymatic degradation at position 11 from the 5′ end, wherein the 3′ end of said sense strand and the 5′ end of said antisense strand form a blunt end and said antisense strand is 1-4 nucleotides longer at its 3′ end than the sense strand, wherein the duplex region which is at least 25 nucleotides in length, and said antisense strand is sufficiently complementary to a target mRNA along at least 19 nt of said antisense strand length to reduce target gene expression when said dsRNA molecule is introduced into a mammalian cell, and wherein dicer cleavage of said dsRNA preferentially results in an siRNA comprising said 3′ end of said antisense strand, thereby reducing expression of the target gene in the mammal, wherein the antisense strand contains at least one thermally destabilizing nucleotide, where the at least one thermally destabilizing nucleotide is in the seed region of the antisense strand (i.e. at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six 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; and (vi) the dsRNA comprises at least four 2′-fluoro modifications; and (vii) the dsRNA has a duplex region of 12-29 nucleotide pairs in length.

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 0 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 0 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 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 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 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 antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven or all eight) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5 or 6 2′-fluoro modifications; (ii) the antisense comprises 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; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at 5′-end of the antisense strand.

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

In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven or all eight) of the following 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 3, 4 or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2′-fluoro modifications; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at 5′-end of the antisense strand.

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

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, WO2007/117686, WO2009/014887, and WO2011/031520 which are hereby incorporated by their entirely.

As described in more detail below, the RNAi agent that contains conjugations of one or more carbohydrate moieties to an 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 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 2-14. These agents may further comprise a ligand.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involves chemically linking to the iRNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA, e.g., into a cell. 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 certain 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. Example 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 α 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. In certain embodiments, the ligand is a multivalent galactose, e.g., an N-acetyl-galactosamine.

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, 03-(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 a cancer cell, endothelial cell, or bone 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, 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 invention 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 iRNAs of the invention 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 invention 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 invention, 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 invention 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. Lipid Conjugates

In certain embodiments, the ligand or conjugate 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 distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen 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, 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 certain 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 certain 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).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, such as a helical cell-permeation agent. In certain embodiments, the agent is amphipathic. An exemplary agent is a peptide such as that or antennapedia. 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: 18). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 19)) 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: 20)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 21)) 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 invention 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. Preferred 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 an iRNA agent to a tumor cell expressing αvβ₃(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).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, an iRNA 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 tri-saccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In certain embodiments, a carbohydrate conjugate comprises a monosaccharide.

In certain 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 conjugated to the 5′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5′ end of the sense strand) via a linker, e.g., a linker as described herein.

In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker. In other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a tetravalent linker.

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

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

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 0 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 certain embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

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In certain embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In certain embodiments, the monosaccharide is an N-acetylgalactosamine, such as

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

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

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In certain embodiments, the RNAi agents of the disclosure may include GalNAc ligands, even if such GalNAc ligands are currently projected to be of limited value for the preferred intrathecal/CNS delivery route(s) of the instant disclosure.

In certain embodiments, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent, e.g., the 5′ end of the sense strand of a dsRNA agent, or the 5′ end of one or both sense strands of a dual targeting RNAi agent as described herein. In certain embodiments, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.

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 or a cell permeation peptide.

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

D. 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.

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. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NRB, 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 certain 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.

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 preferred embodiment, 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 preferred 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. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

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 preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster 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 certain 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 certain 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. Preferred embodiments 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—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

iii. Acid Cleavable Linking Groups

In certain 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 preferred 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). A preferred embodiment is when 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 certain 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 yet another embodiment, 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 alkynylene. 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.

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

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

In certain embodiments of the compositions and methods of the invention, a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

In certain embodiments, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XLV)-(XLVI):

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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;

P^2A, P^2B, P^3A, P^3B, P^4A, P^4B, P^5A, P^5B, P^5C, T^2A, T^2B, T^3A, T^3B, T^4A, T^4B, T^4A, T^5B, T^5Care each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;

Q^2A, Q^2B, Q^3A, Q^3B, Q^4A, Q^4B, Q^5A, Q^5B, Q^5C, 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(R^N), C(R′)═C(R″), C≡C or C(O);

R^2A, R^2B, R^3A, R^3B, R^4A, R^4B, R^5A, R^5B, R^5Care each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^a)C(O), —C(O)—CH(R^a)—NH—, CO, CH═N—O,

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or heterocyclyl; L^2A, L^2B, L^3A, L^3B, L^4A, L^4B, L^5A, L^5Band L^5Crepresent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R^ais 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 (XLIX):

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wherein L^5A, L^5Band L^5Crepresent 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.

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; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, the entire contents of each of which are hereby incorporated herein 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 can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNA agents, 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, or increased binding affinity for the target nucleic acid. An additional region of the iRNA can 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 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 can 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. In Vivo Testing of RPS25 Knockdown

A number of RPS25 mouse models are known in the art and reviewed in, for example, Batra and Lee (2017) Front Cell Neurosci. 11: 196. Such models can be used to demonstrate the in vivo efficacy of the RNAi agents provided herein. Some exemplary models are provided below.

A mouse model carrying an AAV2/9 vector expressing a C9orf72 G4C2-repeat DNA (hexanucleotide repeat expansion (GGGGCC (G4C2) (HRE)) with a 119 base-pair (bp) of the upstream 5′ region and 100 bp of the downstream 3′ region of the human C9orf72 (Chew J., et al. (2015). Science 348 1151-1154).

Another example is a transgenic mouse model carrying a bacterial artificial chromosome (BAC) DNA clone containing a partial human C9orf72 gene region, including exons 1 to 6, a (G4C2) 500 region (SEQ ID NO: 22) and a 141 Kb 5′ upstream region (Peters O. M., et al. (2015). Neuron 88 902-909).

A mouse model carrying a BAC clone containing the human C9orf72 locus, including all 11 exons, a (G4C2) 800 region (SEQ ID NO: 2519), 110 Kb 5′ upstream and 20 Kb 3′ downstream flanking regions of the C9orf72 gene (O'Rourke J. G., et al. (2015). Neuron 88 892-901).

VI. Delivery of a RNAi Agent of the Disclosure

The delivery of a RNAi agent of the disclosure to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having an RPS25-associated disorder, e.g., a nucleotide repeat expansion diseases, such as C9orf72 ALS/FTD, Huntington-Like Syndrome Due To C9orf72 Expansions, Fragile X syndrome (FXS), Myotonic dystrophy (i.e., DM1, and DM2), CAG/polyglutamine disease (e.g., Huntington's disease, Spinal and bulbar muscular atrophy (SBMA), Dentatorubral-pallidoluysian atrophy, Spinocerebellar ataxia type I, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3, Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, Spinocerebellar ataxia type 8, Spinocerebellar ataxia type 12, and Spinocerebellar ataxia type 17), Friedreich ataxia, Unverricht-Lundborg myoclonic epilepsy (EPM1), Oculopharyngeal muscular dystrophy (OPMD), and Fuchs endothelial corneal dystrophy (FECD)) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with a RNAi agent of the disclosure either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising a RNAi agent, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the RNAi agent. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with a RNAi agent of the disclosure (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). For in vivo delivery, factors to consider in order to deliver a RNAi agent include, for example, biological stability of the delivered agent, prevention of non-specific effects, and accumulation of the delivered agent in the target tissue. The non-specific effects of a RNAi agent can be minimized by local administration, for example, by direct injection or implantation into a tissue 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 can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the RNAi agent to be administered. Several studies have shown successful knockdown of gene products when a RNAi agent is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J. et al., (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J. 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, P H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, G T., et al. (2004) Neuroscience 129:521-528; Thakker, E R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). For administering a RNAi agent 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 RNAi agent to the target tissue and avoid undesirable off-target effects (e.g., without wishing to be bound by theory, use of GNAs as described herein has been identified to destabilize the seed region of a dsRNA, resulting in enhanced preference of such dsRNAs for on-target effectiveness, relative to off-target effects, as such off-target effects are significantly weakened by such seed region destabilization). RNAi agents can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, a RNAi agent 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 RNAi agent 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 RNAi agent 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 molecule RNAi agent (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an RNAi agent by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an RNAi agent, 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 a RNAi agent. The formation of vesicles or micelles further prevents degradation of the RNAi agent when administered systemically. Methods for making and administering cationic-RNAi agent 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 RNAi agents 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, a RNAi agent forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of RNAi agents and cyclodextrins can be found in U.S. Pat. No. 7,427, 605, which is herein incorporated by reference in its entirety.

Certain aspects of the instant disclosure relate to a method of reducing the expression of an RPS25 target gene in a cell, comprising contacting said cell with the double-stranded RNAi agent of the disclosure. In one embodiment, the cell is an extrahepatic cell, optionally a CNS cell.

Another aspect of the disclosure relates to a method of reducing the expression of an RPS25 target gene in a subject, comprising administering to the subject the double-stranded RNAi agent of the disclosure.

Another aspect of the disclosure relates to a method of treating a subject having a CNS disorder, comprising administering to the subject a therapeutically effective amount of the double-stranded RPS25-targeting RNAi agent of the disclosure, thereby treating the subject. Exemplary CNS disorders that can be treated by the method of the disclosure include nucleotide repeat expansion diseases, such as C9orf72 ALS/FTD, Huntington-Like Syndrome Due To C9orf72 Expansions, Fragile X syndrome (FXS), Myotonic dystrophy (i.e., DM1, and DM2), CAG/polyglutamine disease (e.g., Huntington's disease, Spinal and bulbar muscular atrophy (SBMA), Dentatorubral-pallidoluysian atrophy, Spinocerebellar ataxia type I, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3, Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, Spinocerebellar ataxia type 8, Spinocerebellar ataxia type 12, and Spinocerebellar ataxia type 17), Friedreich ataxia, Unverricht-Lundborg myoclonic epilepsy (EPM1), Oculopharyngeal muscular dystrophy (OPMD), and Fuchs endothelial corneal dystrophy (FECD).

In one embodiment, the double-stranded RNAi agent is administered intrathecally. By intrathecal administration of the double-stranded RNAi agent, the method can reduce the expression of an RPS25 target gene in a brain (e.g., striatum) or spine tissue, for instance, cortex, cerebellum, cervical spine, lumbar spine, and thoracic spine.

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to modified siRNA compounds. It may be understood, however, that these formulations, compositions and methods can be practiced with other siRNA compounds, e.g., unmodified siRNA compounds, and such practice is within the disclosure. A composition that includes a RNAi agent can be delivered to a subject by a variety of routes. Exemplary routes include: intrathecal, intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, and ocular.

The RNAi agents of the disclosure can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of RNAi agent and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

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 topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the RNAi agent in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the RNAi agent and mechanically introducing the RNA.

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

Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening or flavoring agents can be added.

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

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

In one embodiment, the administration of the siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, composition is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral, or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.

Intrathecal Administration.

In one embodiment, the double-stranded RNAi agent is delivered by intrathecal injection (i.e., injection into the spinal fluid which bathes the brain and spinal cord tissue). Intrathecal injection of RNAi agents into the spinal fluid can be performed as a bolus injection or via minipumps which can be implanted beneath the skin, providing a regular and constant delivery of siRNA into the spinal fluid. The circulation of the spinal fluid from the choroid plexus, where it is produced, down around the spinal chord and dorsal root ganglia and subsequently up past the cerebellum and over the cortex to the arachnoid granulations, where the fluid can exit the CNS, that, depending upon size, stability, and solubility of the compounds injected, molecules delivered intrathecally could hit targets throughout the entire CNS.

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

In some embodiments, the intrathecal administration is via an intrathecal delivery system for a pharmaceutical including a reservoir containing a volume of the pharmaceutical agent, and a pump configured to deliver a portion of the pharmaceutical agent contained in the reservoir. More details about this intrathecal delivery system may be found in WO 2015/116658, which is incorporated by reference in its entirety.

The amount of intrathecally injected RNAi agents may vary from one target gene to another target gene and the appropriate amount that has to be applied may have to be determined individually for each target gene. Typically, this amount ranges from 10 μg to 2 mg, preferably 50 μg to 1500 μg, more preferably 100 μg to 1000 μg.

Vector Encoded RNAi Agents of the Disclosure

RNAi agents targeting the RPS25 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; WO 00/22113, WO 00/22114, and U.S. Pat. No. 6,054,299). Expression is preferably sustained (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., (1995) Proc. Natl. Acad. Sci. USA 92:1292).

The individual strand or strands of a RNAi agent 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 one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

RNAi agent expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of a RNAi agent as described herein. Delivery of RNAi agent 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.

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) SV 40 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 can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of a RNAi agent will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the RNAi agent in target cells. Other aspects to consider for vectors and constructs are known in the art.

VII. Pharmaceutical Compositions of the Invention

The present disclosure also includes pharmaceutical compositions and formulations which include the RNAi agents of the disclosure. In one embodiment, provided herein are pharmaceutical compositions containing an RNAi agent, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the RNAi agent are useful for treating a disease or disorder associated with the expression or activity of RPS25, e.g., nucleotide repeat expansion diseases, such as C9orf72 ALS/FTD, Huntington-Like Syndrome Due To C9orf72 Expansions, Fragile X syndrome (FXS), Myotonic dystrophy (i.e., DM1, and DM2), CAG/polyglutamine disease (e.g., Huntington's disease, Spinal and bulbar muscular atrophy (SBMA), Dentatorubral-pallidoluysian atrophy, Spinocerebellar ataxia type I, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3, Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, Spinocerebellar ataxia type 8, Spinocerebellar ataxia type 12, and Spinocerebellar ataxia type 17), Friedreich ataxia, Unverricht-Lundborg myoclonic epilepsy (EPM1), Oculopharyngeal muscular dystrophy (OPMD), and Fuchs endothelial corneal dystrophy (FECD).

Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV), intramuscular (IM), or for subcutaneous (subQ) delivery. Another example is compositions that are formulated for direct delivery into the CNS, e.g., by intrathecal or intravitreal routes of injection, optionally by infusion into the brain (e.g., striatum), such as by continuous pump infusion.

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

The pharmaceutical compositions of the disclosure may be administered in dosages sufficient to inhibit expression of an RPS25 gene. In general, a suitable dose of an RNAi agent of the disclosure will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day.

A repeat-dose regimen may include administration of a therapeutic amount of a RNAi agent on a regular basis, such as monthly to once every six months. In certain embodiments, the RNAi agent is administered about once per quarter (i.e., about once every three months) to about twice per year.

After an initial treatment regimen (e.g., loading dose), the treatments can be administered on a less frequent basis.

In other embodiments, a single dose of the pharmaceutical compositions can be long lasting, such that subsequent doses are administered at not more than 1, 2, 3, or 4 or more month intervals. In some embodiments of the disclosure, a single dose of the pharmaceutical compositions of the disclosure is administered once per month. In other embodiments of the disclosure, a single dose of the pharmaceutical compositions of the disclosure is administered once per quarter to twice per year.

The skilled artisan will appreciate that certain factors can 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 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.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as C9orf72 ALS/FTD that would benefit from reduction in the expression of RPS25. Such models can be used for in vivo testing of RNAi agents, as well as for determining a therapeutically effective dose. Suitable mouse models are known in the art and include, for example, the mouse models described elsewhere herein.

The pharmaceutical compositions of the present disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral 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.

The RNAi agents can be delivered in a manner to target a particular tissue, such as the CNS (e.g., neuronal, glial or vascular tissue of the brain).

Pharmaceutical compositions and formulations for topical administration can 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 can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the RNAi agents 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). RNAi agents featured in the disclosure can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, RNAi agents can 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 C_1-20alkyl 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. RNAi Agent Formulations Comprising Membranous Molecular Assemblies

A RNAi agent for use in the compositions and methods of the disclosure can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the RNAi agent composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the RNAi agent composition, although in some examples, it may. 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 liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the RNAi agent are delivered into the cell where the RNAi agent can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the RNAi agent to particular cell types.

A liposome containing an RNAi agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic.

Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of RNAi agent.

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

Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham et al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75: 4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161. Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169. These methods are readily adapted to packaging RNAi agent preparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/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. (1987) Biochem. Biophys. Res. Commun., 147:980-985).

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

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

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 cyclosporine A into different layers of the skin (Hu et al., (1994) S.T.P.Pharma. Sci., 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 G_M1, 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., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 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 G_M1, 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 G_M1or 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).

In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.

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 RNAi agents in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of RNAi agent (see, e.g., Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited 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 RNAi agent into the skin. In some implementations, liposomes are used for delivering RNAi agent to epidermal cells and also to enhance the penetration of RNAi agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2,405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. and Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

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 a drug into the dermis of mouse skin. Such formulations with RNAi agent are useful for treating a dermatological disorder.

Liposomes that include RNAi agents can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transfersomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include RNAi agent can be delivered, for example, subcutaneously by infection in order to deliver RNAi agent to keratinocytes in the skin. In order to cross 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. In addition, due to the lipid properties, these transfersomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

Other formulations amenable to the present disclosure are described in U.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008; 61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCT application number PCT/US2007/080331, filed Oct. 3, 2007, also describes formulations that are amenable to the present disclosure.

Transfersomes, yet another type of liposomes, are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can 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 transfersomes-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as those described herein, particularly in emulsions (including microemulsions) and liposomes. The most common 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).

The RNAi agent for use in the methods of the disclosure can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C₈to C₂₂alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phenol or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

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

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

Lipid Particles

RNAi agents, e.g., dsRNAs of in the disclosure may be fully encapsulated in a lipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipid particle. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). LNPs 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). LNPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in 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; United States Patent publication No. 2010/0324120 and WO 96/40964.

In one embodiment, 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. Ranges intermediate to the above recited ranges are also contemplated to be part of the disclosure.

Certain specific LNP formulations for delivery of RNAi agents have been described in the art, including, e.g., “LNP01” formulations as described in, e.g., WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are identified in the table below.

cationic lipid/non-cationic

lipid/cholesterol/PEG-lipid conjugate

Ionizable/Cationic Lipid
Lipid:siRNA ratio

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

dimethylaminopropane (DLinDMA)
cDMA

(57.1/7.1/34.4/1.4)

lipid:siRNA~7:1

2-XTC
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DPPC/Cholesterol/PEG-cDMA

dioxolane (XTC)
57.1/7.1/34.4/1.4

lipid:siRNA~7:1

LNP05
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG

dioxolane (XTC)
57.5/7.5/31.5/3.5

lipid:siRNA~ 6:1

LNP06
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG

dioxolane (XTC)
57.5/7.5/31.5/3.5

lipid:siRNA~11:1

LNP07
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG

dioxolane (XTC)
60/7.5/31/1.5,

lipid:siRNA - 6:1

LNP08
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG

dioxolane (XTC)
60/7.5/31/1.5,

lipid:siRNA~11:1

LNP09
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG

dioxolane (XTC)
50/10/38.5/1.5

Lipid:siRNA 10:1

LNP10
(3aR,5s,6aS)-N,N-dimethyl-2,2-
ALN1 OO/DSPC/Cholesterol/PEG-

di((9Z, 12Z) -octadeca-9,12-
DMG

dienyl)tetrahydro-3aH-
50/10/38.5/1.5

cyclopenta[d][1,3]dioxol-5-amine
Lipid:siRNA 10:1

(ALNI 00)

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

tetraen-19-yl 4-(dimethylamino)butanoate
50/10/38.5/1.5

(MC3)
Lipid:siRNA 10:1

LNP12
1,1-(2-(4-(2-((2-(bis(2-
Tech Gl/DSPC/Cholesterol/PEG-

hydroxydodecyl)amino)ethyl)(2-
DMG

hydroxydodecyl)amino)ethyl)piperazin-1-
50/10/38.5/1.5

yl)ethylazanediyl)didodecan-2-ol (Tech
Lipid:siRNA 10:1

G1)

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 (l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in WO 2009/127060, which is hereby incorporated by reference.

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

MC3 comprising formulations are described, e.g., in United States Patent Publication No. 2010/0324120, the entire contents of which are hereby incorporated by reference.

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

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

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 can 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 enhancer surfactants and chelators. Suitable surfactants include fatty acids or esters or salts thereof, bile acids or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and 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 can 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, U.S. 2003/0027780, 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, intraventricular or intrahepatic administration can include sterile aqueous solutions which can 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 can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the brain when treating APP-associated diseases or disorders.

The pharmaceutical formulations of the present disclosure, which can conveniently be presented in unit dosage form, can 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 of the present disclosure can 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 of the present disclosure can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol or dextran. The suspension can also contain stabilizers.

Additional Formulations

i. Emulsions

The compositions of the present disclosure can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm 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, N.Y.; 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 can 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 can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can 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 can 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 can be incorporated into either phase of the emulsion. Emulsifiers can 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, N.Y.; 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, N.Y.; 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 can 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, N.Y. 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 can 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 can 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, N.Y.; 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, N.Y.; 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.

ii. Microemulsions

In one embodiment of the present disclosure, the compositions of RNAi agents and nucleic acids are formulated as microemulsions. A microemulsion can 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, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; 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, isotropically 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, N.Y.; 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 can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) 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 can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or RNAi agents. 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 RNAi agents and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of RNAi agents and nucleic acids.

Microemulsions of the present disclosure can 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 RNAi agents and nucleic acids of the present disclosure. Penetration enhancers used in the microemulsions of the present disclosure can be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

iii. Microparticles

An RNAi agent of the disclosure may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present disclosure employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly RNAi agents, 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 can 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 can 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, N.Y., 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 (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 RNAi agents 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, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Various fatty acids 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, Mass., 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).

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, N.Y., 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, N.Y., 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, 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 RNAi agents 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 beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 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 Rd., 1990, 14, 43-51).

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 RNAi agents through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- 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 RNAi agents at the cellular level can also be added to the pharmaceutical and other compositions of the present disclosure. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.

Other agents can 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.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can 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 can 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 can 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.

vii. Other Components

The compositions of the present disclosure can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can 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 or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the disclosure include (a) one or more RNAi agents and (b) one or more agents which function by a non-RNAi mechanism and which are useful in treating an RPS25-associated disorder. Examples of such agents include, but are not limited to SSRIs, venlafaxine, bupropion, and atypical antipsychotics.

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 preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the disclosure lies generally within a range of circulating concentrations that include the ED₅₀with little or no toxicity. The dosage can 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 can 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 can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the RNAi agents featured in the disclosure can be administered in combination with other known agents effective in treatment of pathological processes mediated by nucleotide repeat expression. In any event, the administering physician can adjust the amount and timing of RNAi agent administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

VIII. Kits

In certain aspects, the instant disclosure provides kits that include a suitable container containing a pharmaceutical formulation of a siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a ssiRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof). In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for a siRNA compound preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.

IX. Methods for Inhibiting RPS25 Expression

The present disclosure also provides methods of inhibiting expression of an RPS25 gene in a cell. The methods include contacting a cell with an RNAi agent, e.g., double stranded RNAi agent, in an amount effective to inhibit expression of RPS25 in the cell, thereby inhibiting expression of RPS25 in the cell. In certain embodiments of the disclosure, RPS25 is inhibited preferentially in CNS (e.g., brain) cells.

Contacting of a cell with a RNAi agent, e.g., a double stranded RNAi agent, may be done in vitro or in vivo. Contacting a cell in vivo with the RNAi agent includes contacting a cell or group of cells within a subject, e.g., a human subject, with the RNAi agent. Combinations of in vitro and in vivo methods of contacting a cell are also possible.

Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc ligand, or any other ligand that directs the RNAi agent to a site of interest.

The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition. In certain embodiments, a level of inhibition, e.g., for an RNAi agent of the instant disclosure, can be assessed in cell culture conditions, e.g., wherein cells in cell culture are transfected via Lipofectamine™-mediated transfection at a concentration in the vicinity of a cell of 10 nM or less, 1 nM or less, etc. Knockdown of a given RNAi agent can be determined via comparison of pre-treated levels in cell culture versus post-treated levels in cell culture, optionally also comparing against cells treated in parallel with a scrambled or other form of control RNAi agent. Knockdown in cell culture of, e.g., preferably 50% or more, can thereby be identified as indicative of “inhibiting” or “reducing”, “downregulating” or “suppressing”, etc. having occurred. It is expressly contemplated that assessment of targeted mRNA or encoded protein levels (and therefore an extent of “inhibiting”, etc. caused by a RNAi agent of the disclosure) can also be assessed in in vivo systems for the RNAi agents of the instant disclosure, under properly controlled conditions as described in the art.

The phrase “inhibiting expression of an RPS25 gene” or “inhibiting expression of RPS25,” as used herein, includes inhibition of expression of any RPS25 gene (such as, e.g., a mouse RPS25 gene, a rat RPS25 gene, a monkey RPS25 gene, or a human RPS25 gene) as well as variants or mutants of an RPS25 gene that encode an RPS25 protein. Thus, the RPS25 gene may be a wild-type RPS25 gene, a mutant RPS25 gene, or a transgenic RPS25 gene in the context of a genetically manipulated cell, group of cells, or organism.

“Inhibiting expression of an RPS25 gene” includes any level of inhibition of an RPS25 gene, e.g., at least partial suppression of the expression of an RPS25 gene, such as an inhibition by at least 20%. In certain embodiments, inhibition is by at least 30%, at least 40%, preferably at least 50%, at least about 60%, at least 70%, at least about 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; or to below the level of detection of the assay method.

The expression of an RPS25 gene may be assessed based on the level of any variable associated with RPS25 gene expression, e.g., RPS25 mRNA level or RPS25 protein level, or, for example, the level of C9orf72 expanded protein.

Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

In some embodiments of the methods of the disclosure, expression of an RPS25 gene is inhibited by at least 20%, 30%, 40%, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay. In certain embodiments, the methods include a clinically relevant inhibition of expression of RPS25, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of RPS25.

Inhibition of the expression of an RPS25 gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which an RPS25 gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with a RNAi agent of the disclosure, or by administering a RNAi agent of the disclosure to a subject in which the cells are or were present) such that the expression of an RPS25 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s) not treated with a RNAi agent or not treated with a RNAi agent targeted to the gene of interest). The degree of inhibition may be expressed in terms of:

$\frac{(mRNA in control cells) - (mRNA in treated cells)}{(mRNA in control cells)} \cdot 100 %$

In other embodiments, inhibition of the expression of an RPS25 gene may be assessed in terms of a reduction of a parameter that is functionally linked to an RPS25 gene expression, e.g., RPS25 protein expression. RPS25 gene silencing may be determined in any cell expressing RPS25, either endogenous or heterologous from an expression construct, and by any assay known in the art.

Inhibition of the expression of an RPS25 protein may be manifested by a reduction in the level of the RPS25 protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above, for the assessment of mRNA suppression, the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess the inhibition of the expression of an RPS25 gene includes a cell or group of cells that has not yet been contacted with an RNAi agent of the disclosure. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent.

The level of RPS25 mRNA that is expressed by a cell or group of cells may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of expression of RPS25 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the RPS25 gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy™ RNA preparation kits (Qiagen®) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis. Circulating RPS25 mRNA may be detected using methods the described in WO2012/177906, the entire contents of which are hereby incorporated herein by reference.

In some embodiments, the level of expression of RPS25 is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific RPS25 nucleic acid or protein, or fragment thereof. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to RPS25 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix® gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of RPS25 mRNA.

An alternative method for determining the level of expression of RPS25 in a sample involves the process of nucleic acid amplification or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the disclosure, the level of expression of RPS25 is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™ System), by a Dual-Glo® Luciferase assay, or by other art-recognized method for measurement of RPS25 expression or mRNA level.

The expression level of RPS25 mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of RPS25 expression level may also comprise using nucleic acid probes in solution.

In some embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of this PCR method is described and exemplified in the Examples presented herein. Such methods can also be used for the detection of RPS25 nucleic acids.

The level of RPS25 protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like. Such assays can also be used for the detection of proteins indicative of the presence or replication of RPS25 proteins.

In some embodiments, the efficacy of the methods of the disclosure in the treatment of an RPS25-related disease is assessed by a decrease in RPS25 mRNA level (e.g, by assessment of a CSF sample for RPS25 level, by brain biopsy, or otherwise).

In some embodiments of the methods of the disclosure, the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject. The inhibition of expression of RPS25 may be assessed using measurements of the level or change in the level of RPS25 mRNA or RPS25 protein in a sample derived from a specific site within the subject, e.g., CNS cells. In certain embodiments, the methods include a clinically relevant inhibition of expression of RPS25, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of RPS25.

As used herein, the terms detecting or determining a level of an analyte are understood to mean performing the steps to determine if a material, e.g., protein, RNA, is present. As used herein, methods of detecting or determining include detection or determination of an analyte level that is below the level of detection for the method used.

X. Methods of Treating or Preventing RPS25-Associated Diseases

The present disclosure also provides methods of using a RNAi agent of the disclosure or a composition containing a RNAi agent of the disclosure to reduce or inhibit RPS25 expression in a cell. The methods include contacting the cell with a dsRNA of the disclosure and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of an RPS25 gene, thereby inhibiting expression of the RPS25 gene in the cell. Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of RPS25 may be determined by determining the mRNA expression level of RPS25 using methods routine to one of ordinary skill in the art, e.g., northern blotting, qRT-PCR; by determining the protein level of RPS25 using methods routine to one of ordinary skill in the art, such as western blotting, immunological techniques.

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

A cell suitable for treatment using the methods of the disclosure may be any cell that expresses an RPS25 gene. A cell suitable for use in the methods of the disclosure may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a rat cell, or a mouse cell. In one embodiment, the cell is a human cell, e.g., a human CNS cell.

RPS25 expression is inhibited in the cell by at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or about 100%, i.e., to below the level of detection. In preferred embodiments, RPS25 expression is inhibited by at least 50%.

The in vivo methods of the disclosure may include administering to a subject a composition containing a RNAi agent, where the RNAi agent includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the RPS25 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, intravitreal, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection. In certain embodiments, the compositions are administered by intrathecal injection.

In some embodiments, the administration is via a depot injection. A depot injection may release the RNAi agent in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of RPS25, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intracranial, intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the RNAi agent to the CNS.

The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

In one aspect, the present disclosure also provides methods for inhibiting the expression of an RPS25 gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets an RPS25 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the RPS25 gene, thereby inhibiting expression of the RPS25 gene in the cell. Reduction in gene expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, described herein. In one embodiment, a CNS biopsy sample or a cerebrospinal fluid (CSF) sample serves as the tissue material for monitoring the reduction in RPS25 gene or protein expression (or of a proxy therefore).

The present disclosure further provides methods of treatment of a subject in need thereof. The treatment methods of the disclosure include administering an RNAi agent of the disclosure to a subject, e.g., a subject that would benefit from inhibition of RPS25 expression, in a therapeutically effective amount of a RNAi agent targeting an RPS25 gene or a pharmaceutical composition comprising a RNAi agent targeting aRPS25 gene.

In addition, the present disclosure provides methods of preventing, treating or inhibiting the progression of an RPS25-associated disease or disorder (e.g., nucleotide repeat expansion diseases, such as C9orf72 ALS/FTD, Huntington-Like Syndrome Due To C9orf72 Expansions, Fragile X syndrome (FXS), Myotonic dystrophy (i.e., DM1, and DM2), CAG/polyglutamine disease (e.g., Huntington's disease, Spinal and bulbar muscular atrophy (SBMA), Dentatorubral-pallidoluysian atrophy, Spinocerebellar ataxia type I, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3, Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, Spinocerebellar ataxia type 8, Spinocerebellar ataxia type 12, and Spinocerebellar ataxia type 17), Friedreich ataxia, Unverricht-Lundborg myoclonic epilepsy (EPM1), Oculopharyngeal muscular dystrophy (OPMD), and Fuchs endothelial corneal dystrophy (FECD)) in a subject, such as the progression of an RPS25-associated disease or disorder. The methods include administering to the subject a therapeutically effective amount of any of the RNAi agent, e.g., dsRNA agents, or the pharmaceutical composition provided herein, thereby preventing, treating or inhibiting the progression of an RPS25-associated disease or disorder in the subject.

An RNAi agent of the disclosure may be administered as a “free RNAi agent.” A free RNAi agent is administered in the absence of a pharmaceutical composition. The naked RNAi agent may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the RNAi agent can be adjusted such that it is suitable for administering to a subject.

Alternatively, an RNAi agent of the disclosure may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction or inhibition of RPS25 gene expression are those having an RPS25-associated disease.

The disclosure further provides methods for the use of a RNAi agent or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction or inhibition of RPS25 expression, e.g., a subject having an RPS25-associated disorder, in combination with other pharmaceuticals or other therapeutic methods, e.g., with known pharmaceuticals or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, in certain embodiments, an RNAi agent targeting RPS25 is administered in combination with, e.g., an agent useful in treating an RPS25-associated disorder as described elsewhere herein or as otherwise known in the art. For example, additional agents suitable for treating a subject that would benefit from reduction in RPS25 expression, e.g., a subject having an RPS25-associated disorder, may include agents currently used to treat symptoms of RPS25. The RNAi agent and additional therapeutic agents may be administered at the same time or in the same combination, e.g., intrathecally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times or by another method known in the art or described herein.

In one embodiment, the method includes administering a composition featured herein such that expression of the target RPS25 gene is decreased, for at least one month. In preferred embodiments, expression is decreased for at least 2 months, 3 months, or 6 months.

Preferably, the RNAi agents useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target RPS25 gene. Compositions and methods for inhibiting the expression of these genes using RNAi agents can be prepared and performed as described herein.

Administration of the dsRNA according to the methods of the disclosure may result in a reduction of the severity, signs, symptoms, or markers of such diseases or disorders in a patient with an RPS25-associated disorder. By “reduction” in this context is meant a statistically significant or clinically significant decrease in such level. The reduction can be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of an RPS25-associated disorder may be assessed, for example, by periodic monitoring of a subject's. Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of a RNAi agent targeting RPS25 or pharmaceutical composition thereof, “effective against” an RPS25-associated disorder indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating RPS25-associated disorders and the related causes.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given RNAi agent drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale. Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using a RNAi agent or RNAi agent formulation as described herein.

Subjects can be administered a therapeutic amount of dsRNA, such as about 0.01 mg/kg to about 200 mg/kg.

The RNAi agent can be administered intrathecally, via intravitreal injection, or by intravenous infusion over a period of time, on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. Administration of the RNAi agent can reduce RPS25 levels, e.g., in a cell, tissue, blood, CSF sample or other compartment of the patient by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70,% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least about 99% or more. In a preferred embodiment, administration of the RNAi agent can reduce RPS25 levels, e.g., in a cell, tissue, blood, CSF sample or other compartment of the patient by at least 50%.

Before administration of a full dose of the RNAi agent, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Alternatively, the RNAi agent can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired, e.g., monthly dose of RNAi agent to a subject. The injections may be repeated over a period of time. The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeat-dose regimen may include administration of a therapeutic amount of RNAi agent on a regular basis, such as monthly or extending to once a quarter, twice per year, once per year. In certain embodiments, the RNAi agent is administered about once per month to about once per quarter (i.e., about once every three months).

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 invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the RNAi agents and methods featured in the invention, 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.

An informal Sequence Listing is filed herewith and forms part of the specification as filed.

EXAMPLES
Example 1. RNAi Agent Design, Synthesis, Selection, and In Vitro Evaluation

This Example describes methods for the design, synthesis, selection, and in vitro evaluation of RPS25 RNAi agents.

Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Bioinformatics

A set of siRNAs targeting the human small ribosomal protein subunit 25 (RPS25; human NCBI refseqID NM_001028.3; NCBI GeneID: 6230) was designed using custom R and Python scripts. The human NM_001028 REFSEQ mRNA, version 3, has a length of 483 bases.

RPS25 single strands and duplexes were made using routine methods known in the art. A detailed list of the unmodified RPS25 sense and antisense strand sequences is shown in Tables 2, 4, 6, 8, 10 and 13 and a detailed list of the modified RPS25 sense and antisense strand sequences is shown in Tables 3, 5, 7, 9, 11, 12 and 14.

In Vitro Dual-Luciferase and Endogenous Screening Assays

Dual-Glo® Luciferase Assay

Cos-7 cells (ATCC, Manassas, Va.) are grown to near confluence at 37° C. in an atmosphere of 5% CO₂in DMEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Multi-dose experiments are performed at 10 nM and 0.1 nM. siRNA and psiCHECK2-RPS25 (GenBank Accession No. NM_001028.3) plasmid transfections are carried out with a plasmid containing the 3′ untranslated region (UTR). Transfection is carried out by adding 5 μL of siRNA duplexes and 5 μL (5 ng) of psiCHECK2 plasmid per well along with 4.9 μL of Opti-MEM plus 0.1 μL of Lipofectamine 2000 per well (Invitrogen, Carlsbad Calif. cat #13778-150) and then incubated at room temperature for 15 minutes. The mixture is then added to the cells which are re-suspended in 35 μL of fresh complete media. The transfected cells are incubated at 37° C. in an atmosphere of 5% CO₂.

Forty-eight hours after the siRNAs and psiCHECK2 plasmid are transfected; Firefly (transfection control) and Renilla (fused to RPS25 target sequence) luciferase are measured. First, media is removed from cells. Then Firefly luciferase activity is measured by adding 20 μL of Dual-Glo® Luciferase Reagent equal to the culture medium volume to each well and mix. The mixture is incubated at room temperature for 30 minutes before luminescence (500 nm) is measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity is measured by adding 20 μL of room temperature of Dual-Glo® Stop & Glo® Reagent is added to each well and the plates are incubated for 10-15 minutes before luminescence is again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent quenches the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. siRNA activity is determined by normalizing the Renilla (RPS25) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity is then assessed relative to cells that were transfected with the same vector but were not treated with siRNA or were treated with a non-targeting siRNA. All transfections are done with n=4.

Cell Culture and Transfections

Cells are transfected by adding 4.9 μL of Opti-MEM plus 0.1 μL of RNAiMAX per well (Invitrogen, Carlsbad Calif. 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 MEDIA containing ˜5×10³cells are then added to the siRNA mixture. Cells are incubated for 24 hours prior to RNA purification. Experiments are performed at 10 nM and 0.1 nM. Transfection experiments are performed in human hepatoma Hep3B cells (ATCC HB-8064) with EMEM (ATCC catalog no. 30-2003), human neuroblastoma BE(2)-C cells (ATCC CRL-2268) with EMEM:F12 media (Gibco catalog no. 11765054) and mouse neuroblastoma Neuro2A cells (ATCC CCL-131) with EMEM media.

For HeLa cells, cells were transfected by adding 3 μL of Opti-MEM plus 0.1 μL of RNAiMAX per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μL of siRNA duplexes per well, with 4 replicates of each siRNA duplex, into a 96-well plate, and incubated at room temperature for 15 minutes. Forty μL of MEDIA containing ˜1.5×10⁴cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Experiments are performed at 10 nM. Transfection experiments are performed in HeLa cells.

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, Calif., 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 were added to RNA isolated above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 hour incubation at 37° C.

Real Time PCR

Two μL of cDNA were added to a master mix containing 0.5 μL of human or mouse GAPDH TaqMan Probe (ThermoFisher cat 4352934E or 4351309) and 0.5 μL of appropriate RPS25 probe (commercially available, e.g., from Thermo Fisher) and 5 μL Lightcycler 480 probe master mix (Roche Cat #04887301001) 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 with N=4 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. The results of the screening of the dsRNA agents listed in Table 12 at 10 nM in HeLa cells are shown in Table 15.

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

As
adenosine-3′-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

Cs
cytidine-3′-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

Gs
guanosine-3′-phosphorothioate

T
5′-methyluridine-3′-phosphate

Tf
2′-fluoro-5-methyluridine-3′-phosphate

Tfs
2‘-fluoro-5-methyluridine-3‘-phosphorothioate

Ts
5-methyluridine-3′-phosphorothioate

U
Uridine-3′-phosphate

Uf
2′-fluorouridine-3′-phosphate

Ufs
2′-fluorouridine-3′-phosphorothioate

Us
uridine-3′-phosphorothioate

N
anynucleotide,modifiedorunmodified

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′-0-methyl-5-methyluridine-3′-phosphate

ts
2′-0-methyl-5-methyluridine-3′-phosphorothioate

u
2'-O-methyluridine-3′-phosphate

us
2'-O-methyluridine-3′-phosphorothioate

s
phosphorothioatelinkage

L96
N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol

Hyp-(GalNAc-alkyl)3

Y34
2-hydroxymethyl-tetrahydrofurane-4-methoxy-3-phosphate(abasic2'-0Me

furanose)

Y44
invertedabasicDNA(2-hydroxymethyl-tetrahydrofurane-5-phosphate)

(Agn)
Adenosine-glycolnucleicacid(GNA)

L10
N-(cholesterylcarboxamidocaproyl)-4-hydroxyprolinol(Hyp-C6-Chol)

(Cen)
Cytidine-glycolnucleicacid(GNA)

(Ggn)
Guanosine-glycolnucleicacid(GNA)

(Tz)
Thymidine-glycolnucleicacid(GNA)S-Isomer

P
Phosphate

VP
Vinyl-phosphonate

(Aam)
2'-O-(N-methylacetamide)adenosine-3'-phosphate

(Aams)
2'-O-(N-methylacetamide)adenosine-3'-phosphorothioate

(Gam)
2'-O-(N-methylacetamide)guanosine-3'-phosphate

(Gams)
2'-O-(N-methylacetamide)guanosine-3'-phosphorothioate

(Tam)
2'-O-(N-methylacetamide)thymidine-3'-phosphate

(Tams)
2'-O-(N-methylacetamide)thymidine-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-3'-phosphate

dTs
2'-deoxythymidine-3'-phosphorothioate

dU
2'-deoxyuridine

dUs
2'-deoxyuridine-3'-phosphorothioate

(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

(A3m)
3'-O-methyladenosine-2'-phosphate

(A3mx)
3'-O-methyl-xylofuranosyladenosine-2'-phosphate

(G3m)
3'-O-methylguanosine-2'-phosphate

(G3mx)
3'-O-methyl-xylofuranosylguanosine-2'-phosphate

(C3m)
3'-O-methylcytidine-2'-phosphate

(C3mx)
3'-O-methyl-xylofuranosylcytidine-2'-phosphate

(U3m)
3'-0-methyluridine-2'-phosphate

U3mx)
3'-O-methyl-xylofuranosyluridine-2'-phosphate

(m5Cam)
2'-O-(N-methylacetamide)-5-methylcytidine-3'-phosphate

(m5Cams)
2'-O-(N-methylacetamide)-5-methylcytidine-3'-phosphorothioate

(Chd)
2′-O-hexadecyl-cytidine-3′-phosphate

(Chds)
2′-O-hexadecyl-cytidine-3′-phosphorothioate

(Uhd)
2′-O-hexadecyl-uridine-3′-phosphate

(Uhds)
2′-O-hexadecyl-uridine-3′-phosphorothioate

(pshe)
Hydroxyethylphosphorothioate

(Chd)
2′-O-hexadecyl-cytidine-3′-phosphate

(Ahd)
2′-O-hexadecyl-adenosine-3′-phosphate

(Ghd)
2′-O-hexadecyl-guanosine-3′-phosphate

(Uhd)
2′-O-hexadecyl-uridine-3′-phosphate

(C2p)
cytidine-2'-phosphate

(G2p)
guanosine-2'-phosphate

(U2p)
uridine-2'-phosphate

Abbreviation
Nucleotide(s)

(A2p)
adenosine-2'-phosphate

TABLE 2

RPS25 Unmodified Sequences

NM_00

Sense
SEQ
Sense
Antisense
SEQ
Antisense
1028.3

Duplex
Sequence
ID
Oligo
Sequence
ID
Oligo
Target

ID
5′ to 3′
NO:
Name
5′ to 3′
NO:
Name
Site

AD-
CUUUUUGUCC
23
NM_
AAAGAUGUCG
433
NM_
1-19

960501
GACAUCUUU

001028.3_1-
GACAAAAAG

001028.3_1-

19_G19U_s

19_C1A_as

AD-
UUUUUGUCCG
24
NM_
ACAAGAUGUC
434
NM_
2-20

960502
ACAUCUUGU

001028.3_2-
GGACAAAAA

001028.3_2-

20_A19U_s

20_U1A_as

AD-
UUUUGUCCGA
25
NM_
AUCAAGAUGU
435
NM_
3-21

960503
CAUCUUGAU

001028.3_3-
CGGACAAAA

001028.3_3-

21_C19U_s

21_G1A_as

AD-
UUUGUCCGAC
26
NM_
AGUCAAGAUG
436
NM_
4-22

960504
AUCUUGACU

001028.3_4-
UCGGACAAA

001028.3_4-

22_G19U_s

22_C1A_as

AD-
UUGUCCGACA
27
NM_
ACGUCAAGAU
437
NM_
5-23

960505
UCUUGACGU

001028.3_5-
GUCGGACAA

001028.3_5-

23_A19U_s

23_U1A_as

AD-
UGUCCGACAU
28
NM_
AUCGUCAAGA
438
NM_
6-24

960506
CUUGACGAU

001028.3_6-
UGUCGGACA

001028.3_6-

24_G19U_s

24_C1A_as

AD-
GUCCGACAUC
29
NM_
ACUCGUCAAG
439
NM_
7-25

960507
UUGACGAGU

001028.3_7-
AUGUCGGAC

001028.3_7-

25_G19U_s

25_C1A_as

AD-
UCCGACAUCU
30
NM_
ACCUCGUCAA
440
NM_
8-26

960508
UGACGAGGU

001028.3_8-
GAUGUCGGA

001028.3_8-

26_C19U_s

26_G1A_as

AD-
CCGACAUCUU
31
NM_
AGCCUCGUCA
441
NM_
9-27

960509
GACGAGGCU

001028.3_9-
AGAUGUCGG

001028.3_9-

27_s

27_as

AD-
CGACAUCUUG
32
NM_
AAGCCUCGUC
442
NM_
10-28

960510
ACGAGGCUU

001028.3_10-
AAGAUGUCG

001028.3_10-

28_G19U_s

28_C1A_as

AD-
GACAUCUUGA
33
NM_
ACAGCCUCGU
443
NM_
11-29

960511
CGAGGCUGU

001028.3_11-
CAAGAUGUC

001028.3_11-

29_C19U_s

29_G1A_as

AD-
ACAUCUUGAC
34
NM_
AGCAGCCUCG
444
NM_
12-30

960512
GAGGCUGCU

001028.3_12-
UCAAGAUGU

001028.3_12-

30_G19U_s

30_C1A_as

AD-
CAUCUUGACG
35
NM_
ACGCAGCCUC
445
NM_
13-31

960513
AGGCUGCGU

001028.3_13-
GUCAAGAUG

001028.3_13-

31_G19U_s

31_C1A_as

AD-
AUCUUGACGA
36
NM_
ACCGCAGCCU
446
NM_
14-32

960514
GGCUGCGGU

001028.3_14-
CGUCAAGAU

001028.3_14-

32_s

32_as

AD-
UCUUGACGAG
37
NM_
AACCGCAGCC
447
NM_
15-33

960515
GCUGCGGUU

001028.3_15-
UCGUCAAGA

001028.3_15-

33_G19U_s

33_C1A_as

AD-
CUUGACGAGG
38
NM_
ACACCGCAGC
448
NM_
16-34

960516
CUGCGGUGU

001028.3_16-
CUCGUCAAG

001028.3_16-

34_s

34_as

AD-
UUGACGAGGC
39
NM_
AACACCGCAG
449
NM_
17-35

960517
UGCGGUGUU

001028.3_17-
CCUCGUCAA

001028.3_17-

35_C19U_s

35_G1A_as

AD-
UGACGAGGCU
40
NM_
AGACACCGCA
450
NM_
18-36

960518
GCGGUGUCU

001028.3_18-
GCCUCGUCA

001028.3_18-

36_s

36_as

AD-
GACGAGGCUG
41
NM_
AAGACACCGC
451
NM_
19-37

960519
CGGUGUCUU

001028.3_19-
AGCCUCGUC

001028.3_19-

37_G19U_s

37_C1A_as

AD-
ACGAGGCUGC
42
NM_
ACAGACACCG
452
NM_
20-38

960520
GGUGUCUGU

001028.3_20-
CAGCCUCGU

001028.3_20-

38_C19U_s

38_G1A_as

AD-
CGAGGCUGCG
43
NM_
AGCAGACACC
453
NM_
21-39

960521
GUGUCUGCU

001028.3_21-
GCAGCCUCG

001028.3_21-

39_s

39_as

AD-
GAGGCUGCGG
44
NM_
AAGCAGACAC
454
NM_
22-40

960522
UGUCUGCUU

001028.3_22-
CGCAGCCUC

001028.3_22-

40_G19U_s

40_C1A_as

AD-
AGGCUGCGGU
45
NM_
ACAGCAGACA
455
NM_
23-41

960523
GUCUGCUGU

001028.3_23-
CCGCAGCCU

001028.3_23-

41_C19U_s

41_G1A_as

AD-
GGCUGCGGUG
46
NM_
AGCAGCAGAC
456
NM_
24-42

960524
UCUGCUGCU

001028.3_24-
ACCGCAGCC

001028.3_24-

42_s

42_as

AD-
GCUGCGGUGU
47
NM_
AAGCAGCAGA
457
NM_
25-43

960525
CUGCUGCUU

001028.3_25-
CACCGCAGC

001028.3_25-

43_A19U_s

43_U1A_as

AD-
CUGCGGUGUC
48
NM_
AUAGCAGCAG
458
NM_
26-44

960526
UGCUGCUAU

001028.3_26-
ACACCGCAG

001028.3_26-

44_s

44_as

AD-
UGCGGUGUCU
49
NM_
AAUAGCAGCA
459
NM_
27-45

960527
GCUGCUAUU

001028.3_27-
GACACCGCA

001028.3_27-

45_s

45_as

AD-
GCGGUGUCUG
50
NM_
AAAUAGCAGC
460
NM_
28-46

960528
CUGCUAUUU

001028.3_28-
AGACACCGC

001028.3_28-

46_C19U_s

46_G1A_as

AD-
CGGUGUCUGC
51
NM_
AGAAUAGCAG
461
NM_
29-47

960529
UGCUAUUCU

001028.3_29-
CAGACACCG

001028.3_29-

47_s

47_as

AD-
GGUGUCUGCU
52
NM_
AAGAAUAGCA
462
NM_
30-48

960530
GCUAUUCUU

001028.3_30-
GCAGACACC

001028.3_30-

48_C19U_s

48_G1A_as

AD-
GUGUCUGCUG
53
NM_
AGAGAAUAGC
463
NM_
31-49

960531
CUAUUCUCU

001028.3_31-
AGCAGACAC

001028.3_31-

49_C19U_s

49_G1A_as

AD-
UGUCUGCUGC
54
NM_
AGGAGAAUAG
464
NM_
32-50

960532
UAUUCUCCU

001028.3_32-
CAGCAGACA

001028.3_32-

50_G19U_s

50_C1A_as

AD-
GUCUGCUGCU
55
NM_
ACGGAGAAUA
465
NM_
33-51

960533
AUUCUCCGU

001028.3_33-
GCAGCAGAC

001028.3_33-

51_A19U_s

51_U1A_as

AD-
UCUGCUGCUA
56
NM_
AUCGGAGAAU
466
NM_
34-52

960534
UUCUCCGAU

001028.3_34-
AGCAGCAGA

001028.3_34-

52_G19U_s

52_C1A_as

AD-
CUGCUGCUAU
57
NM_
ACUCGGAGAA
467
NM_
35-53

960535
UCUCCGAGU

001028.3_35-
UAGCAGCAG

001028.3_35-

53_C19U_s

53_G1A_as

AD-
UGCUGCUAUU
58
NM_
AGCUCGGAGA
468
NM_
36-54

960536
CUCCGAGCU

001028.3_36-
AUAGCAGCA

001028.3_36-

54_s

54_as

AD-
GCUGCUAUUC
59
NM_
AAGCUCGGAG
469
NM_
37-55

960537
UCCGAGCUU

001028.3_37-
AAUAGCAGC

001028.3_37-

55_s

55_as

AD-
CUGCUAUUCU
60
NM_
AAAGCUCGGA
470
NM_
38-56

960538
CCGAGCUUU

001028.3_38-
GAAUAGCAG

001028.3_38-

56_C19U_s

56_G1A_as

AD-
UGCUAUUCUC
61
NM_
AGAAGCUCGG
471
NM_
39-57

960539
CGAGCUUCU

001028.3_39-
AGAAUAGCA

001028.3_39-

57_G19U_s

57_C1A_as

AD-
GCUAUUCUCC
62
NM_
ACGAAGCUCG
472
NM_
40-58

960540
GAGCUUCGU

001028.3_40-
GAGAAUAGC

001028.3_40-

58_C19U_s

58_G1A_as

AD-
CUAUUCUCCG
63
NM_
AGCGAAGCUC
473
NM_
41-59

960541
AGCUUCGCU

001028.3_41-
GGAGAAUAG

001028.3_41-

59_A19U_s

59_U1A_as

AD-
UAUUCUCCGA
64
NM_
AUGCGAAGCU
474
NM_
42-60

960542
GCUUCGCAU

001028.3_42-
CGGAGAAUA

001028.3_42-

60_A19U_s

60_U1A_as

AD-
AUUCUCCGAG
65
NM_
AUUGCGAAGC
475
NM_
43-61

960543
CUUCGCAAU

001028.3_43-
UCGGAGAAU

001028.3_43-

61_s

61_as

AD-
UUCUCCGAGC
66
NM_
AAUUGCGAAG
476
NM_
44-62

960544
UUCGCAAUU

001028.3_44-
CUCGGAGAA

001028.3_44-

62_G19U_s

62_C1A_as

AD-
UCUCCGAGCU
67
NM_
ACAUUGCGAA
477
NM_
45-63

960545
UCGCAAUGU

001028.3_45-
GCUCGGAGA

001028.3_45-

63_C19U_s

63_G1A_as

AD-
CUCCGAGCUU
68
NM_
AGCAUUGCGA
478
NM_
46-64

960546
CGCAAUGCU

001028.3_46-
AGCUCGGAG

001028.3_46-

64_C19U_s

64_G1A_as

AD-
UCCGAGCUUC
69
NM_
AGGCAUUGCG
479
NM_
47-65

960547
GCAAUGCCU

001028.3_47-
AAGCUCGGA

001028.3_47-

65_G19U_s

65_C1A_as

AD-
CCGAGCUUCG
70
NM_
ACGGCAUUGC
480
NM_
48-66

960548
CAAUGCCGU

001028.3_48-
GAAGCUCGG

001028.3_48-

66_C19U_s

66_G1A_as

AD-
CGAGCUUCGC
71
NM_
AGCGGCAUUG
481
NM_
49-67

960549
AAUGCCGCU

001028.3_49-
CGAAGCUCG

001028.3_49-

67_C19U_s

67_G1A_as

AD-
GAGCUUCGCA
72
NM_
AGGCGGCAUU
482
NM_
50-68

960550
AUGCCGCCU

001028.3_50-
GCGAAGCUC

001028.3_50-

68_s

68_as

AD-
AGCUUCGCAA
73
NM_
AAGGCGGCAU
483
NM_
51-69

960551
UGCCGCCUU

001028.3_51-
UGCGAAGCU

001028.3_51-

69_A19U_s

69_U1A_as

AD-
GCUUCGCAAU
74
NM_
AUAGGCGGCA
484
NM_
52-70

960552
GCCGCCUAU

001028.3_52-
UUGCGAAGC

001028.3_52-

70_A19U_s

70_U1A_as

AD-
CUUCGCAAUG
75
NM_
AUUAGGCGGC
485
NM_
53-71

960553
CCGCCUAAU

001028.3_53-
AUUGCGAAG

001028.3_53-

71_G19U_s

71_C1A_as

AD-
UUCGCAAUGC
76
NM_
ACUUAGGCGG
486
NM_
54-72

960554
CGCCUAAGU

001028.3_54-
CAUUGCGAA

001028.3_54-

72_G19U_s

72_C1A_as

AD-
UCGCAAUGCC
77
NM_
ACCUUAGGCG
487
NM_
55-73

960555
GCCUAAGGU

001028.3_55-
GCAUUGCGA

001028.3_55-

73_A19U_s

73_U1A_as

AD-
CGCAAUGCCG
78
NM_
AUCCUUAGGC
488
NM_
56-74

960556
CCUAAGGAU

001028.3_56-
GGCAUUGCG

001028.3_56-

74_C19U_s

74_G1A_as

AD-
GCAAUGCCGC
79
NM_
AGUCCUUAGG
489
NM_
57-75

960557
CUAAGGACU

001028.3_57-
CGGCAUUGC

001028.3_57-

75_G19U_s

75_C1A_as

AD-
CAAUGCCGCC
80
NM_
ACGUCCUUAG
490
NM_
58-76

960558
UAAGGACGU

001028.3_58-
GCGGCAUUG

001028.3_58-

76_A19U_s

76_U1A_as

AD-
AAUGCCGCCU
81
NM_
AUCGUCCUUA
491
NM_
59-77

960559
AAGGACGAU

001028.3_59-
GGCGGCAUU

001028.3_59-

77_C19U_s

77_G1A_as

AD-
AUGCCGCCUA
82
NM_
AGUCGUCCUU
492
NM_
60-78

960560
AGGACGACU

001028.3_60-
AGGCGGCAU

001028.3_60-

78_A19U_s

78_U1A_as

AD-
UGCCGCCUAA
83
NM_
AUGUCGUCCU
493
NM_
61-79

960561
GGACGACAU

001028.3_61-
UAGGCGGCA

001028.3_61-

79_A19U_s

79_U1A_as

AD-
GCCGCCUAAG
84
NM_
AUUGUCGUCC
494
NM_
62-80

960562
GACGACAAU

001028.3_62-
UUAGGCGGC

001028.3_62-

80_G19U_s

80_C1A_as

AD-
CCGCCUAAGG
85
NM_
ACUUGUCGUC
495
NM_
63-81

960563
ACGACAAGU

001028.3_63-
CUUAGGCGG

001028.3_63-

81_A19U_s

81_U1A_as

AD-
CGCCUAAGGA
86
NM_
AUCUUGUCGU
496
NM_
64-82

960564
CGACAAGAU

001028.3_64-
CCUUAGGCG

001028.3_64-

82_A19U_s

82_U1A_as

AD-
GCCUAAGGAC
87
NM_
AUUCUUGUCG
497
NM_
65-83

960565
GACAAGAAU

001028.3_65-
UCCUUAGGC

001028.3_65-

83_G19U_s

83_C1A_as

AD-
CCUAAGGACG
88
NM_
ACUUCUUGUC
498
NM_
66-84

960566
ACAAGAAGU

001028.3_66-
GUCCUUAGG

001028.3_66-

84_A19U_s

84_U1A_as

AD-
CUAAGGACGA
89
NM_
AUCUUCUUGU
499
NM_
67-85

960567
CAAGAAGAU

001028.3_67-
CGUCCUUAG

001028.3_67-

85_A19U_s

85_U1A_as

AD-
UAAGGACGAC
90
NM_
AUUCUUCUUG
500
NM_
68-86

960568
AAGAAGAAU

001028.3_68-
UCGUCCUUA

001028.3_68-

86_G19U_s

86_C1A_as

AD-
AAGGACGACA
91
NM_
ACUUCUUCUU
501
NM_
69-87

960569
AGAAGAAGU

001028.3_69-
GUCGUCCUU

001028.3_69-

87_A19U_s

87_U1A_as

AD-
AGGACGACAA
92
NM_
AUCUUCUUCU
502
NM_
70-88

960570
GAAGAAGAU

001028.3_70-
UGUCGUCCU

001028.3_70-

88_A19U_s

88_U1A_as

AD-
GGACGACAAG
93
NM_
AUUCUUCUUC
503
NM_
71-89

960571
AAGAAGAAU

001028.3_71-
UUGUCGUCC

001028.3_71-

89_G19U_s

89_C1A_as

AD-
GACGACAAGA
94
NM_
ACUUCUUCUU
504
NM_
72-90

960572
AGAAGAAGU

001028.3_72-
CUUGUCGUC

001028.3_72-

90_G19U_s

90_C1A_as

AD-
ACGACAAGAA
95
NM_
ACCUUCUUCU
505
NM_
73-91

960573
GAAGAAGGU

001028.3_73-
UCUUGUCGU

001028.3_73-

91_A19U_s

91_U1A_as

AD-
CGACAAGAAG
96
NM_
AUCCUUCUUC
506
NM_
74-92

960574
AAGAAGGAU

001028.3_74-
UUCUUGUCG

001028.3_74-

92_C19U_s

92_G1A_as

AD-
GACAAGAAGA
97
NM_
AGUCCUUCUU
507
NM_
75-93

960575
AGAAGGACU

001028.3_75-
CUUCUUGUC

001028.3_75-

93_G19U_s

93_C1A_as

AD-
ACAAGAAGAA
98
NM_
ACGUCCUUCU
508
NM_
76-94

960576
GAAGGACGU

001028.3_76-
UCUUCUUGU

001028.3_76-

94_C19U_s

94_G1A_as

AD-
CAAGAAGAAG
99
NM_
AGCGUCCUUC
509
NM_
77-95

960577
AAGGACGCU

001028.3_77-
UUCUUCUUG

001028.3_77-

95_s

95_as

AD-
AAGAAGAAGA
100
NM_
AAGCGUCCUU
510
NM_
78-96

960578
AGGACGCUU

001028.3_78-
CUUCUUCUU

001028.3_78-

96_G19U_s

96_C1A_as

AD-
AGAAGAAGAA
101
NM_
ACAGCGUCCU
511
NM_
79-97

960579
GGACGCUGU

001028.3_79-
UCUUCUUCU

001028.3_79-

97_G19U_s

97_C1A_as

AD-
GAAGAAGAAG
102
NM_
ACCAGCGUCC
512
NM_
80-98

960580
GACGCUGGU

001028.3_80-
UUCUUCUUC

001028.3_80-

98_A19U_s

98_U1A_as

AD-
AAGAAGAAGG
103
NM_
AUCCAGCGUC
513
NM_
81-99

960581
ACGCUGGAU

001028.3_81-
CUUCUUCUU

001028.3_81-

99_A19U_s

99_U1A_as

AD-
AGAAGAAGGA
104
NM_
AUUCCAGCGU
514
NM_
82-100

960582
CGCUGGAAU

001028.3_82-
CCUUCUUCU

001028.3_82-

100_A19U_s

100_U1A_as

AD-
GAAGAAGGAC
105
NM_
AUUUCCAGCG
515
NM_
83-101

960583
GCUGGAAAU

001028.3_83-
UCCUUCUUC

001028.3_83-

101_G19U_s

101_C1A_as

AD-
AAGAAGGACG
106
NM_
ACUUUCCAGC
516
NM_
84-102

960584
CUGGAAAGU

001028.3_84-
GUCCUUCUU

001028.3_84-

102_s

102_as

AD-
AGAAGGACGC
107
NM_
AACUUUCCAG
517
NM_
85-103

960585
UGGAAAGUU

001028.3_85-
CGUCCUUCU

001028.3_85-

103_C19U_s

103_G1A_as

AD-
GAAGGACGCU
108
NM_
AGACUUUCCA
518
NM_
86-104

960586
GGAAAGUCU

001028.3_86-
GCGUCCUUC

001028.3_86-

104_G19U_s

104_C1A_as

AD-
AAGGACGCUG
109
NM_
ACGACUUUCC
519
NM_
87-105

960587
GAAAGUCGU

001028.3_87-
AGCGUCCUU

001028.3_87-

105_G19U_s

105_C1A_as

AD-
AGGACGCUGG
110
NM_
ACCGACUUUC
520
NM_
88-106

960588
AAAGUCGGU

001028.3_88-
CAGCGUCCU

001028.3_88-

106_C19U_s

106_G1A_as

AD-
GGACGCUGGA
ill
NM_
AGCCGACUUU
521
NM_
89-107

960589
AAGUCGGCU

001028.3_89-
CCAGCGUCC

001028.3_89-

107_C19U_s

107_G1A_as

AD-
GACGCUGGAA
112
NM_
AGGCCGACUU
522
NM_
90-108

960590
AGUCGGCCU

001028.3_90-
UCCAGCGUC

001028.3_90-

108_A19U_s

108_U1A_as

AD-
ACGCUGGAAA
113
NM_
AUGGCCGACU
523
NM_
91-109

960591
GUCGGCCAU

001028.3_91-
UUCCAGCGU

001028.3_91-

109_A19U_s

109_U1A_as

AD-
CGCUGGAAAG
114
NM_
AUUGGCCGAC
524
NM_
92-110

960592
UCGGCCAAU

001028.3_92-
UUUCCAGCG

001028.3_92-

110_G19U_s

110_C1A_as

AD-
GCUGGAAAGU
115
NM_
ACUUGGCCGA
525
NM_
93-111

960593
CGGCCAAGU

001028.3_93-
CUUUCCAGC

001028.3_93-

111_A19U_s

111_U1A_as

AD-
CUGGAAAGUC
116
NM_
AUCUUGGCCG
526
NM_
94-112

960594
GGCCAAGAU

001028.3_94-
ACUUUCCAG

001028.3_94-

112_A19U_s

112_U1A_as

AD-
UGGAAAGUCG
117
NM_
AUUCUUGGCC
527
NM_
95-113

960595
GCCAAGAAU

001028.3_95-
GACUUUCCA

001028.3_95-

113_A19U_s

113_U1A_as

AD-
GGAAAGUCGG
118
NM_
AUUUCUUGGC
528
NM_
96-114

960596
CCAAGAAAU

001028.3_96-
CGACUUUCC

001028.3_96-

114_G19U_s

114_C1A_as

AD-
GAAAGUCGGC
119
NM_
ACUUUCUUGG
529
NM_
97-115

960597
CAAGAAAGU

001028.3_97-
CCGACUUUC

001028.3_97-

115_A19U_s

115_U1A_as

AD-
AAAGUCGGCC
120
NM_
AUCUUUCUUG
530
NM_
98-116

960598
AAGAAAGAU

001028.3_98-
GCCGACUUU

001028.3_98-

116_C19U_s

116_G1A_as

AD-
AAGUCGGCCA
121
NM_
AGUCUUUCUU
531
NM_
99-117

960599
AGAAAGACU

001028.3_99-
GGCCGACUU

001028.3_99-

117_A19U_s

117_U1A_as

AD-
AGUCGGCCAA
122
NM_
AUGUCUUUCU
532
NM_
100-118

960600
GAAAGACAU

001028.3_100-
UGGCCGACU

001028.3_100-

118_A19U_s

118_U1A_as

AD-
GUCGGCCAAG
123
NM_
AUUGUCUUUC
533
NM_
101-119

960601
AAAGACAAU

001028.3_101-
UUGGCCGAC

001028.3_101-

119_A19U_s

119_U1A_as

AD-
UCGGCCAAGA
124
NM_
AUUUGUCUUU
534
NM_
102-120

960602
AAGACAAAU

001028.3_ 102-
CUUGGCCGA

001028.3_102-

120_G19U_s

120_C1A_as

AD-
CGGCCAAGAA
125
NM_
ACUUUGUCUU
535
NM_
103-121

960603
AGACAAAGU

001028.3_103-
UCUUGGCCG

001028.3_103-

121_A19U_s

121_U1A_as

AD-
GGCCAAGAAA
126
NM_
AUCUUUGUCU
536
NM_
104-122

960604
GACAAAGAU

001028.3_ 104-
UUCUUGGCC

001028.3_104-

122_C19U_s

122_G1A_as

AD-
GCCAAGAAAG
127
NM_
AGUCUUUGUC
537
NM_
105-123

960605
ACAAAGACU

001028.3_ 105-
UUUCUUGGC

001028.3_105-

123_C19U_s

123_G1A_as

AD-
CCAAGAAAGA
128
NM_
AGGUCUUUGU
538
NM_
106-124

960606
CAAAGACCU

001028.3_ 106-
CUUUCUUGG

001028.3_106-

124_C19U_s

124_G1A_as

AD-
CAAGAAAGAC
129
NM_
AGGGUCUUUG
539
NM_
107-125

960607
AAAGACCCU

001028.3_ 107-
UCUUUCUUG

001028.3_107-

125_A19U_s

125_U1A_as

AD-
AGAAAGACAA
130
NM_
ACUGGGUCUU
540
NM_
109-127

960608
AGACCCAGU

001028.3_109-
UGUCUUUCU

001028.3_109-

127_s

127_as

AD-
GAAAGACAAA
131
NM_
AACUGGGUCU
541
NM_
110-128

960609
GACCCAGUU

001028.3_110-
UUGUCUUUC

001028.3_110-

128_G19U_s

128_C1A_as

AD-
AAAGACAAAG
132
NM_
ACACUGGGUC
542
NM_
111-129

960610
ACCCAGUGU

001028.3_1 11-
UUUGUCUUU

001028.3_111-

129_A19U_s

129_U1A_as

AD-
AAGACAAAGA
133
NM_
AUCACUGGGU
543
NM_
112-130

960611
CCCAGUGAU

001028.3_112-
CUUUGUCUU

001028.3_112-

130_A19U_s

130_U1A_as

AD-
AGACAAAGAC
134
NM_
AUUCACUGGG
544
NM_
113-131

960612
CCAGUGAAU

001028.3_113-
UCUUUGUCU

001028.3_113-

131_C19U_s

131_G1A_as

AD-
GACAAAGACC
135
NM_
AGUUCACUGG
545
NM_
114-132

960613
CAGUGAACU

001028.3_114-
GUCUUUGUC

001028.3_114-

132_A19U_s

132_U1A_as

AD-
ACAAAGACCC
136
NM_
AUGUUCACUG
546
NM_
115-133

960614
AGUGAACAU

001028.3_115-
GGUCUUUGU

001028.3_115-

133_A19U_s

133_U1A_as

AD-
CAAAGACCCA
137
NM_
AUUGUUCACU
547
NM_
116-134

960615
GUGAACAAU

001028.3_116-
GGGUCUUUG

001028.3_116-

134_A19U_s

134_U1A_as

AD-
AAAGACCCAG
138
NM_
AUUUGUUCAC
548
NM_
117-135

960616
UGAACAAAU

001028.3_117-
UGGGUCUUU

001028.3_117-

135_s

135_as

AD-
AAGACCCAGU
139
NM_
AAUUUGUUCA
549
NM_
118-136

960617
GAACAAAUU

001028.3_118-
CUGGGUCUU

001028.3_118-

136_C19U_s

136_G1A_as

AD-
AGACCCAGUG
140
NM_
AGAUUUGUUC
550
NM_
119-137

960618
AACAAAUCU

001028.3_119-
ACUGGGUCU

001028.3_119-

137_C19U_s

137_G1A_as

AD-
GACCCAGUGA
141
NM_
AGGAUUUGUU
551
NM_
120-138

960619
ACAAAUCCU

001028.3_ 120-
CACUGGGUC

001028.3_120-

138_G19U_s

138_C1A_as

AD-
ACCCAGUGAA
142
NM_
ACGGAUUUGU
552
NM_
121-139

960620
CAAAUCCGU

001028.3_121-
UCACUGGGU

001028.3_121-

139_G19U_s

139_C1A_as

AD-
CCCAGUGAAC
143
NM_
ACCGGAUUUG
553
NM_
122-140

960621
AAAUCCGGU

001028.3_ 122-
UUCACUGGG

001028.3_122-

140_G19U_s

140_C1A_as

AD-
GGGCAAGGCC
144
NM_
AUUCUUUUUG
554
NM_
140-158

960622
AAAAAGAAU

001028.3_ 140-
GCCUUGCCC

001028.3_140-

158_G19U_s

158_C1A_as

AD-
GGCAAGGCCA
145
NM_
ACUUCUUUUU
555
NM_
141-159

960623
AAAAGAAGU

001028.3_141-
GGCCUUGCC

001028.3_141-

159_A19U_s

159_U1A_as

AD-
GCAAGGCCAA
146
NM_
AUCUUCUUUU
556
NM_
142-160

960624
AAAGAAGAU

001028.3_ 142-
UGGCCUUGC

001028.3_142-

160_A19U_s

160_U1A_as

AD-
AGGCCAAAAA
147
NM_
AACUUCUUCU
557
NM_
145-163

960625
GAAGAAGUU

001028.3_ 145-
UUUUGGCCU

001028.3_145-

163_G19U_s

163_C1A_as

AD-
GGCCAAAAAG
148
NM_
ACACUUCUUC
558
NM_
146-164

960626
AAGAAGUGU

001028.3_ 146-
UUUUUGGCC

001028.3_146-

164_G19U_s

164_C1A_as

AD-
GCCAAAAAGA
149
NM_
ACCACUUCUU
559
NM_
147-165

960627
AGAAGUGGU

001028.3_ 147-
CUUUUUGGC

001028.3_147-

165_s

165_as

AD-
CCAAAAAGAA
150
NM_
AACCACUUCU
560
NM_
148-166

960628
GAAGUGGUU

001028.3_148-
UCUUUUUGG

001028.3_148-

166_C19U_s

166_G1A_as

AD-
CAAAAAGAAG
151
NM_
AGACCACUUC
561
NM_
149-167

960629
AAGUGGUCU

001028.3_149-
UUCUUUUUG

001028.3_149-

167_C19U_s

167_G1A_as

AD-
AAAAAGAAGA
152
NM_
AGGACCACUU
562
NM_
150-168

960630
AGUGGUCCU

001028.3_ 150-
CUUCUUUUU

001028.3_150-

168_A19U_s

168_U1A_as

AD-
AAAAGAAGAA
153
NM_
AUGGACCACU
563
NM_
151-169

960631
GUGGUCCAU

001028.3_151-
UCUUCUUUU

001028.3_151-

169_A19U_s

169_U1A_as

AD-
AAAGAAGAAG
154
NM_
AUUGGACCAC
564
NM_
152-170

960632
UGGUCCAAU

001028.3_ 152-
UUCUUCUUU

001028.3_152-

170_A19U_s

170_U1A_as

AD-
AAGAAGAAGU
155
NM_
AUUUGGACCA
565
NM_
153-171

960633
GGUCCAAAU

001028.3_153-
CUUCUUCUU

001028.3_153-

171_G19U_s

171_C1A_as

AD-
AGAAGAAGUG
156
NM_
ACUUUGGACC
566
NM_
154-172

960634
GUCCAAAGU

001028.3_154-
ACUUCUUCU

001028.3_154-

172_G19U_s

172_C1A_as

AD-
GAAGAAGUGG
157
NM_
ACCUUUGGAC
567
NM_
155-173

960635
UCCAAAGGU

001028.3_155-
CACUUCUUC

001028.3_155-

173_C19U_s

173_G1A_as

AD-
AAGAAGUGGU
158
NM_
AGCCUUUGGA
568
NM_
156-174

960636
CCAAAGGCU

001028.3_ 156-
CCACUUCUU

001028.3_156-

174_A19U_s

174_U1A_as

AD-
AGAAGUGGUC
159
NM_
AUGCCUUUGG
569
NM_
157-175

960637
CAAAGGCAU

001028.3_157-
ACCACUUCU

001028.3_157-

175_A19U_s

175_U1A_as

AD-
GAAGUGGUCC
160
NM_
AUUGCCUUUG
570
NM_
158-176

960638
AAAGGCAAU

001028.3_158-
GACCACUUC

001028.3_158-

176_A19U_s

176_U1A_as

AD-
AAGUGGUCCA
161
NM_
AUUUGCCUUU
571
NM_
159-177

960639
AAGGCAAAU

001028.3_159-
GGACCACUU

001028.3_159-

177_G19U_s

177_C1A_as

AD-
AGUGGUCCAA
162
NM_
ACUUUGCCUU
572
NM_
160-178

960640
AGGCAAAGU

001028.3_ 160-
UGGACCACU

001028.3_160-

178_s

178_as

AD-
GUGGUCCAAA
163
NM_
AACUUUGCCU
573
NM_
161-179

960641
GGCAAAGUU

001028.3_161-
UUGGACCAC

001028.3_161-

179_s

179_as

AD-
UGGUCCAAAG
164
NM_
AAACUUUGCC
574
NM_
162-180

960642
GCAAAGUUU

001028.3_ 162-
UUUGGACCA

001028.3_162-

180_C19U_s

180_G1A_as

AD-
GGUCCAAAGG
165
NM_
AGAACUUUGC
575
NM_
163-181

960643
CAAAGUUCU

001028.3_163-
CUUUGGACC

001028.3_163-

181_G19U_s

181_C1A_as

AD-
GUCCAAAGGC
166
NM_
ACGAACUUUG
576
NM_
164-182

960644
AAAGUUCGU

001028.3_ 164-
CCUUUGGAC

001028.3_164-

182_G19U_s

182_C1A_as

AD-
UCCAAAGGCA
167
NM_
ACCGAACUUU
577
NM_
165-183

960645
AAGUUCGGU

001028.3_ 165-
GCCUUUGGA

001028.3_165-

183_G19U_s

183_C1A_as

AD-
CCAAAGGCAA
168
NM_
ACCCGAACUU
578
NM_
166-184

960646
AGUUCGGGU

001028.3_ 166-
UGCCUUUGG

001028.3_166-

184_A19U_s

184_U1A_as

AD-
CAAAGGCAAA
169
NM_
AUCCCGAACU
579
NM_
167-185

960647
GUUCGGGAU

001028.3_ 167-
UUGCCUUUG

001028.3_167-

185_C19U_s

185_G1A_as

AD-
AAAGGCAAAG
170
NM_
AGUCCCGAAC
580
NM_
168-186

960648
UUCGGGACU

001028.3_168-
UUUGCCUUU

001028.3_168-

186_A19U_s

186_U1A_as

AD-
AAGGCAAAGU
171
NM_
AUGUCCCGAA
581
NM_
169-187

960649
UCGGGACAU

001028.3_169-
CUUUGCCUU

001028.3_169-

187_A19U_s

187_U1A_as

AD-
AGGCAAAGUU
172
NM_
AUUGUCCCGA
582
NM_
170-188

960650
CGGGACAAU

001028.3_ 170-
ACUUUGCCU

001028.3_170-

188_G19U_s

188_C1A_as

AD-
GGCAAAGUUC
173
NM_
ACUUGUCCCG
583
NM_
171-189

960651
GGGACAAGU

001028.3_171-
AACUUUGCC

001028.3_171-

189_C19U_s

189_G1A_as

AD-
GCAAAGUUCG
174
NM_
AGCUUGUCCC
584
NM_
172-190

960652
GGACAAGCU

001028.3_ 172-
GAACUUUGC

001028.3_172-

190_s

190_as

AD-
CAAAGUUCGG
175
NM_
AAGCUUGUCC
585
NM_
173-191

960653
GACAAGCUU

001028.3_173-
CGAACUUUG

001028.3_173-

191_C19U_s

191_G1A_as

AD-
AAAGUUCGGG
176
NM_
AGAGCUUGUC
586
NM_
174-192

960654
ACAAGCUCU

001028.3_ 174-
CCGAACUUU

001028.3_174-

192_A19U_s

192_U1A_as

AD-
AAGUUCGGGA
177
NM_
AUGAGCUUGU
587
NM_
175-193

960655
CAAGCUCAU

001028.3_175-
CCCGAACUU

001028.3_175-

193_A19U_s

193_U1A_as

AD-
AGUUCGGGAC
178
NM_
AUUGAGCUUG
588
NM_
176-194

960656
AAGCUCAAU

001028.3_ 176-
UCCCGAACU

001028.3_176-

194_s

194_as

AD-
GUUCGGGACA
179
NM_
AAUUGAGCUU
589
NM_
177-195

960657
AGCUCAAUU

001028.3_177-
GUCCCGAAC

001028.3_177-

195_A19U_s

195_U1A_as

AD-
UUCGGGACAA
180
NM_
AUAUUGAGCU
590
NM_
178-196

960658
GCUCAAUAU

001028.3_178-
UGUCCCGAA

001028.3_178-

196_A19U_s

196_U1A_as

AD-
UCGGGACAAG
181
NM_
AUUAUUGAGC
591
NM_
179-197

960659
CUCAAUAAU

001028.3_179-
UUGUCCCGA

001028.3_179-

197_C19U_s

197_G1A_as

AD-
CGGGACAAGC
182
NM_
AGUUAUUGAG
592
NM_
180-198

960660
UCAAUAACU

001028.3_180-
CUUGUCCCG

001028.3_180-

198_s

198_as

AD-
GGGACAAGCU
183
NM_
AAGUUAUUGA
593
NM_
181-199

960661
CAAUAACUU

001028.3_181-
GCUUGUCCC

001028.3_181-

199_s

199_as

AD-
GGACAAGCUC
184
NM_
AAAGUUAUUG
594
NM_
182-200

960662
AAUAACUUU

001028.3_182-
AGCUUGUCC

001028.3_182-

200_A19U_s

200_U1A_as

AD-
GACAAGCUCA
185
NM_
AUAAGUUAUU
595
NM_
183-201

960663
AUAACUUAU

001028.3_183-
GAGCUUGUC

001028.3_183-

201_G19U_s

201_C1A_as

AD-
ACAAGCUCAA
186
NM_
ACUAAGUUAU
596
NM_
184-202

960664
UAACUUAGU

001028.3_ 184-
UGAGCUUGU

001028.3_184-

202_s

202_as

AD-
CAAGCUCAAU
187
NM_
AACUAAGUUA
597
NM_
185-203

960665
AACUUAGUU

001028.3_185-
UUGAGCUUG

001028.3_185-

203_C19U_s

203_G1A_as

AD-
AAGCUCAAUA
188
NM_
AGACUAAGUU
598
NM_
186-204

960666
ACUUAGUCU

001028.3_186-
AUUGAGCUU

001028.3_186-

204_s

204_as

AD-
AGCUCAAUAA
189
NM_
AAGACUAAGU
599
NM_
187-205

960667
CUUAGUCUU

001028.3_187-
UAUUGAGCU

001028.3_187-

205_s

205_as

AD-
GCUCAAUAAC
190
NM_
AAAGACUAAG
600
NM_
188-206

960668
UUAGUCUUU

001028.3_188-
UUAUUGAGC

001028.3_188-

206_G19U_s

206_C1A_as

AD-
CUCAAUAACU
191
NM_
ACAAGACUAA
601
NM_
189-207

960669
UAGUCUUGU

001028.3_189-
GUUAUUGAG

001028.3_189-

207_s

207_as

AD-
UCAAUAACUU
192
NM_
AACAAGACUA
602
NM_
190-208

960670
AGUCUUGUU

001028.3_190-
AGUUAUUGA

001028.3_190-

208_s

208_as

AD-
CAAUAACUUA
193
NM_
AAACAAGACU
603
NM_
191-209

960671
GUCUUGUUU

001028.3_191-
AAGUUAUUG

001028.3_191-

209_s

209_as

AD-
AAUAACUUAG
194
NM_
AAAACAAGAC
604
NM_
192-210

960672
UCUUGUUUU

001028.3_192-
UAAGUUAUU

001028.3_192-

210_G19U_s

210_C1A_as

AD-
AUAACUUAGU
195
NM_
ACAAACAAGA
605
NM_
193-211

960673
CUUGUUUGU

001028.3_193-
CUAAGUUAU

001028.3_193-

211_A19U_s

211_U1A_as

AD-
UAACUUAGUC
196
NM_
AUCAAACAAG
606
NM_
194-212

960674
UUGUUUGAU

001028.3_ 194-
ACUAAGUUA

001028.3_194-

212_C19U_s

212_G1A_as

AD-
AACUUAGUCU
197
NM_
AGUCAAACAA
607
NM_
195-213

960675
UGUUUGACU

001028.3_195-
GACUAAGUU

001028.3_195-

213_A19U_s

213_U1A_as

AD-
ACUUAGUCUU
198
NM_
AUGUCAAACA
608
NM_
196-214

960676
GUUUGACAU

001028.3_196-
AGACUAAGU

001028.3_196-

214_A19U_s

214_U1A_as

AD-
CUUAGUCUUG
199
NM_
AUUGUCAAAC
609
NM_
197-215

960677
UUUGACAAU

001028.3_197-
AAGACUAAG

001028.3_197-

215_A19U_s

215_U1A_as

AD-
UUAGUCUUGU
200
NM_
AUUUGUCAAA
610
NM_
198-216

960678
UUGACAAAU

001028.3_198-
CAAGACUAA

001028.3_198-

216_G19U_s

216_C1A_as

AD-
UAGUCUUGUU
201
NM_
ACUUUGUCAA
611
NM_
199-217

960679
UGACAAAGU

001028.3_199-
ACAAGACUA

001028.3_199-

217_C19U_s

217_G1A_as

AD-
AGUCUUGUUU
202
NM_
AGCUUUGUCA
612
NM_
200-218

960680
GACAAAGCU

001028.3_200-
AACAAGACU

001028.3_200-

218_s

218_as

AD-
GUCUUGUUUG
203
NM_
AAGCUUUGUC
613
NM_
201-219

960681
ACAAAGCUU

001028.3_201-
AAACAAGAC

001028.3_201-

219_A19U_s

219_U1A_as

AD-
UCUUGUUUGA
204
NM_
AUAGCUUUGU
614
NM_
202-220

960682
CAAAGCUAU

001028.3_202-
CAAACAAGA

001028.3_202-

220_C19U_s

220_G1A_as

AD-
CUUGUUUGAC
205
NM_
AGUAGCUUUG
615
NM_
203-221

960683
AAAGCUACU

001028.3_203-
UCAAACAAG

001028.3_203-

221_C19U_s

221_G1A_as

AD-
UUGUUUGACA
206
NM_
AGGUAGCUUU
616
NM_
204-222

960684
AAGCUACCU

001028.3_204-
GUCAAACAA

001028.3_204-

222_s

222_as

AD-
UGUUUGACAA
207
NM_
AAGGUAGCUU
617
NM_
205-223

960685
AGCUACCUU

001028.3_205-
UGUCAAACA

001028.3_205-

223_A19U_s

223_U1A_as

AD-
GUUUGACAAA
208
NM_
AUAGGUAGCU
618
NM_
206-224

960686
GCUACCUAU

001028.3_206-
UUGUCAAAC

001028.3_206-

224_s

224_as

AD-
UUUGACAAAG
209
NM_
AAUAGGUAGC
619
NM_
207-225

960687
CUACCUAUU

001028.3_207-
UUUGUCAAA

001028.3_207-

225_G19U_s

225_C1A_as

AD-
UUGACAAAGC
210
NM_
ACAUAGGUAG
620
NM_
208-226

960688
UACCUAUGU

001028.3_208-
CUUUGUCAA

001028.3_208-

226_A19U_s

226_U1A_as

AD-
UGACAAAGCU
211
NM_
AUCAUAGGUA
621
NM_
209-227

960689
ACCUAUGAU

001028.3_209-
GCUUUGUCA

001028.3_209-

227_s

227_as

AD-
GACAAAGCUA
212
NM_
AAUCAUAGGU
622
NM_
210-228

960690
CCUAUGAUU

001028.3_210-
AGCUUUGUC

001028.3_210-

228_A19U_s

228_U1A_as

AD-
ACAAAGCUAC
213
NM_
AUAUCAUAGG
623
NM_
211-229

960691
CUAUGAUAU

001028.3_211-
UAGCUUUGU

001028.3_211-

229_A19U_s

229_U1A_as

AD-
CAAAGCUACC
214
NM_
AUUAUCAUAG
624
NM_
212-230

960692
UAUGAUAAU

001028.3_212-
GUAGCUUUG

001028.3_212-

230_A19U_s

230_U1A_as

AD-
AAAGCUACCU
215
NM_
AUUUAUCAUA
625
NM_
213-231

960693
AUGAUAAAU

001028.3_213-
GGUAGCUUU

001028.3_213-

231_C19U_s

231_G1A_as

AD-
AAGCUACCUA
216
NM_
AGUUUAUCAU
626
NM_
214-232

960694
UGAUAAACU

001028.3_214-
AGGUAGCUU

001028.3_214-

232_s

232_as

AD-
AGCUACCUAU
217
NM_
AAGUUUAUCA
627
NM_
215-233

960695
GAUAAACUU

001028.3_215-
UAGGUAGCU

001028.3_215-

233_C19U_s

233_G1A_as

AD-
GCUACCUAUG
218
NM_
AGAGUUUAUC
628
NM_
216-234

960696
AUAAACUCU

001028.3_216-
AUAGGUAGC

001028.3_216-

234_s

234_as

AD-
CUACCUAUGA
219
NM_
AAGAGUUUAU
629
NM_
217-235

960697
UAAACUCUU

001028.3_217-
CAUAGGUAG

001028.3_217-

235_G19U_s

235_C1A_as

AD-
UACCUAUGAU
220
NM_
ACAGAGUUUA
630
NM_
218-236

960698
AAACUCUGU

001028.3_218-
UCAUAGGUA

001028.3_218-

236_s

236_as

AD-
ACCUAUGAUA
221
NM_
AACAGAGUUU
631
NM_
219-237

960699
AACUCUGUU

001028.3_219-
AUCAUAGGU

001028.3_219-

237_A19U_s

237_U1A_as

AD-
CCUAUGAUAA
222
NM_
AUACAGAGUU
632
NM_
220-238

960700
ACUCUGUAU

001028.3_220-
UAUCAUAGG

001028.3_220-

238_A19U_s

238_U1A_as

AD-
CUAUGAUAAA
223
NM_
AUUACAGAGU
633
NM_
221-239

960701
CUCUGUAAU

001028.3_221-
UUAUCAUAG

001028.3_221-

239_G19U_s

239_C1A_as

AD-
UAUGAUAAAC
224
NM_
ACUUACAGAG
634
NM_
222-240

960702
UCUGUAAGU

001028.3_222-
UUUAUCAUA

001028.3_222-

240_G19U_s

240_C1A_as

AD-
AUGAUAAACU
225
NM_
ACCUUACAGA
635
NM_
223-241

960703
CUGUAAGGU

001028.3_223-
GUUUAUCAU

001028.3_223-

241_A19U_s

241_U1A_as

AD-
UGAUAAACUC
226
NM_
AUCCUUACAG
636
NM_
224-242

960704
UGUAAGGAU

001028.3_224-
AGUUUAUCA

001028.3_224-

242_A19U_s

242_U1A_as

AD-
GAUAAACUCU
227
NM_
AUUCCUUACA
637
NM_
225-243

960705
GUAAGGAAU

001028.3_225-
GAGUUUAUC

001028.3_225-

243_G19U_s

243_C1A_as

AD-
AUAAACUCUG
228
NM_
ACUUCCUUAC
638
NM_
226-244

960706
UAAGGAAGU

001028.3_226-
AGAGUUUAU

001028.3_226-

244_s

244_as

AD-
UAAACUCUGU
229
NM_
AACUUCCUUA
639
NM_
227-245

960707
AAGGAAGUU

001028.3_227-
CAGAGUUUA

001028.3_227-

245_s

245_as

AD-
AAACUCUGUA
230
NM_
AAACUUCCUU
640
NM_
228-246

960708
AGGAAGUUU

001028.3_228-
ACAGAGUUU

001028.3_228-

246_C19U_s

246_G1A_as

AD-
AACUCUGUAA
231
NM_
AGAACUUCCU
641
NM_
229-247

960709
GGAAGUUCU

001028.3_229-
UACAGAGUU

001028.3_229-

247_C19U_s

247_G1A_as

AD-
ACUCUGUAAG
232
NM_
AGGAACUUCC
642
NM_
230-248

960710
GAAGUUCCU

001028.3_230-
UUACAGAGU

001028.3_230-

248_C19U_s

248_G1A_as

AD-
CUCUGUAAGG
233
NM_
AGGGAACUUC
643
NM_
231-249

960711
AAGUUCCCU

001028.3_231-
CUUACAGAG

001028.3_231-

249_A19U_s

249_U1A_as

AD-
UCUGUAAGGA
234
NM_
AUGGGAACUU
644
NM_
232-250

960712
AGUUCCCAU

001028.3_232-
CCUUACAGA

001028.3_232-

250_A19U_s

250_U1A_as

AD-
CUGUAAGGAA
235
NM_
AUUGGGAACU
645
NM_
233-251

960713
GUUCCCAAU

001028.3_233-
UCCUUACAG

001028.3_233-

251_C19U_s

251_G1A_as

AD-
UGUAAGGAAG
236
NM_
AGUUGGGAAC
646
NM_
234-252

960714
UUCCCAACU

001028.3_234-
UUCCUUACA

001028.3_234-

252_s

252_as

AD-
GUAAGGAAGU
237
NM_
AAGUUGGGAA
647
NM_
235-253

960715
UCCCAACUU

001028.3_235-
CUUCCUUAC

001028.3_235-

253_A19U_s

253_U1A_as

AD-
UAAGGAAGUU
238
NM_
AUAGUUGGGA
648
NM_
236-254

960716
CCCAACUAU

001028.3_236-
ACUUCCUUA

001028.3_236-

254_s

254_as

AD-
AAGGAAGUUC
239
NM_
AAUAGUUGGG
649
NM_
237-255

960717
CCAACUAUU

001028.3_237-
AACUUCCUU

001028.3_237-

255_A19U_s

255_U1A_as

AD-
AGGAAGUUCC
240
NM_
AUAUAGUUGG
650
NM_
238-256

960718
CAACUAUAU

001028.3_238-
GAACUUCCU

001028.3_238-

256_A19U_s

256_U1A_as

AD-
GGAAGUUCCC
241
NM_
AUUAUAGUUG
651
NM_
239-257

960719
AACUAUAAU

001028.3_239-
GGAACUUCC

001028.3_239-

257_A19U_s

257_U1A_as

AD-
GAAGUUCCCA
242
NM_
AUUUAUAGUU
652
NM_
240-258

960720
ACUAUAAAU

001028.3_240-
GGGAACUUC

001028.3_240-

258_C19U_s

258_G1A_as

AD-
AAGUUCCCAA
243
NM_
AGUUUAUAGU
653
NM_
241-259

960721
CUAUAAACU

001028.3_241-
UGGGAACUU

001028.3_241-

259_s

259_as

AD-
AGUUCCCAAC
244
NM_
AAGUUUAUAG
654
NM_
242-260

960722
UAUAAACUU

001028.3_242-
UUGGGAACU

001028.3_242-

260_s

260_as

AD-
GUUCCCAACU
245
NM_
AAAGUUUAUA
655
NM_
243-261

960723
AUAAACUUU

001028.3_243-
GUUGGGAAC

001028.3_243-

261_A19U_s

261_U1A_as

AD-
UUCCCAACUA
246
NM_
AUAAGUUUAU
656
NM_
244-262

960724
UAAACUUAU

001028.3_244-
AGUUGGGAA

001028.3_244-

262_s

262_as

AD-
UCCCAACUAU
247
NM_
AAUAAGUUUA
657
NM_
245-263

960725
AAACUUAUU

001028.3_245-
UAGUUGGGA

001028.3_245-

263_A19U_s

263_U1A_as

AD-
CCCAACUAUA
248
NM_
AUAUAAGUUU
658
NM_
246-264

960726
AACUUAUAU

001028.3_246-
AUAGUUGGG

001028.3_246-

264_A19U_s

264_U1A_as

AD-
CCAACUAUAA
249
NM_
AUUAUAAGUU
659
NM_
247-265

960727
ACUUAUAAU

001028.3_247-
UAUAGUUGG

001028.3_247-

265_C19U_s

265_G1A_as

AD-
CAACUAUAAA
250
NM_
AGUUAUAAGU
660
NM_
248-266

960728
CUUAUAACU

001028.3_248-
UUAUAGUUG

001028.3_248-

266_C19U_s

266_G1A_as

AD-
AACUAUAAAC
251
NM_
AGGUUAUAAG
661
NM_
249-267

960729
UUAUAACCU

001028.3_249-
UUUAUAGUU

001028.3_249-

267_C19U_s

267_G1A_as

AD-
CCCAGCUGUG
252
NM_
AUCAGAGACC
662
NM_
266-284

960730
GUCUCUGAU

001028.3_266-
ACAGCUGGG

001028.3_266-

284_G19U_s

284_C1A_as

AD-
CCAGCUGUGG
253
NM_
ACUCAGAGAC
663
NM_
267-285

960731
UCUCUGAGU

001028.3_267-
CACAGCUGG

001028.3_267-

285_A19U_s

285_U1A_as

AD-
CAGCUGUGGU
254
NM_
AUCUCAGAGA
664
NM_
268-286

960732
CUCUGAGAU

001028.3_268-
CCACAGCUG

001028.3_268-

286_G19U_s

286_C1A_as

AD-
AGCUGUGGUC
255
NM_
ACUCUCAGAG
665
NM_
269-287

960733
UCUGAGAGU

001028.3_269-
ACCACAGCU

001028.3_269-

287_A19U_s

287_U1A_as

AD-
GCUGUGGUCU
256
NM_
AUCUCUCAGA
666
NM_
270-288

960734
CUGAGAGAU

001028.3_270-
GACCACAGC

001028.3_270-

288_C19U_s

288_G1A_as

AD-
CUGUGGUCUC
257
NM_
AGUCUCUCAG
667
NM_
271-289

960735
UGAGAGACU

001028.3_271-
AGACCACAG

001028.3_271-

289_s

289_as

AD-
UGUGGUCUCU
258
NM_
AAGUCUCUCA
668
NM_
272-290

960736
GAGAGACUU

001028.3_272-
GAGACCACA

001028.3_272-

290_G19U_s

290_C1A_as

AD-
GUGGUCUCUG
259
NM_
ACAGUCUCUC
669
NM_
273-291

960737
AGAGACUGU

001028.3_273-
AGAGACCAC

001028.3_273-

291_A19U_s

291_U1A_as

AD-
UGGUCUCUGA
260
NM_
AUCAGUCUCU
670
NM_
274-292

960738
GAGACUGAU

001028.3_274-
CAGAGACCA

001028.3_274-

292_A19U_s

292_U1A_as

AD-
GGUCUCUGAG
261
NM_
AUUCAGUCUC
671
NM_
275-293

960739
AGACUGAAU

001028.3_275-
UCAGAGACC

001028.3_275-

293_G19U_s

293_C1A_as

AD-
GUCUCUGAGA
262
NM_
ACUUCAGUCU
672
NM_
276-294

960740
GACUGAAGU

001028.3_276-
CUCAGAGAC

001028.3_276-

294_A19U_s

294_U1A_as

AD-
UCUCUGAGAG
263
NM_
AUCUUCAGUC
673
NM_
277-295

960741
ACUGAAGAU

001028.3_277-
UCUCAGAGA

001028.3_277-

295_s

295_as

AD-
CUCUGAGAGA
264
NM_
AAUCUUCAGU
674
NM_
278-296

960742
CUGAAGAUU

001028.3_278-
CUCUCAGAG

001028.3_278-

296_s

296_as

AD-
UCUGAGAGAC
265
NM_
AAAUCUUCAG
675
NM_
279-297

960743
UGAAGAUUU

001028.3_279-
UCUCUCAGA

001028.3_279-

297_C19U_s

297_G1A_as

AD-
CUGAGAGACU
266
NM_
AGAAUCUUCA
676
NM_
280-298

960744
GAAGAUUCU

001028.3_280-
GUCUCUCAG

001028.3_280-

298_G19U_s

298_C1A_as

AD-
UGAGAGACUG
267
NM_
ACGAAUCUUC
677
NM_
281-299

960745
AAGAUUCGU

001028.3_281-
AGUCUCUCA

001028.3_281-

299_A19U_s

299_U1A_as

AD-
GAGAGACUGA
268
NM_
AUCGAAUCUU
678
NM_
282-300

960746
AGAUUCGAU

001028.3_282-
CAGUCUCUC

001028.3_282-

300_G19U_s

300_C1A_as

AD-
AGAGACUGAA
269
NM_
ACUCGAAUCU
679
NM_
283-301

960747
GAUUCGAGU

001028.3_283-
UCAGUCUCU

001028.3_283-

301_G19U_s

301_C1A_as

AD-
GAGACUGAAG
270
NM_
ACCUCGAAUC
680
NM_
284-302

960748
AUUCGAGGU

001028.3_284-
UUCAGUCUC

001028.3_284-

302_C19U_s

302_G1A_as

AD-
AGACUGAAGA
271
NM_
AGCCUCGAAU
681
NM_
285-303

960749
UUCGAGGCU

001028.3_285-
CUUCAGUCU

001028.3_285-

303_s

303_as

AD-
GACUGAAGAU
272
NM_
AAGCCUCGAA
682
NM_
286-304

960750
UCGAGGCUU

001028.3_286-
UCUUCAGUC

001028.3_286-

304_C19U_s

304_G1A_as

AD-
ACUGAAGAUU
273
NM_
AGAGCCUCGA
683
NM_
287-305

960751
CGAGGCUCU

001028.3_287-
AUCUUCAGU

001028.3_287-

305_C19U_s

305_G1A_as

AD-
CUGAAGAUUC
274
NM_
AGGAGCCUCG
684
NM_
288-306

960752
GAGGCUCCU

001028.3_288-
AAUCUUCAG

001028.3_288-

306_C19U_s

306_G1A_as

AD-
UGAAGAUUCG
275
NM_
AGGGAGCCUC
685
NM_
289-307

960753
AGGCUCCCU

001028.3_289-
GAAUCUUCA

001028.3_289-

307_s

307_as

AD-
GAAGAUUCGA
276
NM_
AAGGGAGCCU
686
NM_
290-308

960754
GGCUCCCUU

001028.3_290-
CGAAUCUUC

001028.3_290-

308_G19U_s

308_C1A_as

AD-
AAGAUUCGAG
277
NM_
ACAGGGAGCC
687
NM_
291-309

960755
GCUCCCUGU

001028.3_291-
UCGAAUCUU

001028.3_291-

309_G19U_s

309_C1A_as

AD-
AGAUUCGAGG
278
NM_
ACCAGGGAGC
688
NM_
292-310

960756
CUCCCUGGU

001028.3_292-
CUCGAAUCU

001028.3_292-

310_C19U_s

310_G1A_as

AD-
GAUUCGAGGC
279
NM_
AGCCAGGGAG
689
NM_
293-311

960757
UCCCUGGCU

001028.3_293-
CCUCGAAUC

001028.3_293-

311_C19U_s

311_G1A_as

AD-
AUUCGAGGCU
280
NM_
AGGCCAGGGA
690
NM_
294-312

960758
CCCUGGCCU

001028.3_294-
GCCUCGAAU

001028.3_294-

312_A19U_s

312_U1A_as

AD-
UUCGAGGCUC
281
NM_
AUGGCCAGGG
691
NM_
295-313

960759
CCUGGCCAU

001028.3_295-
AGCCUCGAA

001028.3_295-

313_G19U_s

313_C1A_as

AD-
UCGAGGCUCC
282
NM_
ACUGGCCAGG
692
NM_
296-314

960760
CUGGCCAGU

001028.3_296-
GAGCCUCGA

001028.3_296-

314_G19U_s

314_C1A_as

AD-
CUGGCCAGGG
283
NM_
AAAGGGCUGC
693
NM_
306-324

960761
CAGCCCUUU

001028.3_306-
CCUGGCCAG

001028.3_306-

324_C19U_s

324_G1A_as

AD-
UGGCCAGGGC
284
NM_
AGAAGGGCUG
694
NM_
307-325

960762
AGCCCUUCU

001028.3_307-
CCCUGGCCA

001028.3_307-

325_A19U_s

325_U1A_as

AD-
GGCCAGGGCA
285
NM_
AUGAAGGGCU
695
NM_
308-326

960763
GCCCUUCAU

001028.3_308-
GCCCUGGCC

001028.3_308-

326_G19U_s

326_C1A_as

AD-
GCCAGGGCAG
286
NM_
ACUGAAGGGC
696
NM_
309-327

960764
CCCUUCAGU

001028.3_309-
UGCCCUGGC

001028.3_309-

327_G19U_s

327_C1A_as

AD-
CCAGGGCAGC
287
NM_
ACCUGAAGGG
697
NM_
310-328

960765
CCUUCAGGU

001028.3_310-
CUGCCCUGG

001028.3_310-

328_A19U_s

328_U1A_as

AD-
CAGGGCAGCC
288
NM_
AUCCUGAAGG
698
NM_
311-329

960766
CUUCAGGAU

001028.3_311-
GCUGCCCUG

001028.3_311-

329_G19U_s

329_C1A_as

AD-
AGGGCAGCCC
289
NM_
ACUCCUGAAG
699
NM_
312-330

960767
UUCAGGAGU

001028.3_312-
GGCUGCCCU

001028.3_312-

330_C19U_s

330_G1A_as

AD-
GGGCAGCCCU
290
NM_
AGCUCCUGAA
700
NM_
313-331

960768
UCAGGAGCU

001028.3_313-
GGGCUGCCC

001028.3_313-

331_s

331_as

AD-
GGCAGCCCUU
291
NM_
AAGCUCCUGA
701
NM_
314-332

960769
CAGGAGCUU

001028.3_314-
AGGGCUGCC

001028.3_314-

332_C19U_s

332_G1A_as

AD-
GCAGCCCUUC
292
NM_
AGAGCUCCUG
702
NM_
315-333

960770
AGGAGCUCU

001028.3_315-
AAGGGCUGC

001028.3_315-

333_C19U_s

333_G1A_as

AD-
CAGCCCUUCA
293
NM_
AGGAGCUCCU
703
NM_
316-334

960771
GGAGCUCCU

001028.3_316-
GAAGGGCUG

001028.3_316-

334_s

334_as

AD-
AGCCCUUCAG
294
NM_
AAGGAGCUCC
704
NM_
317-335

960772
GAGCUCCUU

001028.3_317-
UGAAGGGCU

001028.3_317-

335_s

335_as

AD-
GCCCUUCAGG
295
NM_
AAAGGAGCUC
705
NM_
318-336

960773
AGCUCCUUU

001028.3_318-
CUGAAGGGC

001028.3_318-

336_A19U_s

336_U1A_as

AD-
CCCUUCAGGA
296
NM_
AUAAGGAGCU
706
NM_
319-337

960774
GCUCCUUAU

001028.3_319-
CCUGAAGGG

001028.3_319-

337_G19U_s

337_C1A_as

AD-
CCUUCAGGAG
297
NM_
ACUAAGGAGC
707
NM_
320-338

960775
CUCCUUAGU

001028.3_320-
UCCUGAAGG

001028.3_320-

338_s

338_as

AD-
CUUCAGGAGC
298
NM_
AACUAAGGAG
708
NM_
321-339

960776
UCCUUAGUU

001028.3_321-
CUCCUGAAG

001028.3_321-

339_A19U_s

339_U1A_as

AD-
UUCAGGAGCU
299
NM_
AUACUAAGGA
709
NM_
322-340

960777
CCUUAGUAU

001028.3_322-
GCUCCUGAA

001028.3_322-

340_A19U_s

340_U1A_as

AD-
UCAGGAGCUC
300
NM_
AUUACUAAGG
710
NM_
323-341

960778
CUUAGUAAU

001028.3_323-
AGCUCCUGA

001028.3_323-

341_A19U_s

341_U1A_as

AD-
CAGGAGCUCC
301
NM_
AUUUACUAAG
711
NM_
324-342

960779
UUAGUAAAU

001028.3_324-
GAGCUCCUG

001028.3_324-

342_G19U_s

342_C1A_as

AD-
AGGAGCUCCU
302
NM_
ACUUUACUAA
712
NM_
325-343

960780
UAGUAAAGU

001028.3_325-
GGAGCUCCU

001028.3_325-

343_G19U_s

343_C1A_as

AD-
GGAGCUCCUU
303
NM_
ACCUUUACUA
713
NM_
326-344

960781
AGUAAAGGU

001028.3_326-
AGGAGCUCC

001028.3_326-

344_A19U_s

344_U1A_as

AD-
GAGCUCCUUA
304
NM_
AUCCUUUACU
714
NM_
327-345

960782
GUAAAGGAU

001028.3_327-
AAGGAGCUC

001028.3_327-

345_C19U_s

345_G1A_as

AD-
AGCUCCUUAG
305
NM_
AGUCCUUUAC
715
NM_
328-346

960783
UAAAGGACU

001028.3_328-
UAAGGAGCU

001028.3_328-

346_s

346_as

AD-
GCUCCUUAGU
306
NM_
AAGUCCUUUA
716
NM_
329-347

960784
AAAGGACUU

001028.3_329-
CUAAGGAGC

001028.3_329-

347_s

347_as

AD-
CUCCUUAGUA
307
NM_
AAAGUCCUUU
717
NM_
330-348

960785
AAGGACUUU

001028.3_330-
ACUAAGGAG

001028.3_330-

348_A19U_s

348_U1A_as

AD-
UCCUUAGUAA
308
NM_
AUAAGUCCUU
718
NM_
331-349

960786
AGGACUUAU

001028.3_331-
UACUAAGGA

001028.3_331-

349_s

349_as

AD-
CCUUAGUAAA
309
NM_
AAUAAGUCCU
719
NM_
332-350

960787
GGACUUAUU

001028.3_332-
UUACUAAGG

001028.3_332-

350_C19U_s

350_G1A_as

AD-
CUUAGUAAAG
310
NM_
AGAUAAGUCC
720
NM_
333-351

960788
GACUUAUCU

001028.3_333-
UUUACUAAG

001028.3_333-

351_A19U_s

351_U1A_as

AD-
UUAGUAAAGG
311
NM_
AUGAUAAGUC
721
NM_
334-352

960789
ACUUAUCAU

001028.3_334-
CUUUACUAA

001028.3_334-

352_A19U_s

352_U1A_as

AD-
UAGUAAAGGA
312
NM_
AUUGAUAAGU
722
NM_
335-353

960790
CUUAUCAAU

001028.3_335-
CCUUUACUA

001028.3_335-

353_A19U_s

353_U1A_as

AD-
AGUAAAGGAC
313
NM_
AUUUGAUAAG
723
NM_
336-354

960791
UUAUCAAAU

001028.3_336-
UCCUUUACU

001028.3_336-

354_C19U_s

354_G1A_as

AD-
GUAAAGGACU
314
NM_
AGUUUGAUAA
724
NM_
337-355

960792
UAUCAAACU

001028.3_337-
GUCCUUUAC

001028.3_337-

355_s

355_as

AD-
UAAAGGACUU
315
NM_
AAGUUUGAUA
725
NM_
338-356

960793
AUCAAACUU

001028.3_338-
AGUCCUUUA

001028.3_338-

356_G19U_s

356_C1A_as

AD-
AAAGGACUUA
316
NM_
ACAGUUUGAU
726
NM_
339-357

960794
UCAAACUGU

001028.3_339-
AAGUCCUUU

001028.3_339-

357_G19U_s

357_C1A_as

AD-
AAGGACUUAU
317
NM_
ACCAGUUUGA
727
NM_
340-358

960795
CAAACUGGU

001028.3_340-
UAAGUCCUU

001028.3_340-

358_s

358_as

AD-
AGGACUUAUC
318
NM_
AACCAGUUUG
728
NM_
341-359

960796
AAACUGGUU

001028.3_341-
AUAAGUCCU

001028.3_341-

359_s

359_as

AD-
GGACUUAUCA
319
NM_
AAACCAGUUU
729
NM_
342-360

960797
AACUGGUUU

001028.3_342-
GAUAAGUCC

001028.3_342-

360_s

360_as

AD-
GACUUAUCAA
320
NM_
AAAACCAGUU
730
NM_
343-361

960798
ACUGGUUUU

001028.3_343-
UGAUAAGUC

001028.3_343-

361_C19U_s

361_G1A_as

AD-
ACUUAUCAAA
321
NM_
AGAAACCAGU
731
NM_
344-362

960799
CUGGUUUCU

001028.3_344-
UUGAUAAGU

001028.3_344-

362_A19U_s

362_U1A_as

AD-
CUUAUCAAAC
322
NM_
AUGAAACCAG
732
NM_
345-363

960800
UGGUUUCAU

001028.3_345-
UUUGAUAAG

001028.3_345-

363_A19U_s

363_U1A_as

AD-
UUAUCAAACU
323
NM_
AUUGAAACCA
733
NM_
346-364

960801
GGUUUCAAU

001028.3_346-
GUUUGAUAA

001028.3_346-

364_A19U_s

364_U1A_as

AD-
UAUCAAACUG
324
NM_
AUUUGAAACC
734
NM_
347-365

960802
GUUUCAAAU

001028.3_347-
AGUUUGAUA

001028.3_347-

365_G19U_s

365_C1A_as

AD-
AUCAAACUGG
325
NM_
ACUUUGAAAC
735
NM_
348-366

960803
UUUCAAAGU

001028.3_348-
CAGUUUGAU

001028.3_348-

366_C19U_s

366_G1A_as

AD-
UCAAACUGGU
326
NM_
AGCUUUGAAA
736
NM_
349-367

960804
UUCAAAGCU

001028.3_349-
CCAGUUUGA

001028.3_349-

367_A19U_s

367_U1A_as

AD-
CAAACUGGUU
327
NM_
AUGCUUUGAA
737
NM_
350-368

960805
UCAAAGCAU

001028.3_350-
ACCAGUUUG

001028.3_350-

368_C19U_s

368_G1A_as

AD-
AAACUGGUUU
328
NM_
AGUGCUUUGA
738
NM_
351-369

960806
CAAAGCACU

001028.3_351-
AACCAGUUU

001028.3_351-

369_A19U_s

369_U1A_as

AD-
AACUGGUUUC
329
NM_
AUGUGCUUUG
739
NM_
352-370

960807
AAAGCACAU

001028.3_352-
AAACCAGUU

001028.3_352-

370_G19U_s

370_C1A_as

AD-
ACUGGUUUCA
330
NM_
ACUGUGCUUU
740
NM_
353-371

960808
AAGCACAGU

001028.3_353-
GAAACCAGU

001028.3_353-

371_A19U_s

371_U1A_as

AD-
CUGGUUUCAA
331
NM_
AUCUGUGCUU
741
NM_
354-372

960809
AGCACAGAU

001028.3_354-
UGAAACCAG

001028.3_354-

372_G19U_s

372_C1A_as

AD-
UGGUUUCAAA
332
NM_
ACUCUGUGCU
742
NM_
355-373

960810
GCACAGAGU

001028.3_355-
UUGAAACCA

001028.3_355-

373_C19U_s

373_G1A_as

AD-
GGUUUCAAAG
333
NM_
AGCUCUGUGC
743
NM_
356-374

960811
CACAGAGCU

001028.3_356-
UUUGAAACC

001028.3_356-

374_s

374_as

AD-
GUUUCAAAGC
334
NM_
AAGCUCUGUG
744
NM_
357-375

960812
ACAGAGCUU

001028.3_357-
CUUUGAAAC

001028.3_357-

375_C19U_s

375_G1A_as

AD-
UUUCAAAGCA
335
NM_
AGAGCUCUGU
745
NM_
358-376

960813
CAGAGCUCU

001028.3_358-
GCUUUGAAA

001028.3_358-

376_A19U_s

376_U1A_as

AD-
UUCAAAGCAC
336
NM_
AUGAGCUCUG
746
NM_
359-377

960814
AGAGCUCAU

001028.3_359-
UGCUUUGAA

001028.3_359-

377_A19U_s

377_U1A_as

AD-
UCAAAGCACA
337
NM_
AUUGAGCUCU
747
NM_
360-378

960815
GAGCUCAAU

001028.3_360-
GUGCUUUGA

001028.3_360-

378_G19U_s

378_C1A_as

AD-
CAAAGCACAG
338
NM_
ACUUGAGCUC
748
NM_
361-379

960816
AGCUCAAGU

001028.3_361-
UGUGCUUUG

001028.3_361-

379_s

379_as

AD-
AAAGCACAGA
339
NM_
AACUUGAGCU
749
NM_
362-380

960817
GCUCAAGUU

001028.3_362-
CUGUGCUUU

001028.3_362-

380_A19U_s

380_U1A_as

AD-
AAGCACAGAG
340
NM_
AUACUUGAGC
750
NM_
363-381

960818
CUCAAGUAU

001028.3_363-
UCUGUGCUU

001028.3_363-

381_A19U_s

381_U1A_as

AD-
AGCACAGAGC
341
NM_
AUUACUUGAG
751
NM_
364-382

960819
UCAAGUAAU

001028.3_364-
CUCUGUGCU

001028.3_364-

382_s

382_as

AD-
GCACAGAGCU
342
NM_
AAUUACUUGA
752
NM_
365-383

960820
CAAGUAAUU

001028.3_365-
GCUCUGUGC

001028.3_365-

383_s

383_as

AD-
CACAGAGCUC
343
NM_
AAAUUACUUG
753
NM_
366-384

960821
AAGUAAUUU

001028.3_366-
AGCUCUGUG

001028.3_366-

384_s

384_as

AD-
ACAGAGCUCA
344
NM_
AAAAUUACUU
754
NM_
367-385

960822
AGUAAUUUU

001028.3_367-
GAGCUCUGU

001028.3_367-

385_A19U_s

385_U1A_as

AD-
CAGAGCUCAA
345
NM_
AUAAAUUACU
755
NM_
368-386

960823
GUAAUUUAU

001028.3_368-
UGAGCUCUG

001028.3_368-

386_C19U_s

386_G1A_as

AD-
AGAGCUCAAG
346
NM_
AGUAAAUUAC
756
NM_
369-387

960824
UAAUUUACU

001028.3_369-
UUGAGCUCU

001028.3_369-

387_A19U_s

387_U1A_as

AD-
GAGCUCAAGU
347
NM_
AUGUAAAUUA
757
NM_
370-388

960825
AAUUUACAU

001028.3_370-
CUUGAGCUC

001028.3_370-

388_C19U_s

388_G1A_as

AD-
AGCUCAAGUA
348
NM_
AGUGUAAAUU
758
NM_
371-389

960826
AUUUACACU

001028.3_371-
ACUUGAGCU

001028.3_371-

389_C19U_s

389_G1A_as

AD-
GCUCAAGUAA
349
NM_
AGGUGUAAAU
759
NM_
372-390

960827
UUUACACCU

001028.3_372-
UACUUGAGC

001028.3_372-

390_A19U_s

390_U1A_as

AD-
CUCAAGUAAU
350
NM_
AUGGUGUAAA
760
NM_
373-391

960828
UUACACCAU

001028.3_373-
UUACUUGAG

001028.3_373-

391_G19U_s

391_C1A_as

AD-
UCAAGUAAUU
351
NM_
ACUGGUGUAA
761
NM_
374-392

960829
UACACCAGU

001028.3_374-
AUUACUUGA

001028.3_374-

392_A19U_s

392_U1A_as

AD-
CAAGUAAUUU
352
NM_
AUCUGGUGUA
762
NM_
375-393

960830
ACACCAGAU

001028.3_375-
AAUUACUUG

001028.3_375-

393_A19U_s

393_U1A_as

AD-
AAGUAAUUUA
353
NM_
AUUCUGGUGU
763
NM_
376-394

960831
CACCAGAAU

001028.3_376-
AAAUUACUU

001028.3_376-

394_A19U_s

394_U1A_as

AD-
AGUAAUUUAC
354
NM_
AUUUCUGGUG
764
NM_
377-395

960832
ACCAGAAAU

001028.3_377-
UAAAUUACU

001028.3_377-

395_s

395_as

AD-
GUAAUUUACA
355
NM_
AAUUUCUGGU
765
NM_
378-396

960833
CCAGAAAUU

001028.3_378-
GUAAAUUAC

001028.3_378-

396_A19U_s

396_U1A_as

AD-
UAAUUUACAC
356
NM_
AUAUUUCUGG
766
NM_
379-397

960834
CAGAAAUAU

001028.3_379-
UGUAAAUUA

001028.3_379-

397_C19U_s

397_G1A_as

AD-
AAUUUACACC
357
NM_
AGUAUUUCUG
767
NM_
380-398

960835
AGAAAUACU

001028.3_380-
GUGUAAAUU

001028.3_380-

398_C19U_s

398_G1A_as

AD-
AUUUACACCA
358
NM_
AGGUAUUUCU
768
NM_
381-399

960836
GAAAUACCU

001028.3_381-
GGUGUAAAU

001028.3_381-

399_A19U_s

399_U1A_as

AD-
UUUACACCAG
359
NM_
AUGGUAUUUC
769
NM_
382-400

960837
AAAUACCAU

001028.3_382-
UGGUGUAAA

001028.3_382-

400_A19U_s

400_U1A_as

AD-
UUACACCAGA
360
NM_
AUUGGUAUUU
770
NM_
383-401

960838
AAUACCAAU

001028.3_383-
CUGGUGUAA

001028.3_383-

401_G19U_s

401_C1A_as

AD-
UACACCAGAA
361
NM_
ACUUGGUAUU
771
NM_
384-402

960839
AUACCAAGU

001028.3_384-
UCUGGUGUA

001028.3_384-

402_G19U_s

402_C1A_as

AD-
ACACCAGAAA
362
NM_
ACCUUGGUAU
772
NM_
385-403

960840
UACCAAGGU

001028.3_385-
UUCUGGUGU

001028.3_385-

403_G19U_s

403_C1A_as

AD-
CACCAGAAAU
363
NM_
ACCCUUGGUA
773
NM_
386-404

960841
ACCAAGGGU

001028.3_386-
UUUCUGGUG

001028.3_386-

404_s

404_as

AD-
ACCAGAAAUA
364
NM_
AACCCUUGGU
774
NM_
387-405

960842
CCAAGGGUU

001028.3_387-
AUUUCUGGU

001028.3_387-

405_G19U_s

405_C1A_as

AD-
CCAGAAAUAC
365
NM_
ACACCCUUGG
775
NM_
388-406

960843
CAAGGGUGU

001028.3_388-
UAUUUCUGG

001028.3_388-

406_G19U_s

406_C1A_as

AD-
CAGAAAUACC
366
NM_
ACCACCCUUG
776
NM_
389-407

960844
AAGGGUGGU

001028.3_389-
GUAUUUCUG

001028.3_389-

407_A19U_s

407_U1A_as

AD-
AGAAAUACCA
367
NM_
AUCCACCCUU
777
NM_
390-408

960845
AGGGUGGAU

001028.3_390-
GGUAUUUCU

001028.3_390-

408_G19U_s

408_C1A_as

AD-
GAAAUACCAA
368
NM_
ACUCCACCCU
778
NM_
391-409

960846
GGGUGGAGU

001028.3_391-
UGGUAUUUC

001028.3_391-

409_A19U_s

409_U1A_as

AD-
AAAUACCAAG
369
NM_
AUCUCCACCC
779
NM_
392-410

960847
GGUGGAGAU

001028.3_392-
UUGGUAUUU

001028.3_392-

410_s

410_as

AD-
AAUACCAAGG
370
NM_
AAUCUCCACC
780
NM_
393-411

960848
GUGGAGAUU

001028.3_393-
CUUGGUAUU

001028.3_393-

411_G19U_s

411_C1A_as

AD-
AUACCAAGGG
371
NM_
ACAUCUCCAC
781
NM_
394-412

960849
UGGAGAUGU

001028.3_394-
CCUUGGUAU

001028.3_394-

412_C19U_s

412_G1A_as

AD-
UACCAAGGGU
372
NM_
AGCAUCUCCA
782
NM_
395-413

960850
GGAGAUGCU

001028.3_395-
CCCUUGGUA

001028.3_395-

413_s

413_as

AD-
ACCAAGGGUG
373
NM_
AAGCAUCUCC
783
NM_
396-414

960851
GAGAUGCUU

001028.3_396-
ACCCUUGGU

001028.3_396-

414_C19U_s

414_G1A_as

AD-
CCAAGGGUGG
374
NM_
AGAGCAUCUC
784
NM_
397-415

960852
AGAUGCUCU

001028.3_397-
CACCCUUGG

001028.3_397-

415_C19U_s

415_G1A_as

AD-
CAAGGGUGGA
375
NM_
AGGAGCAUCU
785
NM_
398-416

960853
GAUGCUCCU

001028.3_398-
CCACCCUUG

001028.3_398-

416_A19U_s

416_U1A_as

AD-
AAGGGUGGAG
376
NM_
AUGGAGCAUC
786
NM_
399-417

960854
AUGCUCCAU

001028.3_399-
UCCACCCUU

001028.3_399-

417_G19U_s

417_C1A_as

AD-
AGGGUGGAGA
377
NM_
ACUGGAGCAU
787
NM_
400-418

960855
UGCUCCAGU

001028.3_400-
CUCCACCCU

001028.3_400-

418_C19U_s

418_G1A_as

AD-
GGGUGGAGAU
378
NM_
AGCUGGAGCA
788
NM_
401-419

960856
GCUCCAGCU

001028.3_401-
UCUCCACCC

001028.3_401-

419_s

419_as

AD-
GGUGGAGAUG
379
NM_
AAGCUGGAGC
789
NM_
402-420

960857
CUCCAGCUU

001028.3_402-
AUCUCCACC

001028.3_402-

420_G19U_s

420_C1A_as

AD-
GUGGAGAUGC
380
NM_
ACAGCUGGAG
790
NM_
403-421

960858
UCCAGCUGU

001028.3_403-
CAUCUCCAC

001028.3_403-

421_C19U_s

421_G1A_as

AD-
UGGAGAUGCU
381
NM_
AGCAGCUGGA
791
NM_
404-422

960859
CCAGCUGCU

001028.3_404-
GCAUCUCCA

001028.3_404-

422_s

422_as

AD-
GGAGAUGCUC
382
NM_
AAGCAGCUGG
792
NM_
405-423

960860
CAGCUGCUU

001028.3_405-
AGCAUCUCC

001028.3_405-

423_G19U_s

423_C1A_as

AD-
GAGAUGCUCC
383
NM_
ACAGCAGCUG
793
NM_
406-424

960861
AGCUGCUGU

001028.3_406-
GAGCAUCUC

001028.3_406-

424_G19U_s

424_C1A_as

AD-
AGAUGCUCCA
384
NM_
ACCAGCAGCU
794
NM_
407-425

960862
GCUGCUGGU

001028.3_407-
GGAGCAUCU

001028.3_407-

425_s

425_as

AD-
GAUGCUCCAG
385
NM_
AACCAGCAGC
795
NM_
408-426

960863
CUGCUGGUU

001028.3_408-
UGGAGCAUC

001028.3_408-

426_G19U_s

426_C1A_as

AD-
AUGCUCCAGC
386
NM_
ACACCAGCAG
796
NM_
409-427

960864
UGCUGGUGU

001028.3_409-
CUGGAGCAU

001028.3_409-

427_A19U_s

427_U1A_as

AD-
UGCUCCAGCU
387
NM_
AUCACCAGCA
797
NM_
410-428

960865
GCUGGUGAU

001028.3_410-
GCUGGAGCA

001028.3_410-

428_A19U_s

428_U1A_as

AD-
GCUCCAGCUG
388
NM_
AUUCACCAGC
798
NM_
411-429

960866
CUGGUGAAU

001028.3_411-
AGCUGGAGC

001028.3_411-

429_G19U_s

429_C1A_as

AD-
CUCCAGCUGC
389
NM_
ACUUCACCAG
799
NM_
412-430

960867
UGGUGAAGU

001028.3_412-
CAGCUGGAG

001028.3_412-

430_A19U_s

430_U1A_as

AD-
UCCAGCUGCU
390
NM_
AUCUUCACCA
800
NM_
413-431

960868
GGUGAAGAU

001028.3_413-
GCAGCUGGA

001028.3_413-

431_s

431_as

AD-
CCAGCUGCUG
391
NM_
AAUCUUCACC
801
NM_
414-432

960869
GUGAAGAUU

001028.3_414-
AGCAGCUGG

001028.3_414-

432_G19U_s

432_C1A_as

AD-
CAGCUGCUGG
392
NM_
ACAUCUUCAC
802
NM_
415-433

960870
UGAAGAUGU

001028.3_415-
CAGCAGCUG

001028.3_415-

433_C19U_s

433_G1A_as

AD-
AGCUGCUGGU
393
NM_
AGCAUCUUCA
803
NM_
416-434

960871
GAAGAUGCU

001028.3_416-
CCAGCAGCU

001028.3_416-

434_A19U_s

434_U1A_as

AD-
GCUGCUGGUG
394
NM_
AUGCAUCUUC
804
NM_
417-435

960872
AAGAUGCAU

001028.3_417-
ACCAGCAGC

001028.3_417-

435_s

435_as

AD-
CUGCUGGUGA
395
NM_
AAUGCAUCUU
805
NM_
418-436

960873
AGAUGCAUU

001028.3_418-
CACCAGCAG

001028.3_418-

436_G19U_s

436_C1A_as

AD-
UGCUGGUGAA
396
NM_
ACAUGCAUCU
806
NM_
419-437

960874
GAUGCAUGU

001028.3_419-
UCACCAGCA

001028.3_419-

437_A19U_s

437_U1A_as

AD-
GCUGGUGAAG
397
NM_
AUCAUGCAUC
807
NM_
420-438

960875
AUGCAUGAU

001028.3_420-
UUCACCAGC

001028.3_420-

438_A19U_s

438_U1A_as

AD-
CUGGUGAAGA
398
NM_
AUUCAUGCAU
808
NM_
421-439

960876
UGCAUGAAU

001028.3_421-
CUUCACCAG

001028.3_421-

439_s

439_as

AD-
UGGUGAAGAU
399
NM_
AAUUCAUGCA
809
NM_
422-440

960877
GCAUGAAUU

001028.3_422-
UCUUCACCA

001028.3_422-

440_A19U_s

440_U1A_as

AD-
GGUGAAGAUG
400
NM_
AUAUUCAUGC
810
NM_
423-441

960878
CAUGAAUAU

001028.3_423-
AUCUUCACC

001028.3_423-

441_G19U_s

441_C1A_as

AD-
GUGAAGAUGC
401
NM_
ACUAUUCAUG
811
NM_
424-442

960879
AUGAAUAGU

001028.3_424-
CAUCUUCAC

001028.3_424-

442_G19U_s

442_C1A_as

AD-
UGAAGAUGCA
402
NM_
ACCUAUUCAU
812
NM_
425-443

960880
UGAAUAGGU

001028.3_425-
GCAUCUUCA

001028.3_425-

443_s

443_as

AD-
GAAGAUGCAU
403
NM_
AACCUAUUCA
813
NM_
426-444

960881
GAAUAGGUU

001028.3_426-
UGCAUCUUC

001028.3_426-

444_C19U_s

444_G1A_as

AD-
AAGAUGCAUG
404
NM_
AGACCUAUUC
814
NM_
427-445

960882
AAUAGGUCU

001028.3_427-
AUGCAUCUU

001028.3_427-

445_C19U_s

445_G1A_as

AD-
AGAUGCAUGA
405
NM_
AGGACCUAUU
815
NM_
428-446

960883
AUAGGUCCU

001028.3_428-
CAUGCAUCU

001028.3_428-

446_A19U_s

446_U1A_as

AD-
GAUGCAUGAA
406
NM_
AUGGACCUAU
816
NM_
429-447

960884
UAGGUCCAU

001028.3_429-
UCAUGCAUC

001028.3_429-

447_A19U_s

447_U1A_as

AD-
AUGCAUGAAU
407
NM_
AUUGGACCUA
817
NM_
430-448

960885
AGGUCCAAU

001028.3_430-
UUCAUGCAU

001028.3_430-

448_C19U_s

448_G1A_as

AD-
UGCAUGAAUA
408
NM_
AGUUGGACCU
818
NM_
431-449

960886
GGUCCAACU

001028.3_431-
AUUCAUGCA

001028.3_431-

449_C19U_s

449_G1A_as

AD-
GCAUGAAUAG
409
NM_
AGGUUGGACC
819
NM_
432-450

960887
GUCCAACCU

001028.3_432-
UAUUCAUGC

001028.3_432-

450_A19U_s

450_U1A_as

AD-
CAUGAAUAGG
410
NM_
AUGGUUGGAC
820
NM_
433-451

960888
UCCAACCAU

001028.3_433-
CUAUUCAUG

001028.3_433-

451_G19U_s

451_C1A_as

AD-
AUGAAUAGGU
411
NM_
ACUGGUUGGA
821
NM_
434-452

960889
CCAACCAGU

001028.3_434-
CCUAUUCAU

001028.3_434-

452_C19U_s

452_G1A_as

AD-
UGAAUAGGUC
412
NM_
AGCUGGUUGG
822
NM_
435-453

960890
CAACCAGCU

001028.3_435-
ACCUAUUCA

001028.3_435-

453_s

453_as

AD-
GAAUAGGUCC
413
NM_
AAGCUGGUUG
823
NM_
436-454

960891
AACCAGCUU

001028.3_436-
GACCUAUUC

001028.3_436-

454_G19U_s

454_C1A_as

AD-
AAUAGGUCCA
414
NM_
ACAGCUGGUU
824
NM_
437-455

960892
ACCAGCUGU

001028.3_437-
GGACCUAUU

001028.3_437-

455_s

455_as

AD-
AUAGGUCCAA
415
NM_
AACAGCUGGU
825
NM_
438-456

960893
CCAGCUGUU

001028.3_438-
UGGACCUAU

001028.3_438-

456_A19U_s

456_U1A_as

AD-
UAGGUCCAAC
416
NM_
AUACAGCUGG
826
NM_
439-457

960894
CAGCUGUAU

001028.3_439-
UUGGACCUA

001028.3_439-

457_C19U_s

457_G1A_as

AD-
AGGUCCAACC
417
NM_
AGUACAGCUG
827
NM_
440-458

960895
AGCUGUACU

001028.3_440-
GUUGGACCU

001028.3_440-

458_A19U_s

458_U1A_as

AD-
GGUCCAACCA
418
NM_
AUGUACAGCU
828
NM_
441-459

960896
GCUGUACAU

001028.3_441-
GGUUGGACC

001028.3_441-

459_s

459_as

AD-
GUCCAACCAG
419
NM_
AAUGUACAGC
829
NM_
442-460

960897
CUGUACAUU

001028.3_442-
UGGUUGGAC

001028.3_442-

460_s

460_as

AD-
UCCAACCAGC
420
NM_
AAAUGUACAG
830
NM_
443-461

960898
UGUACAUUU

001028.3_443-
CUGGUUGGA

001028.3_443-

461_s

461_as

AD-
CCAACCAGCU
421
NM_
AAAAUGUACA
831
NM_
444-462

960899
GUACAUUUU

001028.3_444-
GCUGGUUGG

001028.3_444-

462_G19U_s

462_C1A_as

AD-
CAACCAGCUG
422
NM_
ACAAAUGUAC
832
NM_
445-463

960900
UACAUUUGU

001028.3_445-
AGCUGGUUG

001028.3_445-

463_G19U_s

463_C1A_as

AD-
AACCAGCUGU
423
NM_
ACCAAAUGUA
833
NM_
446-464

960901
ACAUUUGGU

001028.3_446-
CAGCUGGUU

001028.3_446-

464_A19U_s

464_U1A_as

AD-
ACCAGCUGUA
424
NM_
AUCCAAAUGU
834
NM_
447-465

960902
CAUUUGGAU

001028.3_447-
ACAGCUGGU

001028.3_447-

465_A19U_s

465_U1A_as

AD-
CCAGCUGUAC
425
NM_
AUUCCAAAUG
835
NM_
448-466

960903
AUUUGGAAU

001028.3_448-
UACAGCUGG

001028.3_448-

466_A19U_s

466_U1A_as

AD-
CAGCUGUACA
426
NM_
AUUUCCAAAU
836
NM_
449-467

960904
UUUGGAAAU

001028.3_449-
GUACAGCUG

001028.3_449-

467_A19U_s

467_U1A_as

AD-
AGCUGUACAU
427
NM_
AUUUUCCAAA
837
NM_
450-468

960905
UUGGAAAAU

001028.3_450-
UGUACAGCU

001028.3_450-

468_A19U_s

468_U1A_as

AD-
GCUGUACAUU
428
NM_
AUUUUUCCAA
838
NM_
451-469

960906
UGGAAAAAU

001028.3_451-
AUGUACAGC

001028.3_451-

469_s

469_as

AD-
CUGUACAUUU
429
NM_
AAUUUUUCCA
839
NM_
452-470

960907
GGAAAAAUU

001028.3_452-
AAUGUACAG

001028.3_452-

470_A19U_s

470_U1A_as

AD-
UGUACAUUUG
430
NM_
AUAUUUUUCC
840
NM_
453-471

960908
GAAAAAUAU

001028.3_453-
AAAUGUACA

001028.3_453-

471_A19U_s

471_U1A_as

AD-
GUACAUUUGG
431
NM_
AUUAUUUUUC
841
NM_
454-472

960909
AAAAAUAAU

001028.3_454-
CAAAUGUAC

001028.3_454-

472_A19U_s

472_U1A_as

AD-
CAUUUGGAAA
432
NM_
AGUUUUAUUU
842
NM_
457-475

960910
AAUAAAACU

001028.3_457-
UUCCAAAUG

001028.3_457-

475_s

475_as

TABLE 3

RPS25 Modified duplex Sequences

Sense Oligo
SEQ
Antisense
SEQ
NM_001028.3

Duplex
Sequence
ID
OligoSequence
ID
Target

ID
5′ to 3′
NO:
5′ to 3′
NO:
Site

AD-
CUUUUUGUCCGAC
843
AAAGAUGUCGGAC
1253
1-19

960501
AUCUUUdTdT

AAAAAGdTdT

AD-
UUUUUGUCCGACA
844
ACAAGAUGUCGGA
1254
2-20

960502
UCUUGUdTdT

CAAAAAdTdT

AD-
UUUUGUCCGACAU
845
AUCAAGAUGUCGG
1255
3-21

960503
CUUGAUdTdT

ACAAAAdTdT

AD-
UUUGUCCGACAUC
846
AGUCAAGAUGUCG
1256
4-22

960504
UUGACUdTdT

GACAAAdTdT

AD-
UUGUCCGACAUCU
847
ACGUCAAGAUGUC
1257
5-23

960505
UGACGUdTdT

GGACAAdTdT

AD-
UGUCCGACAUCUU
848
AUCGUCAAGAUGU
1258
6-24

960506
GACGAUdTdT

CGGACAdTdT

AD-
GUCCGACAUCUUG
849
ACUCGUCAAGAUG
1259
7-25

960507
ACGAGUdTdT

UCGGACdTdT

AD-
UCCGACAUCUUGA
850
ACCUCGUCAAGAU
1260
8-26

960508
CGAGGUdTdT

GUCGGAdTdT

AD-
CCGACAUCUUGAC
851
AGCCUCGUCAAGA
1261
9-27

960509
GAGGCUdTdT

UGUCGGdTdT

AD-
CGACAUCUUGACG
852
AAGCCUCGUCAAG
1262
10-28

960510
AGGCUUdTdT

AUGUCGdTdT

AD-
GACAUCUUGACGA
853
ACAGCCUCGUCAA
1263
11-29

960511
GGCUGUdTdT

GAUGUCdTdT

AD-
ACAUCUUGACGAG
854
AGCAGCCUCGUCA
1264
12-30

960512
GCUGCUdTdT

AGAUGUdTdT

AD-
CAUCUUGACGAGG
855
ACGCAGCCUCGUC
1265
13-31

960513
CUGCGUdTdT

AAGAUGdTdT

AD-
AUCUUGACGAGGC
856
ACCGCAGCCUCGU
1266
14-32

960514
UGCGGUdTdT

CAAGAUdTdT

AD-
UCUUGACGAGGCU
857
AACCGCAGCCUCG
1267
15-33

960515
GCGGUUdTdT

UCAAGAdTdT

AD-
CUUGACGAGGCUG
858
ACACCGCAGCCUC
1268
16-34

960516
CGGUGUdTdT

GUCAAGdTdT

AD-
UUGACGAGGCUGC
859
AACACCGCAGCCU
1269
17-35

960517
GGUGUUdTdT

CGUCAAdTdT

AD-
UGACGAGGCUGCG
860
AGACACCGCAGCC
1270
18-36

960518
GUGUCUdTdT

UCGUCAdTdT

AD-
GACGAGGCUGCGG
861
AAGACACCGCAGC
1271
19-37

960519
UGUCUUdTdT

CUCGUCdTdT

AD-
ACGAGGCUGCGGU
862
ACAGACACCGCAG
1272
20-38

960520
GUCUGUdTdT

CCUCGUdTdT

AD-
CGAGGCUGCGGUG
863
AGCAGACACCGCA
1273
21-39

960521
UCUGCUdTdT

GCCUCGdTdT

AD-
GAGGCUGCGGUGU
864
AAGCAGACACCGC
1274
22-40

960522
CUGCUUdTdT

AGCCUCdTdT

AD-
AGGCUGCGGUGUC
865
ACAGCAGACACCG
1275
23-41

960523
UGCUGUdTdT

CAGCCUdTdT

AD-
GGCUGCGGUGUCU
866
AGCAGCAGACACC
1276
24-42

960524
GCUGCUdTdT

GCAGCCdTdT

AD-
GCUGCGGUGUCUG
867
AAGCAGCAGACAC
1277
25-43

960525
CUGCUUdTdT

CGCAGCdTdT

AD-
CUGCGGUGUCUGC
868
AUAGCAGCAGACA
1278
26-44

960526
UGCUAUdTdT

CCGCAGdTdT

AD-
UGCGGUGUCUGCU
869
AAUAGCAGCAGAC
1279
27-45

960527
GCUAUUdTdT

ACCGCAdTdT

AD-
GCGGUGUCUGCUG
870
AAAUAGCAGCAGA
1280
28-46

960528
CUAUUUdTdT

CACCGCdTdT

AD-
CGGUGUCUGCUGC
871
AGAAUAGCAGCAG
1281
29-47

960529
UAUUCUdTdT

ACACCGdTdT

AD-
GGUGUCUGCUGCU
872
AAGAAUAGCAGCA
1282
30-48

960530
AUUCUUdTdT

GACACCdTdT

AD-
GUGUCUGCUGCUA
873
AGAGAAUAGCAGC
1283
31-49

960531
UUCUCUdTdT

AGACACdTdT

AD-
UGUCUGCUGCUAU
874
AGGAGAAUAGCAG
1284
32-50

960532
UCUCCUdTdT

CAGACAdTdT

AD-
GUCUGCUGCUAUU
875
ACGGAGAAUAGCA
1285
33-51

960533
CUCCGUdTdT

GCAGACdTdT

AD-
UCUGCUGCUAUUC
876
AUCGGAGAAUAGC
1286
34-52

960534
UCCGAUdTdT

AGCAGAdTdT

AD-
CUGCUGCUAUUCU
877
ACUCGGAGAAUAG
1287
35-53

960535
CCGAGUdTdT

CAGCAGdTdT

AD-
UGCUGCUAUUCUC
878
AGCUCGGAGAAUA
1288
36-54

960536
CGAGCUdTdT

GCAGCAdTdT

AD-
GCUGCUAUUCUCC
879
AAGCUCGGAGAAU
1289
37-55

960537
GAGCUUdTdT

AGCAGCdTdT

AD-
CUGCUAUUCUCCG
880
AAAGCUCGGAGAA
1290
38-56

960538
AGCUUUdTdT

UAGCAGdTdT

AD-
UGCUAUUCUCCGA
881
AGAAGCUCGGAGA
1291
39-57

960539
GCUUCUdTdT

AUAGCAdTdT

AD-
GCUAUUCUCCGAG
882
ACGAAGCUCGGAG
1292
40-58

960540
CUUCGUdTdT

AAUAGCdTdT

AD-
CUAUUCUCCGAGC
883
AGCGAAGCUCGGA
1293
41-59

960541
UUCGCUdTdT

GAAUAGdTdT

AD-
UAUUCUCCGAGCU
884
AUGCGAAGCUCGG
1294
42-60

960542
UCGCAUdTdT

AGAAUAdTdT

AD-
AUUCUCCGAGCUU
885
AUUGCGAAGCUCG
1295
43-61

960543
CGCAAUdTdT

GAGAAUdTdT

AD-
UUCUCCGAGCUUC
886
AAUUGCGAAGCUC
1296
44-62

960544
GCAAUUdTdT

GGAGAAdTdT

AD-
UCUCCGAGCUUCG
887
ACAUUGCGAAGCU
1297
45-63

960545
CAAUGUdTdT

CGGAGAdTdT

AD-
CUCCGAGCUUCGC
888
AGCAUUGCGAAGC
1298
46-64

960546
AAUGCUdTdT

UCGGAGdTdT

AD-
UCCGAGCUUCGCA
889
AGGCAUUGCGAAG
1299
47-65

960547
AUGCCUdTdT

CUCGGAdTdT

AD-
CCGAGCUUCGCAA
890
ACGGCAUUGCGAA
1300
48-66

960548
UGCCGUdTdT

GCUCGGdTdT

AD-
CGAGCUUCGCAAU
891
AGCGGCAUUGCGA
1301
49-67

960549
GCCGCUdTdT

AGCUCGdTdT

AD-
GAGCUUCGCAAUG
892
AGGCGGCAUUGCG
1302
50-68

960550
CCGCCUdTdT

AAGCUCdTdT

AD-
AGCUUCGCAAUGC
893
AAGGCGGCAUUGC
1303
51-69

960551
CGCCUUdTdT

GAAGCUdTdT

AD-
GCUUCGCAAUGCC
894
AUAGGCGGCAUUG
1304
52-70

960552
GCCUAUdTdT

CGAAGCdTdT

AD-
CUUCGCAAUGCCG
895
AUUAGGCGGCAUU
1305
53-71

960553
CCUAAUdTdT

GCGAAGdTdT

AD-
UUCGCAAUGCCGC
896
ACUUAGGCGGCAU
1306
54-72

960554
CUAAGUdTdT

UGCGAAdTdT

AD-
UCGCAAUGCCGCC
897
ACCUUAGGCGGCA
1307
55-73

960555
UAAGGUdTdT

UUGCGAdTdT

AD-
CGCAAUGCCGCCU
898
AUCCUUAGGCGGC
1308
56-74

960556
AAGGAUdTdT

AUUGCGdTdT

AD-
GCAAUGCCGCCUA
899
AGUCCUUAGGCGG
1309
57-75

960557
AGGACUdTdT

CAUUGCdTdT

AD-
CAAUGCCGCCUAA
900
ACGUCCUUAGGCG
1310
58-76

960558
GGACGUdTdT

GCAUUGdTdT

AD-
AAUGCCGCCUAAG
901
AUCGUCCUUAGGC
1311
59-77

960559
GACGAUdTdT

GGCAUUdTdT

AD-
AUGCCGCCUAAGG
902
AGUCGUCCUUAGG
1312
60-78

960560
ACGACUdTdT

CGGCAUdTdT

AD-
UGCCGCCUAAGGA
903
AUGUCGUCCUUAG
1313
61-79

960561
CGACAUdTdT

GCGGCAdTdT

AD-
GCCGCCUAAGGAC
904
AUUGUCGUCCUUA
1314
62-80

960562
GACAAUdTdT

GGCGGCdTdT

AD-
CCGCCUAAGGACG
905
ACUUGUCGUCCUU
1315
63-81

960563
ACAAGUdTdT

AGGCGGdTdT

AD-
CGCCUAAGGACGA
906
AUCUUGUCGUCCU
1316
64-82

960564
CAAGAUdTdT

UAGGCGdTdT

AD-
GCCUAAGGACGAC
907
AUUCUUGUCGUCC
1317
65-83

960565
AAGAAUdTdT

UUAGGCdTdT

AD-
CCUAAGGACGACA
908
ACUUCUUGUCGUC
1318
66-84

960566
AGAAGUdTdT

CUUAGGdTdT

AD-
CUAAGGACGACAA
909
AUCUUCUUGUCGU
1319
67-85

960567
GAAGAUdTdT

CCUUAGdTdT

AD-
UAAGGACGACAAG
910
AUUCUUCUUGUCG
1320
68-86

960568
AAGAAUdTdT

UCCUUAdTdT

AD-
AAGGACGACAAGA
911
ACUUCUUCUUGUC
1321
69-87

960569
AGAAGUdTdT

GUCCUUdTdT

AD-
AGGACGACAAGAA
912
AUCUUCUUCUUGU
1322
70-88

960570
GAAGAUdTdT

CGUCCUdTdT

AD-
GGACGACAAGAAG
913
AUUCUUCUUCUUG
1323
71-89

960571
AAGAAUdTdT

UCGUCCdTdT

AD-
GACGACAAGAAGA
914
ACUUCUUCUUCUU
1324
72-90

960572
AGAAGUdTdT

GUCGUCdTdT

AD-
ACGACAAGAAGAA
915
ACCUUCUUCUUCU
1325
73-91

960573
GAAGGUdTdT

UGUCGUdTdT

AD-
CGACAAGAAGAAG
916
AUCCUUCUUCUUC
1326
74-92

960574
AAGGAUdTdT

UUGUCGdTdT

AD-
GACAAGAAGAAGA
917
AGUCCUUCUUCUU
1327
75-93

960575
AGGACUdTdT

CUUGUCdTdT

AD-
ACAAGAAGAAGAA
918
ACGUCCUUCUUCU
1328
76-94

960576
GGACGUdTdT

UCUUGUdTdT

AD-
CAAGAAGAAGAAG
919
AGCGUCCUUCUUC
1329
77-95

960577
GACGCUdTdT

UUCUUGdTdT

AD-
AAGAAGAAGAAGG
920
AAGCGUCCUUCUU
1330
78-96

960578
ACGCUUdTdT

CUUCUUdTdT

AD-
AGAAGAAGAAGGA
921
ACAGCGUCCUUCU
1331
79-97

960579
CGCUGUdTdT

UCUUCUdTdT

AD-
GAAGAAGAAGGAC
922
ACCAGCGUCCUUC
1332
80-98

960580
GCUGGUdTdT

UUCUUCdTdT

AD-
AAGAAGAAGGACG
923
AUCCAGCGUCCUU
1333
81-99

960581
CUGGAUdTdT

CUUCUUdTdT

AD-
AGAAGAAGGACGC
924
AUUCCAGCGUCCU
1334
82-100

960582
UGGAAUdTdT

UCUUCUdTdT

AD-
GAAGAAGGACGCU
925
AUUUCCAGCGUCC
1335
83-101

960583
GGAAAUdTdT

UUCUUCdTdT

AD-
AAGAAGGACGCUG
926
ACUUUCCAGCGUC
1336
84-102

960584
GAAAGUdTdT

CUUCUUdTdT

AD-
AGAAGGACGCUGG
927
AACUUUCCAGCGU
1337
85-103

960585
AAAGUUdTdT

CCUUCUdTdT

AD-
GAAGGACGCUGGA
928
AGACUUUCCAGCG
1338
86-104

960586
AAGUCUdTdT

UCCUUCdTdT

AD-
AAGGACGCUGGAA
929
ACGACUUUCCAGC
1339
87-105

960587
AGUCGUdTdT

GUCCUUdTdT

AD-
AGGACGCUGGAAA
930
ACCGACUUUCCAG
1340
88-106

960588
GUCGGUdTdT

CGUCCUdTdT

AD-
GGACGCUGGAAAG
931
AGCCGACUUUCCA
1341
89-107

960589
UCGGCUdTdT

GCGUCCdTdT

AD-
GACGCUGGAAAGU
932
AGGCCGACUUUCC
1342
90-108

960590
CGGCCUdTdT

AGCGUCdTdT

AD-
ACGCUGGAAAGUC
933
AUGGCCGACUUUC
1343
91-109

960591
GGCCAUdTdT

CAGCGUdTdT

AD-
CGCUGGAAAGUCG
934
AUUGGCCGACUUU
1344
92-110

960592
GCCAAUdTdT

CCAGCGdTdT

AD-
GCUGGAAAGUCGG
935
ACUUGGCCGACUU
1345
93-111

960593
CCAAGUdTdT

UCCAGCdTdT

AD-
CUGGAAAGUCGGC
936
AUCUUGGCCGACU
1346
94-112

960594
CAAGAUdTdT

UUCCAGdTdT

AD-
UGGAAAGUCGGCC
937
AUUCUUGGCCGAC
1347
95-113

960595
AAGAAUdTdT

UUUCCAdTdT

AD-
GGAAAGUCGGCCA
938
AUUUCUUGGCCGA
1348
96-114

960596
AGAAAUdTdT

CUUUCCdTdT

AD-
GAAAGUCGGCCAA
939
ACUUUCUUGGCCG
1349
97-115

960597
GAAAGUdTdT

ACUUUCdTdT

AD-
AAAGUCGGCCAAG
940
AUCUUUCUUGGCC
1350
98-116

960598
AAAGAUdTdT

GACUUUdTdT

AD-
AAGUCGGCCAAGA
941
AGUCUUUCUUGGC
1351
99-117

960599
AAGACUdTdT

CGACUUdTdT

AD-
AGUCGGCCAAGAA
942
AUGUCUUUCUUGG
1352
100-118

960600
AGACAUdTdT

CCGACUdTdT

AD-
GUCGGCCAAGAAA
943
AUUGUCUUUCUUG
1353
101-119

960601
GACAAUdTdT

GCCGACdTdT

AD-
UCGGCCAAGAAAG
944
AUUUGUCUUUCUU
1354
102-120

960602
ACAAAUdTdT

GGCCGAdTdT

AD-
CGGCCAAGAAAGA
945
ACUUUGUCUUUCU
1355
103-121

960603
CAAAGUdTdT

UGGCCGdTdT

AD-
GGCCAAGAAAGAC
946
AUCUUUGUCUUUC
1356
104-122

960604
AAAGAUdTdT

UUGGCCdTdT

AD-
GCCAAGAAAGACA
947
AGUCUUUGUCUUU
1357
105-123

960605
AAGACUdTdT

CUUGGCdTdT

AD-
CCAAGAAAGACAA
948
AGGUCUUUGUCUU
1358
106-124

960606
AGACCUdTdT

UCUUGGdTdT

AD-
CAAGAAAGACAAA
949
AGGGUCUUUGUCU
1359
107-125

960607
GACCCUdTdT

UUCUUGdTdT

AD-
AGAAAGACAAAGA
950
ACUGGGUCUUUGU
1360
109-127

960608
CCCAGUdTdT

CUUUCUdTdT

AD-
GAAAGACAAAGAC
951
AACUGGGUCUUUG
1361
110-128

960609
CCAGUUdTdT

UCUUUCdTdT

AD-
AAAGACAAAGACC
952
ACACUGGGUCUUU
1362
111-129

960610
CAGUGUdTdT

GUCUUUdTdT

AD-
AAGACAAAGACCC
953
AUCACUGGGUCUU
1363
112-130

960611
AGUGAUdTdT

UGUCUUdTdT

AD-
AGACAAAGACCCA
954
AUUCACUGGGUCU
1364
113-131

960612
GUGAAUdTdT

UUGUCUdTdT

AD-
GACAAAGACCCAG
955
AGUUCACUGGGUC
1365
114-132

960613
UGAACUdTdT

UUUGUCdTdT

AD-
ACAAAGACCCAGU
956
AUGUUCACUGGGU
1366
115-133

960614
GAACAUdTdT

CUUUGUdTdT

AD-
CAAAGACCCAGUG
957
AUUGUUCACUGGG
1367
116-134

960615
AACAAUdTdT

UCUUUGdTdT

AD-
AAAGACCCAGUGA
958
AUUUGUUCACUGG
1368
117-135

960616
ACAAAUdTdT

GUCUUUdTdT

AD-
AAGACCCAGUGAA
959
AAUUUGUUCACUG
1369
118-136

960617
CAAAUUdTdT

GGUCUUdTdT

AD-
AGACCCAGUGAAC
960
AGAUUUGUUCACU
1370
119-137

960618
AAAUCUdTdT

GGGUCUdTdT

AD-
GACCCAGUGAACA
961
AGGAUUUGUUCAC
1371
120-138

960619
AAUCCUdTdT

UGGGUCdTdT

AD-
ACCCAGUGAACAA
962
ACGGAUUUGUUCA
1372
121-139

960620
AUCCGUdTdT

CUGGGUdTdT

AD-
CCCAGUGAACAAA
963
ACCGGAUUUGUUC
1373
122-140

960621
UCCGGUdTdT

ACUGGGdTdT

AD-
GGGCAAGGCCAAA
964
AUUCUUUUUGGCC
1374
140-158

960622
AAGAAUdTdT

UUGCCCdTdT

AD-
GGCAAGGCCAAAA
965
ACUUCUUUUUGGC
1375
141-159

960623
AGAAGUdTdT

CUUGCCdTdT

AD-
GCAAGGCCAAAAA
966
AUCUUCUUUUUGG
1376
142-160

960624
GAAGAUdTdT

CCUUGCdTdT

AD-
AGGCCAAAAAGAA
967
AACUUCUUCUUUU
1377
145-163

960625
GAAGUUdTdT

UGGCCUdTdT

AD-
GGCCAAAAAGAAG
968
ACACUUCUUCUUU
1378
146-164

960626
AAGUGUdTdT

UUGGCCdTdT

AD-
GCCAAAAAGAAGA
969
ACCACUUCUUCUU
1379
147-165

960627
AGUGGUdTdT

UUUGGCdTdT

AD-
CCAAAAAGAAGAA
970
AACCACUUCUUCU
1380
148-166

960628
GUGGUUdTdT

UUUUGGdTdT

AD-
CAAAAAGAAGAAG
971
AGACCACUUCUUC
1381
149-167

960629
UGGUCUdTdT

UUUUUGdTdT

AD-
AAAAAGAAGAAGU
972
AGGACCACUUCUU
1382
150-168

960630
GGUCCUdTdT

CUUUUUdTdT

AD-
AAAAGAAGAAGUG
973
AUGGACCACUUCU
1383
151-169

960631
GUCCAUdTdT

UCUUUUdTdT

AD-
AAAGAAGAAGUGG
974
AUUGGACCACUUC
1384
152-170

960632
UCCAAUdTdT

UUCUUUdTdT

AD-
AAGAAGAAGUGGU
975
AUUUGGACCACUU
1385
153-171

960633
CCAAAUdTdT

CUUCUUdTdT

AD-
AGAAGAAGUGGUC
976
ACUUUGGACCACU
1386
154-172

960634
CAAAGUdTdT

UCUUCUdTdT

AD-
GAAGAAGUGGUCC
977
ACCUUUGGACCAC
1387
155-173

960635
AAAGGUdTdT

UUCUUCdTdT

AD-
AAGAAGUGGUCCA
978
AGCCUUUGGACCA
1388
156-174

960636
AAGGCUdTdT

CUUCUUdTdT

AD-
AGAAGUGGUCCAA
979
AUGCCUUUGGACC
1389
157-175

960637
AGGCAUdTdT

ACUUCUdTdT

AD-
GAAGUGGUCCAAA
980
AUUGCCUUUGGAC
1390
158-176

960638
GGCAAUdTdT

CACUUCdTdT

AD-
AAGUGGUCCAAAG
981
AUUUGCCUUUGGA
1391
159-177

960639
GCAAAUdTdT

CCACUUdTdT

AD-
AGUGGUCCAAAGG
982
ACUUUGCCUUUGG
1392
160-178

960640
CAAAGUdTdT

ACCACUdTdT

AD-
GUGGUCCAAAGGC
983
AACUUUGCCUUUG
1393
161-179

960641
AAAGUUdTdT

GACCACdTdT

AD-
UGGUCCAAAGGCA
984
AAACUUUGCCUUU
1394
162-180

960642
AAGUUUdTdT

GGACCAdTdT

AD-
GGUCCAAAGGCAA
985
AGAACUUUGCCUU
1395
163-181

960643
AGUUCUdTdT

UGGACCdTdT

AD-
GUCCAAAGGCAAA
986
ACGAACUUUGCCU
1396
164-182

960644
GUUCGUdTdT

UUGGACdTdT

AD-
UCCAAAGGCAAAG
987
ACCGAACUUUGCC
1397
165-183

960645
UUCGGUdTdT

UUUGGAdTdT

AD-
CCAAAGGCAAAGU
988
ACCCGAACUUUGC
1398
166-184

960646
UCGGGUdTdT

CUUUGGdTdT

AD-
CAAAGGCAAAGUU
989
AUCCCGAACUUUG
1399
167-185

960647
CGGGAUdTdT

CCUUUGdTdT

AD-
AAAGGCAAAGUUC
990
AGUCCCGAACUUU
1400
168-186

960648
GGGACUdTdT

GCCUUUdTdT

AD-
AAGGCAAAGUUCG
991
AUGUCCCGAACUU
1401
169-187

960649
GGACAUdTdT

UGCCUUdTdT

AD-
AGGCAAAGUUCGG
992
AUUGUCCCGAACU
1402
170-188

960650
GACAAUdTdT

UUGCCUdTdT

AD-
GGCAAAGUUCGGG
993
ACUUGUCCCGAAC
1403
171-189

960651
ACAAGUdTdT

UUUGCCdTdT

AD-
GCAAAGUUCGGGA
994
AGCUUGUCCCGAA
1404
172-190

960652
CAAGCUdTdT

CUUUGCdTdT

AD-
CAAAGUUCGGGAC
995
AAGCUUGUCCCGA
1405
173-191

960653
AAGCUUdTdT

ACUUUGdTdT

AD-
AAAGUUCGGGACA
996
AGAGCUUGUCCCG
1406
174-192

960654
AGCUCUdTdT

AACUUUdTdT

AD-
AAGUUCGGGACAA
997
AUGAGCUUGUCCC
1407
175-193

960655
GCUCAUdTdT

GAACUUdTdT

AD-
AGUUCGGGACAAG
998
AUUGAGCUUGUCC
1408
176-194

960656
CUCAAUdTdT

CGAACUdTdT

AD-
GUUCGGGACAAGC
999
AAUUGAGCUUGUC
1409
177-195

960657
UCAAUUdTdT

CCGAACdTdT

AD-
UUCGGGACAAGCU
1000
AUAUUGAGCUUGU
1410
178-196

960658
CAAUAUdTdT

CCCGAAdTdT

AD-
UCGGGACAAGCUC
1001
AUUAUUGAGCUUG
1411
179-197

960659
AAUAAUdTdT

UCCCGAdTdT

AD-
CGGGACAAGCUCA
1002
AGUUAUUGAGCUU
1412
180-198

960660
AUAACUdTdT

GUCCCGdTdT

AD-
GGGACAAGCUCAA
1003
AAGUUAUUGAGCU
1413
181-199

960661
UAACUUdTdT

UGUCCCdTdT

AD-
GGACAAGCUCAAU
1004
AAAGUUAUUGAGC
1414
182-200

960662
AACUUUdTdT

UUGUCCdTdT

AD-
GACAAGCUCAAUA
1005
AUAAGUUAUUGAG
1415
183-201

960663
ACUUAUdTdT

CUUGUCdTdT

AD-
ACAAGCUCAAUAA
1006
ACUAAGUUAUUGA
1416
184-202

960664
CUUAGUdTdT

GCUUGUdTdT

AD-
CAAGCUCAAUAAC
1007
AACUAAGUUAUUG
1417
185-203

960665
UUAGUUdTdT

AGCUUGdTdT

AD-
AAGCUCAAUAACU
1008
AGACUAAGUUAUU
1418
186-204

960666
UAGUCUdTdT

GAGCUUdTdT

AD-
AGCUCAAUAACUU
1009
AAGACUAAGUUAU
1419
187-205

960667
AGUCUUdTdT

UGAGCUdTdT

AD-
GCUCAAUAACUUA
1010
AAAGACUAAGUUA
1420
188-206

960668
GUCUUUdTdT

UUGAGCdTdT

AD-
CUCAAUAACUUAG
1011
ACAAGACUAAGUU
1421
189-207

960669
UCUUGUdTdT

AUUGAGdTdT

AD-
UCAAUAACUUAGU
1012
AACAAGACUAAGU
1422
190-208

960670
CUUGUUdTdT

UAUUGAdTdT

AD-
CAAUAACUUAGUC
1013
AAACAAGACUAAG
1423
191-209

960671
UUGUUUdTdT

UUAUUGdTdT

AD-
AAUAACUUAGUCU
1014
AAAACAAGACUAA
1424
192-210

960672
UGUUUUdTdT

GUUAUUdTdT

AD-
AUAACUUAGUCUU
1015
ACAAACAAGACUA
1425
193-211

960673
GUUUGUdTdT

AGUUAUdTdT

AD-
UAACUUAGUCUUG
1016
AUCAAACAAGACU
1426
194-212

960674
UUUGAUdTdT

AAGUUAdTdT

AD-
AACUUAGUCUUGU
1017
AGUCAAACAAGAC
1427
195-213

960675
UUGACUdTdT

UAAGUUdTdT

AD-
ACUUAGUCUUGUU
1018
AUGUCAAACAAGA
1428
196-214

960676
UGACAUdTdT

CUAAGUdTdT

AD-
CUUAGUCUUGUUU
1019
AUUGUCAAACAAG
1429
197-215

960677
GACAAUdTdT

ACUAAGdTdT

AD-
UUAGUCUUGUUUG
1020
AUUUGUCAAACAA
1430
198-216

960678
ACAAAUdTdT

GACUAAdTdT

AD-
UAGUCUUGUUUGA
1021
ACUUUGUCAAACA
1431
199-217

960679
CAAAGUdTdT

AGACUAdTdT

AD-
AGUCUUGUUUGAC
1022
AGCUUUGUCAAAC
1432
200-218

960680
AAAGCUdTdT

AAGACUdTdT

AD-
GUCUUGUUUGACA
1023
AAGCUUUGUCAAA
1433
201-219

960681
AAGCUUdTdT

CAAGACdTdT

AD-
UCUUGUUUGACAA
1024
AUAGCUUUGUCAA
1434
202-220

960682
AGCUAUdTdT

ACAAGAdTdT

AD-
CUUGUUUGACAAA
1025
AGUAGCUUUGUCA
1435
203-221

960683
GCUACUdTdT

AACAAGdTdT

AD-
UUGUUUGACAAAG
1026
AGGUAGCUUUGUC
1436
204-222

960684
CUACCUdTdT

AAACAAdTdT

AD-
UGUUUGACAAAGC
1027
AAGGUAGCUUUGU
1437
205-223

960685
UACCUUdTdT

CAAACAdTdT

AD-
GUUUGACAAAGCU
1028
AUAGGUAGCUUUG
1438
206-224

960686
ACCUAUdTdT

UCAAACdTdT

AD-
UUUGACAAAGCUA
1029
AAUAGGUAGCUUU
1439
207-225

960687
CCUAUUdTdT

GUCAAAdTdT

AD-
UUGACAAAGCUAC
1030
ACAUAGGUAGCUU
1440
208-226

960688
CUAUGUdTdT

UGUCAAdTdT

AD-
UGACAAAGCUACC
1031
AUCAUAGGUAGCU
1441
209-227

960689
UAUGAUdTdT

UUGUCAdTdT

AD-
GACAAAGCUACCU
1032
AAUCAUAGGUAGC
1442
210-228

960690
AUGAUUdTdT

UUUGUCdTdT

AD-
ACAAAGCUACCUA
1033
AUAUCAUAGGUAG
1443
211-229

960691
UGAUAUdTdT

CUUUGUdTdT

AD-
CAAAGCUACCUAU
1034
AUUAUCAUAGGUA
1444
212-230

960692
GAUAAUdTdT

GCUUUGdTdT

AD-
AAAGCUACCUAUG
1035
AUUUAUCAUAGGU
1445
213-231

960693
AUAAAUdTdT

AGCUUUdTdT

AD-
AAGCUACCUAUGA
1036
AGUUUAUCAUAGG
1446
214-232

960694
UAAACUdTdT

UAGCUUdTdT

AD-
AGCUACCUAUGAU
1037
AAGUUUAUCAUAG
1447
215-233

960695
AAACUUdTdT

GUAGCUdTdT

AD-
GCUACCUAUGAUA
1038
AGAGUUUAUCAUA
1448
216-234

960696
AACUCUdTdT

GGUAGCdTdT

AD-
CUACCUAUGAUAA
1039
AAGAGUUUAUCAU
1449
217-235

960697
ACUCUUdTdT

AGGUAGdTdT

AD-
UACCUAUGAUAAA
1040
ACAGAGUUUAUCA
1450
218-236

960698
CUCUGUdTdT

UAGGUAdTdT

AD-
ACCUAUGAUAAAC
1041
AACAGAGUUUAUC
1451
219-237

960699
UCUGUUdTdT

AUAGGUdTdT

AD-
CCUAUGAUAAACU
1042
AUACAGAGUUUAU
1452
220-238

960700
CUGUAUdTdT

CAUAGGdTdT

AD-
CUAUGAUAAACUC
1043
AUUACAGAGUUUA
1453
221-239

960701
UGUAAUdTdT

UCAUAGdTdT

AD-
UAUGAUAAACUCU
1044
ACUUACAGAGUUU
1454
222-240

960702
GUAAGUdTdT

AUCAUAdTdT

AD-
AUGAUAAACUCUG
1045
ACCUUACAGAGUU
1455
223-241

960703
UAAGGUdTdT

UAUCAUdTdT

AD-
UGAUAAACUCUGU
1046
AUCCUUACAGAGU
1456
224-242

960704
AAGGAUdTdT

UUAUCAdTdT

AD-
GAUAAACUCUGUA
1047
AUUCCUUACAGAG
1457
225-243

960705
AGGAAUdTdT

UUUAUCdTdT

AD-
AUAAACUCUGUAA
1048
ACUUCCUUACAGA
1458
226-244

960706
GGAAGUdTdT

GUUUAUdTdT

AD-
UAAACUCUGUAAG
1049
AACUUCCUUACAG
1459
227-245

960707
GAAGUUdTdT

AGUUUAdTdT

AD-
AAACUCUGUAAGG
1050
AAACUUCCUUACA
1460
228-246

960708
AAGUUUdTdT

GAGUUUdTdT

AD-
AACUCUGUAAGGA
1051
AGAACUUCCUUAC
1461
229-247

960709
AGUUCUdTdT

AGAGUUdTdT

AD-
ACUCUGUAAGGAA
1052
AGGAACUUCCUUA
1462
230-248

960710
GUUCCUdTdT

CAGAGUdTdT

AD-
CUCUGUAAGGAAG
1053
AGGGAACUUCCUU
1463
231-249

960711
UUCCCUdTdT

ACAGAGdTdT

AD-
UCUGUAAGGAAGU
1054
AUGGGAACUUCCU
1464
232-250

960712
UCCCAUdTdT

UACAGAdTdT

AD-
CUGUAAGGAAGUU
1055
AUUGGGAACUUCC
1465
233-251

960713
CCCAAUdTdT

UUACAGdTdT

AD-
UGUAAGGAAGUUC
1056
AGUUGGGAACUUC
1466
234-252

960714
CCAACUdTdT

CUUACAdTdT

AD-
GUAAGGAAGUUCC
1057
AAGUUGGGAACUU
1467
235-253

960715
CAACUUdTdT

CCUUACdTdT

AD-
UAAGGAAGUUCCC
1058
AUAGUUGGGAACU
1468
236-254

960716
AACUAUdTdT

UCCUUAdTdT

AD-
AAGGAAGUUCCCA
1059
AAUAGUUGGGAAC
1469
237-255

960717
ACUAUUdTdT

UUCCUUdTdT

AD-
AGGAAGUUCCCAA
1060
AUAUAGUUGGGAA
1470
238-256

960718
CUAUAUdTdT

CUUCCUdTdT

AD-
GGAAGUUCCCAAC
1061
AUUAUAGUUGGGA
1471
239-257

960719
UAUAAUdTdT

ACUUCCdTdT

AD-
GAAGUUCCCAACU
1062
AUUUAUAGUUGGG
1472
240-258

960720
AUAAAUdTdT

AACUUCdTdT

AD-
AAGUUCCCAACUA
1063
AGUUUAUAGUUGG
1473
241-259

960721
UAAACUdTdT

GAACUUdTdT

AD-
AGUUCCCAACUAU
1064
AAGUUUAUAGUUG
1474
242-260

960722
AAACUUdTdT

GGAACUdTdT

AD-
GUUCCCAACUAUA
1065
AAAGUUUAUAGUU
1475
243-261

960723
AACUUUdTdT

GGGAACdTdT

AD-
UUCCCAACUAUAA
1066
AUAAGUUUAUAGU
1476
244-262

960724
ACUUAUdTdT

UGGGAAdTdT

AD-
UCCCAACUAUAAA
1067
AAUAAGUUUAUAG
1477
245-263

960725
CUUAUUdTdT

UUGGGAdTdT

AD-
CCCAACUAUAAAC
1068
AUAUAAGUUUAUA
1478
246-264

960726
UUAUAUdTdT

GUUGGGdTdT

AD-
CCAACUAUAAACU
1069
AUUAUAAGUUUAU
1479
247-265

960727
UAUAAUdTdT

AGUUGGdTdT

AD-
CAACUAUAAACUU
1070
AGUUAUAAGUUUA
1480
248-266

960728
AUAACUdTdT

UAGUUGdTdT

AD-
AACUAUAAACUUA
1071
AGGUUAUAAGUUU
1481
249-267

960729
UAACCUdTdT

AUAGUUdTdT

AD-
CCCAGCUGUGGUC
1072
AUCAGAGACCACA
1482
266-284

960730
UCUGAUdTdT

GCUGGGdTdT

AD-
CCAGCUGUGGUCU
1073
ACUCAGAGACCAC
1483
267-285

960731
CUGAGUdTdT

AGCUGGdTdT

AD-
CAGCUGUGGUCUC
1074
AUCUCAGAGACCA
1484
268-286

960732
UGAGAUdTdT

CAGCUGdTdT

AD-
AGCUGUGGUCUCU
1075
ACUCUCAGAGACC
1485
269-287

960733
GAGAGUdTdT

ACAGCUdTdT

AD-
GCUGUGGUCUCUG
1076
AUCUCUCAGAGAC
1486
270-288

960734
AGAGAUdTdT

CACAGCdTdT

AD-
CUGUGGUCUCUGA
1077
AGUCUCUCAGAGA
1487
271-289

960735
GAGACUdTdT

CCACAGdTdT

AD-
UGUGGUCUCUGAG
1078
AAGUCUCUCAGAG
1488
272-290

960736
AGACUUdTdT

ACCACAdTdT

AD-
GUGGUCUCUGAGA
1079
ACAGUCUCUCAGA
1489
273-291

960737
GACUGUdTdT

GACCACdTdT

AD-
UGGUCUCUGAGAG
1080
AUCAGUCUCUCAG
1490
274-292

960738
ACUGAUdTdT

AGACCAdTdT

AD-
GGUCUCUGAGAGA
1081
AUUCAGUCUCUCA
1491
275-293

960739
CUGAAUdTdT

GAGACCdTdT

AD-
GUCUCUGAGAGAC
1082
ACUUCAGUCUCUC
1492
276-294

960740
UGAAGUdTdT

AGAGACdTdT

AD-
UCUCUGAGAGACU
1083
AUCUUCAGUCUCU
1493
277-295

960741
GAAGAUdTdT

CAGAGAdTdT

AD-
CUCUGAGAGACUG
1084
AAUCUUCAGUCUC
1494
278-296

960742
AAGAUUdTdT

UCAGAGdTdT

AD-
UCUGAGAGACUGA
1085
AAAUCUUCAGUCU
1495
279-297

960743
AGAUUUdTdT

CUCAGAdTdT

AD-
CUGAGAGACUGAA
1086
AGAAUCUUCAGUC
1496
280-298

960744
GAUUCUdTdT

UCUCAGdTdT

AD-
UGAGAGACUGAAG
1087
ACGAAUCUUCAGU
1497
281-299

960745
AUUCGUdTdT

CUCUCAdTdT

AD-
GAGAGACUGAAGA
1088
AUCGAAUCUUCAG
1498
282-300

960746
UUCGAUdTdT

UCUCUCdTdT

AD-
AGAGACUGAAGAU
1089
ACUCGAAUCUUCA
1499
283-301

960747
UCGAGUdTdT

GUCUCUdTdT

AD-
GAGACUGAAGAUU
1090
ACCUCGAAUCUUC
1500
284-302

960748
CGAGGUdTdT

AGUCUCdTdT

AD-
AGACUGAAGAUUC
1091
AGCCUCGAAUCUU
1501
285-303

960749
GAGGCUdTdT

CAGUCUdTdT

AD-
GACUGAAGAUUCG
1092
AAGCCUCGAAUCU
1502
286-304

960750
AGGCUUdTdT

UCAGUCdTdT

AD-
ACUGAAGAUUCGA
1093
AGAGCCUCGAAUC
1503
287-305

960751
GGCUCUdTdT

UUCAGUdTdT

AD-
CUGAAGAUUCGAG
1094
AGGAGCCUCGAAU
1504
288-306

960752
GCUCCUdTdT

CUUCAGdTdT

AD-
UGAAGAUUCGAGG
1095
AGGGAGCCUCGAA
1505
289-307

960753
CUCCCUdTdT

UCUUCAdTdT

AD-
GAAGAUUCGAGGC
1096
AAGGGAGCCUCGA
1506
290-308

960754
UCCCUUdTdT

AUCUUCdTdT

AD-
AAGAUUCGAGGCU
1097
ACAGGGAGCCUCG
1507
291-309

960755
CCCUGUdTdT

AAUCUUdTdT

AD-
AGAUUCGAGGCUC
1098
ACCAGGGAGCCUC
1508
292-310

960756
CCUGGUdTdT

GAAUCUdTdT

AD-
GAUUCGAGGCUCC
1099
AGCCAGGGAGCCU
1509
293-311

960757
CUGGCUdTdT

CGAAUCdTdT

AD-
AUUCGAGGCUCCC
1100
AGGCCAGGGAGCC
1510
294-312

960758
UGGCCUdTdT

UCGAAUdTdT

AD-
UUCGAGGCUCCCU
1101
AUGGCCAGGGAGC
1511
295-313

960759
GGCCAUdTdT

CUCGAAdTdT

AD-
UCGAGGCUCCCUG
1102
ACUGGCCAGGGAG
1512
296-314

960760
GCCAGUdTdT

CCUCGAdTdT

AD-
CUGGCCAGGGCAG
1103
AAAGGGCUGCCCU
1513
306-324

960761
CCCUUUdTdT

GGCCAGdTdT

AD-
UGGCCAGGGCAGC
1104
AGAAGGGCUGCCC
1514
307-325

960762
CCUUCUdTdT

UGGCCAdTdT

AD-
GGCCAGGGCAGCC
1105
AUGAAGGGCUGCC
1515
308-326

960763
CUUCAUdTdT

CUGGCCdTdT

AD-
GCCAGGGCAGCCC
1106
ACUGAAGGGCUGC
1516
309-327

960764
UUCAGUdTdT

CCUGGCdTdT

AD-
CCAGGGCAGCCCU
1107
ACCUGAAGGGCUG
1517
310-328

960765
UCAGGUdTdT

CCCUGGdTdT

AD-
CAGGGCAGCCCUU
1108
AUCCUGAAGGGCU
1518
311-329

960766
CAGGAUdTdT

GCCCUGdTdT

AD-
AGGGCAGCCCUUC
1109
ACUCCUGAAGGGC
1519
312-330

960767
AGGAGUdTdT

UGCCCUdTdT

AD-
GGGCAGCCCUUCA
1110
AGCUCCUGAAGGG
1520
313-331

960768
GGAGCUdTdT

CUGCCCdTdT

AD-
GGCAGCCCUUCAG
1111
AAGCUCCUGAAGG
1521
314-332

960769
GAGCUUdTdT

GCUGCCdTdT

AD-
GCAGCCCUUCAGG
1112
AGAGCUCCUGAAG
1522
315-333

960770
AGCUCUdTdT

GGCUGCdTdT

AD-
CAGCCCUUCAGGA
1113
AGGAGCUCCUGAA
1523
316-334

960771
GCUCCUdTdT

GGGCUGdTdT

AD-
AGCCCUUCAGGAG
1114
AAGGAGCUCCUGA
1524
317-335

960772
CUCCUUdTdT

AGGGCUdTdT

AD-
GCCCUUCAGGAGC
1115
AAAGGAGCUCCUG
1525
318-336

960773
UCCUUUdTdT

AAGGGCdTdT

AD-
CCCUUCAGGAGCU
1116
AUAAGGAGCUCCU
1526
319-337

960774
CCUUAUdTdT

GAAGGGdTdT

AD-
CCUUCAGGAGCUC
1117
ACUAAGGAGCUCC
1527
320-338

960775
CUUAGUdTdT

UGAAGGdTdT

AD-
CUUCAGGAGCUCC
1118
AACUAAGGAGCUC
1528
321-339

960776
UUAGUUdTdT

CUGAAGdTdT

AD-
UUCAGGAGCUCCU
1119
AUACUAAGGAGCU
1529
322-340

960777
UAGUAUdTdT

CCUGAAdTdT

AD-
UCAGGAGCUCCUU
1120
AUUACUAAGGAGC
1530
323-341

960778
AGUAAUdTdT

UCCUGAdTdT

AD-
CAGGAGCUCCUUA
1121
AUUUACUAAGGAG
1531
324-342

960779
GUAAAUdTdT

CUCCUGdTdT

AD-
AGGAGCUCCUUAG
1122
ACUUUACUAAGGA
1532
325-343

960780
UAAAGUdTdT

GCUCCUdTdT

AD-
GGAGCUCCUUAGU
1123
ACCUUUACUAAGG
1533
326-344

960781
AAAGGUdTdT

AGCUCCdTdT

AD-
GAGCUCCUUAGUA
1124
AUCCUUUACUAAG
1534
327-345

960782
AAGGAUdTdT

GAGCUCdTdT

AD-
AGCUCCUUAGUAA
1125
AGUCCUUUACUAA
1535
328-346

960783
AGGACUdTdT

GGAGCUdTdT

AD-
GCUCCUUAGUAAA
1126
AAGUCCUUUACUA
1536
329-347

960784
GGACUUdTdT

AGGAGCdTdT

AD-
CUCCUUAGUAAAG
1127
AAAGUCCUUUACU
1537
330-348

960785
GACUUUdTdT

AAGGAGdTdT

AD-
UCCUUAGUAAAGG
1128
AUAAGUCCUUUAC
1538
331-349

960786
ACUUAUdTdT

UAAGGAdTdT

AD-
CCUUAGUAAAGGA
1129
AAUAAGUCCUUUA
1539
332-350

960787
CUUAUUdTdT

CUAAGGdTdT

AD-
CUUAGUAAAGGAC
1130
AGAUAAGUCCUUU
1540
333-351

960788
UUAUCUdTdT

ACUAAGdTdT

AD-
UUAGUAAAGGACU
1131
AUGAUAAGUCCUU
1541
334-352

960789
UAUCAUdTdT

UACUAAdTdT

AD-
UAGUAAAGGACUU
1132
AUUGAUAAGUCCU
1542
335-353

960790
AUCAAUdTdT

UUACUAdTdT

AD-
AGUAAAGGACUUA
1133
AUUUGAUAAGUCC
1543
336-354

960791
UCAAAUdTdT

UUUACUdTdT

AD-
GUAAAGGACUUAU
1134
AGUUUGAUAAGUC
1544
337-355

960792
CAAACUdTdT

CUUUACdTdT

AD-
UAAAGGACUUAUC
1135
AAGUUUGAUAAGU
1545
338-356

960793
AAACUUdTdT

CCUUUAdTdT

AD-
AAAGGACUUAUCA
1136
ACAGUUUGAUAAG
1546
339-357

960794
AACUGUdTdT

UCCUUUdTdT

AD-
AAGGACUUAUCAA
1137
ACCAGUUUGAUAA
1547
340-358

960795
ACUGGUdTdT

GUCCUUdTdT

AD-
AGGACUUAUCAAA
1138
AACCAGUUUGAUA
1548
341-359

960796
CUGGUUdTdT

AGUCCUdTdT

AD-
GGACUUAUCAAAC
1139
AAACCAGUUUGAU
1549
342-360

960797
UGGUUUdTdT

AAGUCCdTdT

AD-
GACUUAUCAAACU
1140
AAAACCAGUUUGA
1550
343-361

960798
GGUUUUdTdT

UAAGUCdTdT

AD-
ACUUAUCAAACUG
1141
AGAAACCAGUUUG
1551
344-362

960799
GUUUCUdTdT

AUAAGUdTdT

AD-
CUUAUCAAACUGG
1142
AUGAAACCAGUUU
1552
345-363

960800
UUUCAUdTdT

GAUAAGdTdT

AD-
UUAUCAAACUGGU
1143
AUUGAAACCAGUU
1553
346-364

960801
UUCAAUdTdT

UGAUAAdTdT

AD-
UAUCAAACUGGUU
1144
AUUUGAAACCAGU
1554
347-365

960802
UCAAAUdTdT

UUGAUAdTdT

AD-
AUCAAACUGGUUU
1145
ACUUUGAAACCAG
1555
348-366

960803
CAAAGUdTdT

UUUGAUdTdT

AD-
UCAAACUGGUUUC
1146
AGCUUUGAAACCA
1556
349-367

960804
AAAGCUdTdT

GUUUGAdTdT

AD-
CAAACUGGUUUCA
1147
AUGCUUUGAAACC
1557
350-368

960805
AAGCAUdTdT

AGUUUGdTdT

AD-
AAACUGGUUUCAA
1148
AGUGCUUUGAAAC
1558
351-369

960806
AGCACUdTdT

CAGUUUdTdT

AD-
AACUGGUUUCAAA
1149
AUGUGCUUUGAAA
1559
352-370

960807
GCACAUdTdT

CCAGUUdTdT

AD-
ACUGGUUUCAAAG
1150
ACUGUGCUUUGAA
1560
353-371

960808
CACAGUdTdT

ACCAGUdTdT

AD-
CUGGUUUCAAAGC
1151
AUCUGUGCUUUGA
1561
354-372

960809
ACAGAUdTdT

AACCAGdTdT

AD-
UGGUUUCAAAGCA
1152
ACUCUGUGCUUUG
1562
355-373

960810
CAGAGUdTdT

AAACCAdTdT

AD-
GGUUUCAAAGCAC
1153
AGCUCUGUGCUUU
1563
356-374

960811
AGAGCUdTdT

GAAACCdTdT

AD-
GUUUCAAAGCACA
1154
AAGCUCUGUGCUU
1564
357-375

960812
GAGCUUdTdT

UGAAACdTdT

AD-
UUUCAAAGCACAG
1155
AGAGCUCUGUGCU
1565
358-376

960813
AGCUCUdTdT

UUGAAAdTdT

AD-
UUCAAAGCACAGA
1156
AUGAGCUCUGUGC
1566
359-377

960814
GCUCAUdTdT

UUUGAAdTdT

AD-
UCAAAGCACAGAG
1157
AUUGAGCUCUGUG
1567
360-378

960815
CUCAAUdTdT

CUUUGAdTdT

AD-
CAAAGCACAGAGC
1158
ACUUGAGCUCUGU
1568
361-379

960816
UCAAGUdTdT

GCUUUGdTdT

AD-
AAAGCACAGAGCU
1159
AACUUGAGCUCUG
1569
362-380

960817
CAAGUUdTdT

UGCUUUdTdT

AD-
AAGCACAGAGCUC
1160
AUACUUGAGCUCU
1570
363-381

960818
AAGUAUdTdT

GUGCUUdTdT

AD-
AGCACAGAGCUCA
1161
AUUACUUGAGCUC
1571
364-382

960819
AGUAAUdTdT

UGUGCUdTdT

AD-
GCACAGAGCUCAA
1162
AAUUACUUGAGCU
1572
365-383

960820
GUAAUUdTdT

CUGUGCdTdT

AD-
CACAGAGCUCAAG
1163
AAAUUACUUGAGC
1573
366-384

960821
UAAUUUdTdT

UCUGUGdTdT

AD-
ACAGAGCUCAAGU
1164
AAAAUUACUUGAG
1574
367-385

960822
AAUUUUdTdT

CUCUGUdTdT

AD-
CAGAGCUCAAGUA
1165
AUAAAUUACUUGA
1575
368-386

960823
AUUUAUdTdT

GCUCUGdTdT

AD-
AGAGCUCAAGUAA
1166
AGUAAAUUACUUG
1576
369-387

960824
UUUACUdTdT

AGCUCUdTdT

AD-
GAGCUCAAGUAAU
1167
AUGUAAAUUACUU
1577
370-388

960825
UUACAUdTdT

GAGCUCdTdT

AD-
AGCUCAAGUAAUU
1168
AGUGUAAAUUACU
1578
371-389

960826
UACACUdTdT

UGAGCUdTdT

AD-
GCUCAAGUAAUUU
1169
AGGUGUAAAUUAC
1579
372-390

960827
ACACCUdTdT

UUGAGCdTdT

AD-
CUCAAGUAAUUUA
1170
AUGGUGUAAAUUA
1580
373-391

960828
CACCAUdTdT

CUUGAGdTdT

AD-
UCAAGUAAUUUAC
1171
ACUGGUGUAAAUU
1581
374-392

960829
ACCAGUdTdT

ACUUGAdTdT

AD-
CAAGUAAUUUACA
1172
AUCUGGUGUAAAU
1582
375-393

960830
CCAGAUdTdT

UACUUGdTdT

AD-
AAGUAAUUUACAC
1173
AUUCUGGUGUAAA
1583
376-394

960831
CAGAAUdTdT

UUACUUdTdT

AD-
AGUAAUUUACACC
1174
AUUUCUGGUGUAA
1584
377-395

960832
AGAAAUdTdT

AUUACUdTdT

AD-
GUAAUUUACACCA
1175
AAUUUCUGGUGUA
1585
378-396

960833
GAAAUUdTdT

AAUUACdTdT

AD-
UAAUUUACACCAG
1176
AUAUUUCUGGUGU
1586
379-397

960834
AAAUAUdTdT

AAAUUAdTdT

AD-
AAUUUACACCAGA
1177
AGUAUUUCUGGUG
1587
380-398

960835
AAUACUdTdT

UAAAUUdTdT

AD-
AUUUACACCAGAA
1178
AGGUAUUUCUGGU
1588
381-399

960836
AUACCUdTdT

GUAAAUdTdT

AD-
UUUACACCAGAAA
1179
AUGGUAUUUCUGG
1589
382-400

960837
UACCAUdTdT

UGUAAAdTdT

AD-
UUACACCAGAAAU
1180
AUUGGUAUUUCUG
1590
383-401

960838
ACCAAUdTdT

GUGUAAdTdT

AD-
UACACCAGAAAUA
1181
ACUUGGUAUUUCU
1591
384-402

960839
CCAAGUdTdT

GGUGUAdTdT

AD-
ACACCAGAAAUAC
1182
ACCUUGGUAUUUC
1592
385-403

960840
CAAGGUdTdT

UGGUGUdTdT

AD-
CACCAGAAAUACC
1183
ACCCUUGGUAUUU
1593
386-404

960841
AAGGGUdTdT

CUGGUGdTdT

AD-
ACCAGAAAUACCA
1184
AACCCUUGGUAUU
1594
387-405

960842
AGGGUUdTdT

UCUGGUdTdT

AD-
CCAGAAAUACCAA
1185
ACACCCUUGGUAU
1595
388-406

960843
GGGUGUdTdT

UUCUGGdTdT

AD-
CAGAAAUACCAAG
1186
ACCACCCUUGGUA
1596
389-407

960844
GGUGGUdTdT

UUUCUGdTdT

AD-
AGAAAUACCAAGG
1187
AUCCACCCUUGGU
1597
390-408

960845
GUGGAUdTdT

AUUUCUdTdT

AD-
GAAAUACCAAGGG
1188
ACUCCACCCUUGG
1598
391-409

960846
UGGAGUdTdT

UAUUUCdTdT

AD-
AAAUACCAAGGGU
1189
AUCUCCACCCUUG
1599
392-410

960847
GGAGAUdTdT

GUAUUUdTdT

AD-
AAUACCAAGGGUG
1190
AAUCUCCACCCUU
1600
393-411

960848
GAGAUUdTdT

GGUAUUdTdT

AD-
AUACCAAGGGUGG
1191
ACAUCUCCACCCU
1601
394-412

960849
AGAUGUdTdT

UGGUAUdTdT

AD-
UACCAAGGGUGGA
1192
AGCAUCUCCACCC
1602
395-413

960850
GAUGCUdTdT

UUGGUAdTdT

AD-
ACCAAGGGUGGAG
1193
AAGCAUCUCCACC
1603
396-414

960851
AUGCUUdTdT

CUUGGUdTdT

AD-
CCAAGGGUGGAGA
1194
AGAGCAUCUCCAC
1604
397-415

960852
UGCUCUdTdT

CCUUGGdTdT

AD-
CAAGGGUGGAGAU
1195
AGGAGCAUCUCCA
1605
398-416

960853
GCUCCUdTdT

CCCUUGdTdT

AD-
AAGGGUGGAGAUG
1196
AUGGAGCAUCUCC
1606
399-417

960854
CUCCAUdTdT

ACCCUUdTdT

AD-
AGGGUGGAGAUGC
1197
ACUGGAGCAUCUC
1607
400-418

960855
UCCAGUdTdT

CACCCUdTdT

AD-
GGGUGGAGAUGCU
1198
AGCUGGAGCAUCU
1608
401-419

960856
CCAGCUdTdT

CCACCCdTdT

AD-
GGUGGAGAUGCUC
1199
AAGCUGGAGCAUC
1609
402-420

960857
CAGCUUdTdT

UCCACCdTdT

AD-
GUGGAGAUGCUCC
1200
ACAGCUGGAGCAU
1610
403-421

960858
AGCUGUdTdT

CUCCACdTdT

AD-
UGGAGAUGCUCCA
1201
AGCAGCUGGAGCA
1611
404-422

960859
GCUGCUdTdT

UCUCCAdTdT

AD-
GGAGAUGCUCCAG
1202
AAGCAGCUGGAGC
1612
405-423

960860
CUGCUUdTdT

AUCUCCdTdT

AD-
GAGAUGCUCCAGC
1203
ACAGCAGCUGGAG
1613
406-424

960861
UGCUGUdTdT

CAUCUCdTdT

AD-
AGAUGCUCCAGCU
1204
ACCAGCAGCUGGA
1614
407-425

960862
GCUGGUdTdT

GCAUCUdTdT

AD-
GAUGCUCCAGCUG
1205
AACCAGCAGCUGG
1615
408-426

960863
CUGGUUdTdT

AGCAUCdTdT

AD-
AUGCUCCAGCUGC
1206
ACACCAGCAGCUG
1616
409-427

960864
UGGUGUdTdT

GAGCAUdTdT

AD-
UGCUCCAGCUGCU
1207
AUCACCAGCAGCU
1617
410-428

960865
GGUGAUdTdT

GGAGCAdTdT

AD-
GCUCCAGCUGCUG
1208
AUUCACCAGCAGC
1618
411-429

960866
GUGAAUdTdT

UGGAGCdTdT

AD-
CUCCAGCUGCUGG
1209
ACUUCACCAGCAG
1619
412-430

960867
UGAAGUdTdT

CUGGAGdTdT

AD-
UCCAGCUGCUGGU
1210
AUCUUCACCAGCA
1620
413-431

960868
GAAGAUdTdT

GCUGGAdTdT

AD-
CCAGCUGCUGGUG
1211
AAUCUUCACCAGC
1621
414-432

960869
AAGAUUdTdT

AGCUGGdTdT

AD-
CAGCUGCUGGUGA
1212
ACAUCUUCACCAG
1622
415-433

960870
AGAUGUdTdT

CAGCUGdTdT

AD-
AGCUGCUGGUGAA
1213
AGCAUCUUCACCA
1623
416-434

960871
GAUGCUdTdT

GCAGCUdTdT

AD-
GCUGCUGGUGAAG
1214
AUGCAUCUUCACC
1624
417-435

960872
AUGCAUdTdT

AGCAGCdTdT

AD-
CUGCUGGUGAAGA
1215
AAUGCAUCUUCAC
1625
418-436

960873
UGCAUUdTdT

CAGCAGdTdT

AD-
UGCUGGUGAAGAU
1216
ACAUGCAUCUUCA
1626
419-437

960874
GCAUGUdTdT

CCAGCAdTdT

AD-
GCUGGUGAAGAUG
1217
AUCAUGCAUCUUC
1627
420-438

960875
CAUGAUdTdT

ACCAGCdTdT

AD-
CUGGUGAAGAUGC
1218
AUUCAUGCAUCUU
1628
421-439

960876
AUGAAUdTdT

CACCAGdTdT

AD-
UGGUGAAGAUGCA
1219
AAUUCAUGCAUCU
1629
422-440

960877
UGAAUUdTdT

UCACCAdTdT

AD-
GGUGAAGAUGCAU
1220
AUAUUCAUGCAUC
1630
423-441

960878
GAAUAUdTdT

UUCACCdTdT

AD-
GUGAAGAUGCAUG
1221
ACUAUUCAUGCAU
1631
424-442

960879
AAUAGUdTdT

CUUCACdTdT

AD-
UGAAGAUGCAUGA
1222
ACCUAUUCAUGCA
1632
425-443

960880
AUAGGUdTdT

UCUUCAdTdT

AD-
GAAGAUGCAUGAA
1223
AACCUAUUCAUGC
1633
426-444

960881
UAGGUUdTdT

AUCUUCdTdT

AD-
AAGAUGCAUGAAU
1224
AGACCUAUUCAUG
1634
427-445

960882
AGGUCUdTdT

CAUCUUdTdT

AD-
AGAUGCAUGAAUA
1225
AGGACCUAUUCAU
1635
428-446

960883
GGUCCUdTdT

GCAUCUdTdT

AD-
GAUGCAUGAAUAG
1226
AUGGACCUAUUCA
1636
429-447

960884
GUCCAUdTdT

UGCAUCdTdT

AD-
AUGCAUGAAUAGG
1227
AUUGGACCUAUUC
1637
430-448

960885
UCCAAUdTdT

AUGCAUdTdT

AD-
UGCAUGAAUAGGU
1228
AGUUGGACCUAUU
1638
431-449

960886
CCAACUdTdT

CAUGCAdTdT

AD-
GCAUGAAUAGGUC
1229
AGGUUGGACCUAU
1639
432-450

960887
CAACCUdTdT

UCAUGCdTdT

AD-
CAUGAAUAGGUCC
1230
AUGGUUGGACCUA
1640
433-451

960888
AACCAUdTdT

UUCAUGdTdT

AD-
AUGAAUAGGUCCA
1231
ACUGGUUGGACCU
1641
434-452

960889
ACCAGUdTdT

AUUCAUdTdT

AD-
UGAAUAGGUCCAA
1232
AGCUGGUUGGACC
1642
435-453

960890
CCAGCUdTdT

UAUUCAdTdT

AD-
GAAUAGGUCCAAC
1233
AAGCUGGUUGGAC
1643
436-454

960891
CAGCUUdTdT

CUAUUCdTdT

AD-
AAUAGGUCCAACC
1234
ACAGCUGGUUGGA
1644
437-455

960892
AGCUGUdTdT

CCUAUUdTdT

AD-
AUAGGUCCAACCA
1235
AACAGCUGGUUGG
1645
438-456

960893
GCUGUUdTdT

ACCUAUdTdT

AD-
UAGGUCCAACCAG
1236
AUACAGCUGGUUG
1646
439-457

960894
CUGUAUdTdT

GACCUAdTdT

AD-
AGGUCCAACCAGC
1237
AGUACAGCUGGUU
1647
440-458

960895
UGUACUdTdT

GGACCUdTdT

AD-
GGUCCAACCAGCU
1238
AUGUACAGCUGGU
1648
441-459

960896
GUACAUdTdT

UGGACCdTdT

AD-
GUCCAACCAGCUG
1239
AAUGUACAGCUGG
1649
442-460

960897
UACAUUdTdT

UUGGACdTdT

AD-
UCCAACCAGCUGU
1240
AAAUGUACAGCUG
1650
443-461

960898
ACAUUUdTdT

GUUGGAdTdT

AD-
CCAACCAGCUGUA
1241
AAAAUGUACAGCU
1651
444-462

960899
CAUUUUdTdT

GGUUGGdTdT

AD-
CAACCAGCUGUAC
1242
ACAAAUGUACAGC
1652
445-463

960900
AUUUGUdTdT

UGGUUGdTdT

AD-
AACCAGCUGUACA
1243
ACCAAAUGUACAG
1653
446-464

960901
UUUGGUdTdT

CUGGUUdTdT

AD-
ACCAGCUGUACAU
1244
AUCCAAAUGUACA
1654
447-465

960902
UUGGAUdTdT

GCUGGUdTdT

AD-
CCAGCUGUACAUU
1245
AUUCCAAAUGUAC
1655
448-466

960903
UGGAAUdTdT

AGCUGGdTdT

AD-
CAGCUGUACAUUU
1246
AUUUCCAAAUGUA
1656
449-467

960904
GGAAAUdTdT

CAGCUGdTdT

AD-
AGCUGUACAUUUG
1247
AUUUUCCAAAUGU
1657
450-468

960905
GAAAAUdTdT

ACAGCUdTdT

AD-
GCUGUACAUUUGG
1248
AUUUUUCCAAAUG
1658
451-469

960906
AAAAAUdTdT

UACAGCdTdT

AD-
CUGUACAUUUGGA
1249
AAUUUUUCCAAAU
1659
452-470

960907
AAAAUUdTdT

GUACAGdTdT

AD-
UGUACAUUUGGAA
1250
AUAUUUUUCCAAA
1660
453-471

960908
AAAUAUdTdT

UGUACAdTdT

AD-
GUACAUUUGGAAA
1251
AUUAUUUUUCCAA
1661
454-472

960909
AAUAAUdTdT

AUGUACdTdT

AD-
CAUUUGGAAAAAU
1252
AGUUUUAUUUUUC
1662
457-475

960910
AAAACUdTdT

CAAAUGdTdT

TABLE 4

RPS25 Unmodified duplex Sequences

Start
End

Site in
Site in
Sense
SEQ
Antisense
SEQ

SEQ

NM_
NM_
Oligo Sequence
ID
Oligo Sequence
ID
Target Sequence
ID

001028.3
00128.3
5’ to 3’
NO:
5’ to 3’
NO:
5’ to 3’
NO:

1
19
CUUUUUGUCCGACAUCUUG
1663
CAAGAUGUCGGACAAAAAG
1774
CTTTTTGTCCGACATCTTG
1885

3
21
UUUUGUCCGACAUCUUGAC
1664
GUCAAGAUGUCGGACAAAA
1775
TTTTGTCCGACATCTTGAC
1886

6
24
UGUCCGACAUCUUGACGAG
1665
CUCGUCAAGAUGUCGGACA
1776
TGTCCGACATCTTGACGAG
1887

9
27
CCGACAUCUUGACGAGGCU
31
AGCCUCGUCAAGAUGUCGG
441
CCGACATCTTGACGAGGCT
1888

12
30
ACAUCUUGACGAGGCUGCG
1666
CGCAGCCUCGUCAAGAUGU
1777
ACATCTTGACGAGGCTGCG
1889

16
34
CUUGACGAGGCUGCGGUGU
38
ACACCGCAGCCUCGUCAAG
448
CTTGACGAGGCTGCGGTGT
1890

22
40
GAGGCUGCGGUGUCUGCUG
1667
CAGCAGACACCGCAGCCUC
1778
GAGGCTGCGGTGTCTGCTG
1891

25
43
GCUGCGGUGUCUGCUGCUA
1668
UAGCAGCAGACACCGCAGC
1779
GCTGCGGTGTCTGCTGCTA
1892

29
47
CGGUGUCUGCUGCUAUUCU
51
AGAAUAGCAGCAGACACCG
461
CGGTGTCTGCTGCTATTCT
1893

31
49
GUGUCUGCUGCUAUUCUCC
1669
GGAGAAUAGCAGCAGACAC
1780
GTGTCTGCTGCTATTCTCC
1894

33
51
GUCUGCUGCUAUUCUCCGA
1670
UCGGAGAAUAGCAGCAGAC
1781
GTCTGCTGCTATTCTCCGA
1895

37
55
GCUGCUAUUCUCCGAGCUU
59
AAGCUCGGAGAAUAGCAGC
469
GCTGCTATTCTCCGAGCTT
1896

42
60
UAUUCUCCGAGCUUCGCAA
1671
UUGCGAAGCUCGGAGAAUA
1782
TATTCTCCGAGCTTCGCAA
1897

45
63
UCUCCGAGCUUCGCAAUGC
1672
GCAUUGCGAAGCUCGGAGA
1783
TCTCCGAGCTTCGCAATGC
1898

48
66
CCGAGCUUCGCAAUGCCGC
1673
GCGGCAUUGCGAAGCUCGG
1784
CCGAGCTTCGCAATGCCGC
1899

53
71
CUUCGCAAUGCCGCCUAAG
1674
CUUAGGCGGCAUUGCGAAG
1785
CTTCGCAATGCCGCCTAAG
1900

54
72
UUCGCAAUGCCGCCUAAGG
1675
CCUUAGGCGGCAUUGCGAA
1786
TTCGCAATGCCGCCTAAGG
1901

60
78
AUGCCGCCUAAGGACGACA
1676
UGUCGUCCUUAGGCGGCAU
1787
ATGCCGCCTAAGGACGACA
1902

62
80
GCCGCCUAAGGACGACAAG
1677
CUUGUCGUCCUUAGGCGGC
1788
GCCGCCTAAGGACGACAAG
1903

64
82
CGCCUAAGGACGACAAGAA
1678
UUCUUGUCGUCCUUAGGCG
1789
CGCCTAAGGACGACAAGAA
1904

70
88
AGGACGACAAGAAGAAGAA
1679
UUCUUCUUCUUGUCGUCCU
1790
AGGACGACAAGAAGAAGAA
2520

71
89
GGACGACAAGAAGAAGAAG
1680
CUUCUUCUUCUUGUCGUCC
1791
GGACGACAAGAAGAAGAAG
2521

76
94
ACAAGAAGAAGAAGGACGC
1681
GCGUCCUUCUUCUUCUUGU
1792
ACAAGAAGAAGAAGGACGC
2522

79
97
AGAAGAAGAAGGACGCUGG
1682
CCAGCGUCCUUCUUCUUCU
1793
AGAAGAAGAAGGACGCTGG
1905

83
101
GAAGAAGGACGCUGGAAAG
1683
CUUUCCAGCGUCCUUCUUC
1794
GAAGAAGGACGCTGGAAAG
1906

85
103
AGAAGGACGCUGGAAAGUC
1684
GACUUUCCAGCGUCCUUCU
1795
AGAAGGACGCTGGAAAGTC
1907

91
109
ACGCUGGAAAGUCGGCCAA
1685
UUGGCCGACUUUCCAGCGU
1796
ACGCTGGAAAGTCGGCCAA
1908

94
112
CUGGAAAGUCGGCCAAGAA
1686
UUCUUGGCCGACUUUCCAG
1797
CTGGAAAGTCGGCCAAGAA
1909

96
114
GGAAAGUCGGCCAAGAAAG
1687
CUUUCUUGGCCGACUUUCC
1798
GGAAAGTCGGCCAAGAAAG
1910

101
119
GUCGGCCAAGAAAGACAAA
1688
UUUGUCUUUCUUGGCCGAC
1799
GTCGGCCAAGAAAGACAAA
1911

103
121
CGGCCAAGAAAGACAAAGA
1689
UCUUUGUCUUUCUUGGCCG
1800
CGGCCAAGAAAGACAAAGA
2523

107
125
CAAGAAAGACAAAGACCCA
1690
UGGGUCUUUGUCUUUCUUG
1801
CAAGAAAGACAAAGACCCA
2524

109
127
AGAAAGACAAAGACCCAGU
130
ACUGGGUCUUUGUCUUUCU
540
AGAAAGACAAAGACCCAGT
1912

115
133
ACAAAGACCCAGUGAACAA
1691
UUGUUCACUGGGUCUUUGU
1802
ACAAAGACCCAGTGAACAA
1913

116
134
CAAAGACCCAGUGAACAAA
1692
UUUGUUCACUGGGUCUUUG
1803
CAAAGACCCAGTGAACAAA
1914

120
138
GACCCAGUGAACAAAUCCG
1693
CGGAUUUGUUCACUGGGUC
1804
GACCCAGTGAACAAATCCG
1915

125
143
AGUGAACAAAUCCGGGGGC
1694
GCCCCCGGAUUUGUUCACU
1805
AGTGAACAAATCCGGGGGC
1916

127
145
UGAACAAAUCCGGGGGCAA
1695
UUGCCCCCGGAUUUGUUCA
1806
TGAACAAATCCGGGGGCAA
1917

130
148
ACAAAUCCGGGGGCAAGGC
1696
GCCUUGCCCCCGGAUUUGU
1807
ACAAATCCGGGGGCAAGGC
1918

136
154
CCGGGGGCAAGGCCAAAAA
1697
UUUUUGGCCUUGCCCCCGG
1808
CCGGGGGCAAGGCCAAAAA
2525

140
158
GGGCAAGGCCAAAAAGAAG
1698
CUUCUUUUUGGCCUUGCCC
1809
GGGCAAGGCCAAAAAGAAG
2526

142
160
GCAAGGCCAAAAAGAAGAA
1699
UUCUUCUUUUUGGCCUUGC
1810
GCAAGGCCAAAAAGAAGAA
2527

146
164
GGCCAAAAAGAAGAAGUGG
1700
CCACUUCUUCUUUUUGGCC
1811
GGCCAAAAAGAAGAAGTGG
1919

148
166
CCAAAAAGAAGAAGUGGUC
1701
GACCACUUCUUCUUUUUGG
1812
CCAAAAAGAAGAAGTGGTC
1920

151
169
AAAAGAAGAAGUGGUCCAA
1702
UUGGACCACUUCUUCUUUU
1813
AAAAGAAGAAGTGGTCCAA
1921

154
172
AGAAGAAGUGGUCCAAAGG
1703
CCUUUGGACCACUUCUUCU
1814
AGAAGAAGTGGTCCAAAGG
1922

160
178
AGUGGUCCAAAGGCAAAGU
162
ACUUUGCCUUUGGACCACU
572
AGTGGTCCAAAGGCAAAGT
1923

163
181
GGUCCAAAGGCAAAGUUCG
1704
CGAACUUUGCCUUUGGACC
1815
GGTCCAAAGGCAAAGTTCG
1924

165
183
UCCAAAGGCAAAGUUCGGG
1705
CCCGAACUUUGCCUUUGGA
1816
TCCAAAGGCAAAGTTCGGG
1925

169
187
AAGGCAAAGUUCGGGACAA
1706
UUGUCCCGAACUUUGCCUU
1817
AAGGCAAAGTTCGGGACAA
1926

173
191
CAAAGUUCGGGACAAGCUC
1707
GAGCUUGUCCCGAACUUUG
1818
CAAAGTTCGGGACAAGCTC
1927

178
196
UUCGGGACAAGCUCAAUAA
1708
UUAUUGAGCUUGUCCCGAA
1819
TTCGGGACAAGCTCAATAA
1928

181
199
GGGACAAGCUCAAUAACUU
183
AAGUUAUUGAGCUUGUCCC
593
GGGACAAGCTCAATAACTT
1929

182
200
GGACAAGCUCAAUAACUUA
1709
UAAGUUAUUGAGCUUGUCC
1820
GGACAAGCTCAATAACTTA
1930

188
206
GCUCAAUAACUUAGUCUUG
1710
CAAGACUAAGUUAUUGAGC
1821
GCTCAATAACTTAGTCTTG
1931

189
207
CUCAAUAACUUAGUCUUGU
191
ACAAGACUAAGUUAUUGAG
601
CTCAATAACTTAGTCTTGT
1932

192
210
AAUAACUUAGUCUUGUUUG
1711
CAAACAAGACUAAGUUAUU
1822
AATAACTTAGTCTTGTTTG
1933

197
215
CUUAGUCUUGUUUGACAAA
1712
UUUGUCAAACAAGACUAAG
1823
CTTAGTCTTGTTTGACAAA
1934

200
218
AGUCUUGUUUGACAAAGCU
202
AGCUUUGUCAAACAAGACU
612
AGTCTTGTTTGACAAAGCT
1935

203
221
CUUGUUUGACAAAGCUACC
1713
GGUAGCUUUGUCAAACAAG
1824
CTTGTTTGACAAAGCTACC
1936

206
224
GUUUGACAAAGCUACCUAU
208
AUAGGUAGCUUUGUCAAAC
618
GTTTGACAAAGCTACCTAT
1937

212
230
CAAAGCUACCUAUGAUAAA
1714
UUUAUCAUAGGUAGCUUUG
1825
CAAAGCTACCTATGATAAA
1938

216
234
GCUACCUAUGAUAAACUCU
218
AGAGUUUAUCAUAGGUAGC
628
GCTACCTATGATAAACTCT
1939

217
235
CUACCUAUGAUAAACUCUG
1715
CAGAGUUUAUCAUAGGUAG
1826
CTACCTATGATAAACTCTG
1940

220
238
CCUAUGAUAAACUCUGUAA
1716
UUACAGAGUUUAUCAUAGG
1827
CCTATGATAAACTCTGTAA
1941

224
242
UGAUAAACUCUGUAAGGAA
1717
UUCCUUACAGAGUUUAUCA
1828
TGATAAACTCTGTAAGGAA
1942

229
247
AACUCUGUAAGGAAGUUCC
1718
GGAACUUCCUUACAGAGUU
1829
AACTCTGTAAGGAAGTTCC
1943

231
249
CUCUGUAAGGAAGUUCCCA
1719
UGGGAACUUCCUUACAGAG
1830
CTCTGTAAGGAAGTTCCCA
1944

236
254
UAAGGAAGUUCCCAACUAU
238
AUAGUUGGGAACUUCCUUA
648
TAAGGAAGTTCCCAACTAT
1945

239
257
GGAAGUUCCCAACUAUAAA
1720
UUUAUAGUUGGGAACUUCC
1831
GGAAGTTCCCAACTATAAA
1946

243
261
GUUCCCAACUAUAAACUUA
1721
UAAGUUUAUAGUUGGGAAC
1832
GTTCCCAACTATAAACTTA
1947

245
263
UCCCAACUAUAAACUUAUA
1722
UAUAAGUUUAUAGUUGGGA
1833
TCCCAACTATAAACTTATA
1948

248
266
CAACUAUAAACUUAUAACC
1723
GGUUAUAAGUUUAUAGUUG
1834
CAACTATAAACTTATAACC
1949

254
272
UAAACUUAUAACCCCAGCU
1724
AGCUGGGGUUAUAAGUUUA
1835
TAAACTTATAACCCCAGCT
1950

255
273
AAACUUAUAACCCCAGCUG
1725
CAGCUGGGGUUAUAAGUUU
1836
AAACTTATAACCCCAGCTG
1951

258
276
CUUAUAACCCCAGCUGUGG
1726
CCACAGCUGGGGUUAUAAG
1837
CTTATAACCCCAGCTGTGG
1952

264
282
ACCCCAGCUGUGGUCUCUG
1727
CAGAGACCACAGCUGGGGU
1838
ACCCCAGCTGTGGTCTCTG
1953

267
285
CCAGCUGUGGUCUCUGAGA
1728
UCUCAGAGACCACAGCUGG
1839
CCAGCTGTGGTCTCTGAGA
1954

271
289
CUGUGGUCUCUGAGAGACU
257
AGUCUCUCAGAGACCACAG
667
CTGTGGTCTCTGAGAGACT
1955

274
292
UGGUCUCUGAGAGACUGAA
1729
UUCAGUCUCUCAGAGACCA
1840
TGGTCTCTGAGAGACTGAA
1956

278
296
CUCUGAGAGACUGAAGAUU
264
AAUCUUCAGUCUCUCAGAG
674
CTCTGAGAGACTGAAGATT
1957

279
297
UCUGAGAGACUGAAGAUUC
1730
GAAUCUUCAGUCUCUCAGA
1841
TCTGAGAGACTGAAGATTC
1958

282
300
GAGAGACUGAAGAUUCGAG
1731
CUCGAAUCUUCAGUCUCUC
1842
GAGAGACTGAAGATTCGAG
1959

287
305
ACUGAAGAUUCGAGGCUCC
1732
GGAGCCUCGAAUCUUCAGU
1843
ACTGAAGATTCGAGGCTCC
1960

289
307
UGAAGAUUCGAGGCUCCCU
275
AGGGAGCCUCGAAUCUUCA
685
TGAAGATTCGAGGCTCCCT
1961

293
311
GAUUCGAGGCUCCCUGGCC
1733
GGCCAGGGAGCCUCGAAUC
1844
GATTCGAGGCTCCCTGGCC
1962

298
316
GAGGCUCCCUGGCCAGGGC
1734
GCCCUGGCCAGGGAGCCUC
1845
GAGGCTCCCTGGCCAGGGC
1963

302
320
CUCCCUGGCCAGGGCAGCC
1735
GGCUGCCCUGGCCAGGGAG
1846
CTCCCTGGCCAGGGCAGCC
1964

306
324
CUGGCCAGGGCAGCCCUUC
1736
GAAGGGCUGCCCUGGCCAG
1847
CTGGCCAGGGCAGCCCTTC
1965

308
326
GGCCAGGGCAGCCCUUCAG
1737
CUGAAGGGCUGCCCUGGCC
1848
GGCCAGGGCAGCCCTTCAG
1966

313
331
GGGCAGCCCUUCAGGAGCU
290
AGCUCCUGAAGGGCUGCCC
700
GGGCAGCCCTTCAGGAGCT
1967

316
334
CAGCCCUUCAGGAGCUCCU
293
AGGAGCUCCUGAAGGGCUG
703
CAGCCCTTCAGGAGCTCCT
1968

318
336
GCCCUUCAGGAGCUCCUUA
1738
UAAGGAGCUCCUGAAGGGC
1849
GCCCTTCAGGAGCTCCTTA
1969

323
341
UCAGGAGCUCCUUAGUAAA
1739
UUUACUAAGGAGCUCCUGA
1850
TCAGGAGCTCCTTAGTAAA
1970

326
344
GGAGCUCCUUAGUAAAGGA
1740
UCCUUUACUAAGGAGCUCC
1851
GGAGCTCCTTAGTAAAGGA
1971

330
348
CUCCUUAGUAAAGGACUUA
1741
UAAGUCCUUUACUAAGGAG
1852
CTCCTTAGTAAAGGACTTA
1972

333
351
CUUAGUAAAGGACUUAUCA
1742
UGAUAAGUCCUUUACUAAG
1853
CTTAGTAAAGGACTTATCA
1973

335
353
UAGUAAAGGACUUAUCAAA
1743
UUUGAUAAGUCCUUUACUA
1854
TAGTAAAGGACTTATCAAA
1974

340
358
AAGGACUUAUCAAACUGGU
317
ACCAGUUUGAUAAGUCCUU
727
AAGGACTTATCAAACTGGT
1975

343
361
GACUUAUCAAACUGGUUUC
1744
GAAACCAGUUUGAUAAGUC
1855
GACTTATCAAACTGGTTTC
1976

345
363
CUUAUCAAACUGGUUUCAA
1745
UUGAAACCAGUUUGAUAAG
1856
CTTATCAAACTGGTTTCAA
1977

348
366
AUCAAACUGGUUUCAAAGC
1746
GCUUUGAAACCAGUUUGAU
1857
ATCAAACTGGTTTCAAAGC
1978

353
371
ACUGGUUUCAAAGCACAGA
1747
UCUGUGCUUUGAAACCAGU
1858
ACTGGTTTCAAAGCACAGA
1979

358
376
UUUCAAAGCACAGAGCUCA
1748
UGAGCUCUGUGCUUUGAAA
1859
TTTCAAAGCACAGAGCTCA
1980

359
377
UUCAAAGCACAGAGCUCAA
1749
UUGAGCUCUGUGCUUUGAA
1860
TTCAAAGCACAGAGCTCAA
1981

365
383
GCACAGAGCUCAAGUAAUU
342
AAUUACUUGAGCUCUGUGC
752
GCACAGAGCTCAAGTAATT
1982

368
386
CAGAGCUCAAGUAAUUUAC
1750
GUAAAUUACUUGAGCUCUG
1861
CAGAGCTCAAGTAATTTAC
1983

369
387
AGAGCUCAAGUAAUUUACA
1751
UGUAAAUUACUUGAGCUCU
1862
AGAGCTCAAGTAATTTACA
1984

373
391
CUCAAGUAAUUUACACCAG
1752
CUGGUGUAAAUUACUUGAG
1863
CTCAAGTAATTTACACCAG
1985

378
396
GUAAUUUACACCAGAAAUA
1753
UAUUUCUGGUGUAAAUUAC
1864
GTAATTTACACCAGAAATA
1986

379
397
UAAUUUACACCAGAAAUAC
1754
GUAUUUCUGGUGUAAAUUA
1865
TAATTTACACCAGAAATAC
1987

384
402
UACACCAGAAAUACCAAGG
1755
CCUUGGUAUUUCUGGUGUA
1866
TACACCAGAAATACCAAGG
1988

387
405
ACCAGAAAUACCAAGGGUG
1756
CACCCUUGGUAUUUCUGGU
1867
ACCAGAAATACCAAGGGTG
1989

390
408
AGAAAUACCAAGGGUGGAG
1757
CUCCACCCUUGGUAUUUCU
1868
AGAAATACCAAGGGTGGAG
1990

393
411
AAUACCAAGGGUGGAGAUG
1758
CAUCUCCACCCUUGGUAUU
1869
AATACCAAGGGTGGAGATG
1991

399
417
AAGGGUGGAGAUGCUCCAG
1759
CUGGAGCAUCUCCACCCUU
1870
AAGGGTGGAGATGCTCCAG
1992

402
420
GGUGGAGAUGCUCCAGCUG
1760
CAGCUGGAGCAUCUCCACC
1871
GGTGGAGATGCTCCAGCTG
1993

404
422
UGGAGAUGCUCCAGCUGCU
381
AGCAGCUGGAGCAUCUCCA
791
TGGAGATGCTCCAGCTGCT
1994

410
428
UGCUCCAGCUGCUGGUGAA
1761
UUCACCAGCAGCUGGAGCA
1872
TGCTCCAGCTGCTGGTGAA
1995

411
429
GCUCCAGCUGCUGGUGAAG
1762
CUUCACCAGCAGCUGGAGC
1873
GCTCCAGCTGCTGGTGAAG
1996

417
435
GCUGCUGGUGAAGAUGCAU
394
AUGCAUCUUCACCAGCAGC
804
GCTGCTGGTGAAGATGCAT
1997

419
437
UGCUGGUGAAGAUGCAUGA
1763
UCAUGCAUCUUCACCAGCA
1874
TGCTGGTGAAGATGCATGA
1998

423
441
GGUGAAGAUGCAUGAAUAG
1764
CUAUUCAUGCAUCUUCACC
1875
GGTGAAGATGCATGAATAG
1999

426
444
GAAGAUGCAUGAAUAGGUC
1765
GACCUAUUCAUGCAUCUUC
1876
GAAGATGCATGAATAGGTC
2000

430
448
AUGCAUGAAUAGGUCCAAC
1766
GUUGGACCUAUUCAUGCAU
1877
ATGCATGAATAGGTCCAAC
2001

432
450
GCAUGAAUAGGUCCAACCA
1767
UGGUUGGACCUAUUCAUGC
1878
GCATGAATAGGTCCAACCA
2002

435
453
UGAAUAGGUCCAACCAGCU
412
AGCUGGUUGGACCUAUUCA
822
TGAATAGGTCCAACCAGCT
2003

441
459
GGUCCAACCAGCUGUACAU
418
AUGUACAGCUGGUUGGACC
828
GGTCCAACCAGCTGTACAT
2004

444
462
CCAACCAGCUGUACAUUUG
1768
CAAAUGUACAGCUGGUUGG
1879
CCAACCAGCTGTACATTTG
2005

448
466
CCAGCUGUACAUUUGGAAA
1769
UUUCCAAAUGUACAGCUGG
1880
CCAGCTGTACATTTGGAAA
2006

451
469
GCUGUACAUUUGGAAAAAU
428
AUUUUUCCAAAUGUACAGC
838
GCTGTACATTTGGAAAAAT
2007

454
472
GUACAUUUGGAAAAAUAAA
1770
UUUAUUUUUCCAAAUGUAC
1881
GTACATTTGGAAAAATAAA
2008

456
474
ACAUUUGGAAAAAUAAAAC
1771
GUUUUAUUUUUCCAAAUGU
1882
ACATTTGGAAAAATAAAAC
2009

462
480
GGAAAAAUAAAACUUUAUU
1772
AAUAAAGUUUUAUUUUUCC
1883
GGAAAAATAAAACTTTATT
2010

465
483
AAAAUAAAACUUUAUUAAA
1773
UUUAAUAAAGUUUUAUUUU
1884
AAAATAAAACTTTATTAAA
2011

TABLE 5

RPS25 Modified duplex Sequences

Start
End

Site in
Site in

SEQ
Sense
SEQ
Antisense
SEQ

NM_
NM_
Target Sequence
ID
Oligo Sequence
ID
Oligo Sequence
ID

001028.3
00128.3
5’ to 3’
NO:
5’ to 3’
NO:
5’ to 3’
NO:

1
19
CTTTTTGTCCGACATCTTG
1885
CUUUUUGUCCGACAUCUUGdTdT
2012
CAAGAUGUCGGACAAAAAGdTdT
2123

3
21
TTTTGTCCGACATCTTGAC
1886
UUUUGUCCGACAUCUUGACdTdT
2013
GUCAAGAUGUCGGACAAAAdTdT
2124

6
24
TGTCCGACATCTTGACGAG
1887
UGUCCGACAUCUUGACGAGdTdT
2014
CUCGUCAAGAUGUCGGACAdTdT
2125

9
27
CCGACATCTTGACGAGGCT
1888
CCGACAUCUUGACGAGGCUdTdT
851
AGCCUCGUCAAGAUGUCGGdTdT
1261

12
30
ACATCTTGACGAGGCTGCG
1889
ACAUCUUGACGAGGCUGCGdTdT
2015
CGCAGCCUCGUCAAGAUGUdTdT
2126

16
34
CTTGACGAGGCTGCGGTGT
1890
CUUGACGAGGCUGCGGUGUdTdT
858
ACACCGCAGCCUCGUCAAGdTdT
1268

22
40
GAGGCTGCGGTGTCTGCTG
1891
GAGGCUGCGGUGUCUGCUGdTdT
2016
CAGCAGACACCGCAGCCUCdTdT
2127

25
43
GCTGCGGTGTCTGCTGCTA
1892
GCUGCGGUGUCUGCUGCUAdTdT
2017
UAGCAGCAGACACCGCAGCdTdT
2128

29
47
CGGTGTCTGCTGCTATTCT
1893
CGGUGUCUGCUGCUAUUCUdTdT
871
AGAAUAGCAGCAGACACCGdTdT
1281

31
49
GTGTCTGCTGCTATTCTCC
1894
GUGUCUGCUGCUAUUCUCCdTdT
2018
GGAGAAUAGCAGCAGACACdTdT
2129

33
51
GTCTGCTGCTATTCTCCGA
1895
GUCUGCUGCUAUUCUCCGAdTdT
2019
UCGGAGAAUAGCAGCAGACdTdT
2130

37
55
GCTGCTATTCTCCGAGCTT
1896
GCUGCUAUUCUCCGAGCUUdTdT
879
AAGCUCGGAGAAUAGCAGCdTdT
1289

42
60
TATTCTCCGAGCTTCGCAA
1897
UAUUCUCCGAGCUUCGCAAdTdT
2020
UUGCGAAGCUCGGAGAAUAdTdT
2131

45
63
TCTCCGAGCTTCGCAATGC
1898
UCUCCGAGCUUCGCAAUGCdTdT
2021
GCAUUGCGAAGCUCGGAGAdTdT
2132

48
66
CCGAGCTTCGCAATGCCGC
1899
CCGAGCUUCGCAAUGCCGCdTdT
2022
GCGGCAUUGCGAAGCUCGGdTdT
2133

53
71
CTTCGCAATGCCGCCTAAG
1900
CUUCGCAAUGCCGCCUAAGdTdT
2023
CUUAGGCGGCAUUGCGAAGdTdT
2134

54
72
TTCGCAATGCCGCCTAAGG
1901
UUCGCAAUGCCGCCUAAGGdTdT
2024
CCUUAGGCGGCAUUGCGAAdTdT
2135

60
78
ATGCCGCCTAAGGACGACA
1902
AUGCCGCCUAAGGACGACAdTdT
2025
UGUCGUCCUUAGGCGGCAUdTdT
2136

62
80
GCCGCCTAAGGACGACAAG
1903
GCCGCCUAAGGACGACAAGdTdT
2026
CUUGUCGUCCUUAGGCGGCdTdT
2137

64
82
CGCCTAAGGACGACAAGAA
1904
CGCCUAAGGACGACAAGAAdTdT
2027
UUCUUGUCGUCCUUAGGCGdTdT
2138

70
88
AGGACGACAAGAAGAAGAA
2520
AGGACGACAAGAAGAAGAAdTdT
2028
UUCUUCUUCUUGUCGUCCUdTdT
2139

71
89
GGACGACAAGAAGAAGAAG
2521
GGACGACAAGAAGAAGAAGdTdT
2029
CUUCUUCUUCUUGUCGUCCdTdT
2140

76
94
ACAAGAAGAAGAAGGACGC
2522
ACAAGAAGAAGAAGGACGCdTdT
2030
GCGUCCUUCUUCUUCUUGUdTdT
2141

79
97
AGAAGAAGAAGGACGCTGG
1905
AGAAGAAGAAGGACGCUGGdTdT
2031
CCAGCGUCCUUCUUCUUCUdTdT
2142

83
101
GAAGAAGGACGCTGGAAAG
1906
GAAGAAGGACGCUGGAAAGdTdT
2032
CUUUCCAGCGUCCUUCUUCdTdT
2143

85
103
AGAAGGACGCTGGAAAGTC
1907
AGAAGGACGCUGGAAAGUCdTdT
2033
GACUUUCCAGCGUCCUUCUdTdT
2144

91
109
ACGCTGGAAAGTCGGCCAA
1908
ACGCUGGAAAGUCGGCCAAdTdT
2034
UUGGCCGACUUUCCAGCGUdTdT
2145

94
112
CTGGAAAGTCGGCCAAGAA
1909
CUGGAAAGUCGGCCAAGAAdTdT
2035
UUCUUGGCCGACUUUCCAGdTdT
2146

96
114
GGAAAGTCGGCCAAGAAAG
1910
GGAAAGUCGGCCAAGAAAGdTdT
2036
CUUUCUUGGCCGACUUUCCdTdT
2147

101
119
GTCGGCCAAGAAAGACAAA
1911
GUCGGCCAAGAAAGACAAAdTdT
2037
UUUGUCUUUCUUGGCCGACdTdT
2148

103
121
CGGCCAAGAAAGACAAAGA
2523
CGGCCAAGAAAGACAAAGAdTdT
2038
UCUUUGUCUUUCUUGGCCGdTdT
2149

107
125
CAAGAAAGACAAAGACCCA
2524
CAAGAAAGACAAAGACCCAdTdT
2039
UGGGUCUUUGUCUUUCUUGdTdT
2150

109
127
AGAAAGACAAAGACCCAGT
1912
AGAAAGACAAAGACCCAGUdTdT
950
ACUGGGUCUUUGUCUUUCUdTdT
1360

115
133
ACAAAGACCCAGTGAACAA
1913
ACAAAGACCCAGUGAACAAdTdT
2040
UUGUUCACUGGGUCUUUGUdTdT
2151

116
134
CAAAGACCCAGTGAACAAA
1914
CAAAGACCCAGUGAACAAAdTdT
2041
UUUGUUCACUGGGUCUUUGdTdT
2152

120
138
GACCCAGTGAACAAATCCG
1915
GACCCAGUGAACAAAUCCGdTdT
2042
CGGAUUUGUUCACUGGGUCdTdT
2153

125
143
AGTGAACAAATCCGGGGGC
1916
AGUGAACAAAUCCGGGGGCdTdT
2043
GCCCCCGGAUUUGUUCACUdTdT
2154

127
145
TGAACAAATCCGGGGGCAA
1917
UGAACAAAUCCGGGGGCAAdTdT
2044
UUGCCCCCGGAUUUGUUCAdTdT
2155

130
148
ACAAATCCGGGGGCAAGGC
1918
ACAAAUCCGGGGGCAAGGCdTdT
2045
GCCUUGCCCCCGGAUUUGUdTdT
2156

136
154
CCGGGGGCAAGGCCAAAAA
2525
CCGGGGGCAAGGCCAAAAAdTdT
2046
UUUUUGGCCUUGCCCCCGGdTdT
2157

140
158
GGGCAAGGCCAAAAAGAAG
2526
GGGCAAGGCCAAAAAGAAGdTdT
2047
CUUCUUUUUGGCCUUGCCCdTdT
2158

142
160
GCAAGGCCAAAAAGAAGAA
2527
GCAAGGCCAAAAAGAAGAAdTdT
2048
UUCUUCUUUUUGGCCUUGCdTdT
2159

146
164
GGCCAAAAAGAAGAAGTGG
1919
GGCCAAAAAGAAGAAGUGGdTdT
2049
CCACUUCUUCUUUUUGGCCdTdT
2160

148
166
CCAAAAAGAAGAAGTGGTC
1920
CCAAAAAGAAGAAGUGGUCdTdT
2050
GACCACUUCUUCUUUUUGGdTdT
2161

151
169
AAAAGAAGAAGTGGTCCAA
1921
AAAAGAAGAAGUGGUCCAAdTdT
2051
UUGGACCACUUCUUCUUUUdTdT
2162

154
172
AGAAGAAGTGGTCCAAAGG
1922
AGAAGAAGUGGUCCAAAGGdTdT
2052
CCUUUGGACCACUUCUUCUdTdT
2163

160
178
AGTGGTCCAAAGGCAAAGT
1923
AGUGGUCCAAAGGCAAAGUdTdT
982
ACUUUGCCUUUGGACCACUdTdT
1392

163
181
GGTCCAAAGGCAAAGTTCG
1924
GGUCCAAAGGCAAAGUUCGdTdT
2053
CGAACUUUGCCUUUGGACCdTdT
2164

165
183
TCCAAAGGCAAAGTTCGGG
1925
UCCAAAGGCAAAGUUCGGGdTdT
2054
CCCGAACUUUGCCUUUGGAdTdT
2165

169
187
AAGGCAAAGTTCGGGACAA
1926
AAGGCAAAGUUCGGGACAAdTdT
2055
UUGUCCCGAACUUUGCCUUdTdT
2166

173
191
CAAAGTTCGGGACAAGCTC
1927
CAAAGUUCGGGACAAGCUCdTdT
2056
GAGCUUGUCCCGAACUUUGdTdT
2167

178
196
TTCGGGACAAGCTCAATAA
1928
UUCGGGACAAGCUCAAUAAdTdT
2057
UUAUUGAGCUUGUCCCGAAdTdT
2168

181
199
GGGACAAGCTCAATAACTT
1929
GGGACAAGCUCAAUAACUUdTdT
1003
AAGUUAUUGAGCUUGUCCCdTdT
1413

182
200
GGACAAGCTCAATAACTTA
1930
GGACAAGCUCAAUAACUUAdTdT
2058
UAAGUUAUUGAGCUUGUCCdTdT
2169

188
206
GCTCAATAACTTAGTCTTG
1931
GCUCAAUAACUUAGUCUUGdTdT
2059
CAAGACUAAGUUAUUGAGCdTdT
2170

189
207
CTCAATAACTTAGTCTTGT
1932
CUCAAUAACUUAGUCUUGUdTdT
1011
ACAAGACUAAGUUAUUGAGdTdT
1421

192
210
AATAACTTAGTCTTGTTTG
1933
AAUAACUUAGUCUUGUUUGdTdT
2060
CAAACAAGACUAAGUUAUUdTdT
2171

197
215
CTTAGTCTTGTTTGACAAA
1934
CUUAGUCUUGUUUGACAAAdTdT
2061
UUUGUCAAACAAGACUAAGdTdT
2172

200
218
AGTCTTGTTTGACAAAGCT
1935
AGUCUUGUUUGACAAAGCUdTdT
1022
AGCUUUGUCAAACAAGACUdTdT
1432

203
221
CTTGTTTGACAAAGCTACC
1936
CUUGUUUGACAAAGCUACCdTdT
2062
GGUAGCUUUGUCAAACAAGdTdT
2173

206
224
GTTTGACAAAGCTACCTAT
1937
GUUUGACAAAGCUACCUAUdTdT
1028
AUAGGUAGCUUUGUCAAACdTdT
1438

212
230
CAAAGCTACCTATGATAAA
1938
CAAAGCUACCUAUGAUAAAdTdT
2063
UUUAUCAUAGGUAGCUUUGdTdT
2174

216
234
GCTACCTATGATAAACTCT
1939
GCUACCUAUGAUAAACUCUdTdT
1038
AGAGUUUAUCAUAGGUAGCdTdT
1448

217
235
CTACCTATGATAAACTCTG
1940
CUACCUAUGAUAAACUCUGdTdT
2064
CAGAGUUUAUCAUAGGUAGdTdT
2175

220
238
CCTATGATAAACTCTGTAA
1941
CCUAUGAUAAACUCUGUAAdTdT
2065
UUACAGAGUUUAUCAUAGGdTdT
2176

224
242
TGATAAACTCTGTAAGGAA
1942
UGAUAAACUCUGUAAGGAAdTdT
2066
UUCCUUACAGAGUUUAUCAdTdT
2177

229
247
AACTCTGTAAGGAAGTTCC
1943
AACUCUGUAAGGAAGUUCCdTdT
2067
GGAACUUCCUUACAGAGUUdTdT
2178

231
249
CTCTGTAAGGAAGTTCCCA
1944
CUCUGUAAGGAAGUUCCCAdTdT
2068
UGGGAACUUCCUUACAGAGdTdT
2179

236
254
TAAGGAAGTTCCCAACTAT
1945
UAAGGAAGUUCCCAACUAUdTdT
1058
AUAGUUGGGAACUUCCUUAdTdT
1468

239
257
GGAAGTTCCCAACTATAAA
1946
GGAAGUUCCCAACUAUAAAdTdT
2069
UUUAUAGUUGGGAACUUCCdTdT
2180

243
261
GTTCCCAACTATAAACTTA
1947
GUUCCCAACUAUAAACUUAdTdT
2070
UAAGUUUAUAGUUGGGAACdTdT
2181

245
263
TCCCAACTATAAACTTATA
1948
UCCCAACUAUAAACUUAUAdTdT
2071
UAUAAGUUUAUAGUUGGGAdTdT
2182

248
266
CAACTATAAACTTATAACC
1949
CAACUAUAAACUUAUAACCdTdT
2072
GGUUAUAAGUUUAUAGUUGdTdT
2183

254
272
TAAACTTATAACCCCAGCT
1950
UAAACUUAUAACCCCAGCUdTdT
2073
AGCUGGGGUUAUAAGUUUAdTdT
2184

255
273
AAACTTATAACCCCAGCTG
1951
AAACUUAUAACCCCAGCUGdTdT
2074
CAGCUGGGGUUAUAAGUUUdTdT
2185

258
276
CTTATAACCCCAGCTGTGG
1952
CUUAUAACCCCAGCUGUGGdTdT
2075
CCACAGCUGGGGUUAUAAGdTdT
2186

264
282
ACCCCAGCTGTGGTCTCTG
1953
ACCCCAGCUGUGGUCUCUGdTdT
2076
CAGAGACCACAGCUGGGGUdTdT
2187

267
285
CCAGCTGTGGTCTCTGAGA
1954
CCAGCUGUGGUCUCUGAGAdTdT
2077
UCUCAGAGACCACAGCUGGdTdT
2188

271
289
CTGTGGTCTCTGAGAGACT
1955
CUGUGGUCUCUGAGAGACUdTdT
1077
AGUCUCUCAGAGACCACAGdTdT
1487

274
292
TGGTCTCTGAGAGACTGAA
1956
UGGUCUCUGAGAGACUGAAdTdT
2078
UUCAGUCUCUCAGAGACCAdTdT
2189

278
296
CTCTGAGAGACTGAAGATT
1957
CUCUGAGAGACUGAAGAUUdTdT
1084
AAUCUUCAGUCUCUCAGAGdTdT
1494

279
297
TCTGAGAGACTGAAGATTC
1958
UCUGAGAGACUGAAGAUUCdTdT
2079
GAAUCUUCAGUCUCUCAGAdTdT
2190

282
300
GAGAGACTGAAGATTCGAG
1959
GAGAGACUGAAGAUUCGAGdTdT
2080
CUCGAAUCUUCAGUCUCUCdTdT
2191

287
305
ACTGAAGATTCGAGGCTCC
1960
ACUGAAGAUUCGAGGCUCCdTdT
2081
GGAGCCUCGAAUCUUCAGUdTdT
2192

289
307
TGAAGATTCGAGGCTCCCT
1961
UGAAGAUUCGAGGCUCCCUdTdT
1095
AGGGAGCCUCGAAUCUUCAdTdT
1505

293
311
GATTCGAGGCTCCCTGGCC
1962
GAUUCGAGGCUCCCUGGCCdTdT
2082
GGCCAGGGAGCCUCGAAUCdTdT
2193

298
316
GAGGCTCCCTGGCCAGGGC
1963
GAGGCUCCCUGGCCAGGGCdTdT
2083
GCCCUGGCCAGGGAGCCUCdTdT
2194

302
320
CTCCCTGGCCAGGGCAGCC
1964
CUCCCUGGCCAGGGCAGCCdTdT
2084
GGCUGCCCUGGCCAGGGAGdTdT
2195

306
324
CTGGCCAGGGCAGCCCTTC
1965
CUGGCCAGGGCAGCCCUUCdTdT
2085
GAAGGGCUGCCCUGGCCAGdTdT
2196

308
326
GGCCAGGGCAGCCCTTCAG
1966
GGCCAGGGCAGCCCUUCAGdTdT
2086
CUGAAGGGCUGCCCUGGCCdTdT
2197

313
331
GGGCAGCCCTTCAGGAGCT
1967
GGGCAGCCCUUCAGGAGCUdTdT
1110
AGCUCCUGAAGGGCUGCCCdTdT
1520

316
334
CAGCCCTTCAGGAGCTCCT
1968
CAGCCCUUCAGGAGCUCCUdTdT
1113
AGGAGCUCCUGAAGGGCUGdTdT
1523

318
336
GCCCTTCAGGAGCTCCTTA
1969
GCCCUUCAGGAGCUCCUUAdTdT
2087
UAAGGAGCUCCUGAAGGGCdTdT
2198

323
341
TCAGGAGCTCCTTAGTAAA
1970
UCAGGAGCUCCUUAGUAAAdTdT
2088
UUUACUAAGGAGCUCCUGAdTdT
2199

326
344
GGAGCTCCTTAGTAAAGGA
1971
GGAGCUCCUUAGUAAAGGAdTdT
2089
UCCUUUACUAAGGAGCUCCdTdT
2200

330
348
CTCCTTAGTAAAGGACTTA
1972
CUCCUUAGUAAAGGACUUAdTdT
2090
UAAGUCCUUUACUAAGGAGdTdT
2201

333
351
CTTAGTAAAGGACTTATCA
1973
CUUAGUAAAGGACUUAUCAdTdT
2091
UGAUAAGUCCUUUACUAAGdTdT
2202

335
353
TAGTAAAGGACTTATCAAA
1974
UAGUAAAGGACUUAUCAAAdTdT
2092
UUUGAUAAGUCCUUUACUAdTdT
2203

340
358
AAGGACTTATCAAACTGGT
1975
AAGGACUUAUCAAACUGGUdTdT
1137
ACCAGUUUGAUAAGUCCUUdTdT
1547

343
361
GACTTATCAAACTGGTTTC
1976
GACUUAUCAAACUGGUUUCdTdT
2093
GAAACCAGUUUGAUAAGUCdTdT
2204

345
363
CTTATCAAACTGGTTTCAA
1977
CUUAUCAAACUGGUUUCAAdTdT
2094
UUGAAACCAGUUUGAUAAGdTdT
2205

348
366
ATCAAACTGGTTTCAAAGC
1978
AUCAAACUGGUUUCAAAGCdTdT
2095
GCUUUGAAACCAGUUUGAUdTdT
2206

353
371
ACTGGTTTCAAAGCACAGA
1979
ACUGGUUUCAAAGCACAGAdTdT
2096
UCUGUGCUUUGAAACCAGUdTdT
2207

358
376
TTTCAAAGCACAGAGCTCA
1980
UUUCAAAGCACAGAGCUCAdTdT
2097
UGAGCUCUGUGCUUUGAAAdTdT
2208

359
377
TTCAAAGCACAGAGCTCAA
1981
UUCAAAGCACAGAGCUCAAdTdT
2098
UUGAGCUCUGUGCUUUGAAdTdT
2209

365
383
GCACAGAGCTCAAGTAATT
1982
GCACAGAGCUCAAGUAAUUdTdT
1162
AAUUACUUGAGCUCUGUGCdTdT
1572

368
386
CAGAGCTCAAGTAATTTAC
1983
CAGAGCUCAAGUAAUUUACdTdT
2099
GUAAAUUACUUGAGCUCUGdTdT
2210

369
387
AGAGCTCAAGTAATTTACA
1984
AGAGCUCAAGUAAUUUACAdTdT
2100
UGUAAAUUACUUGAGCUCUdTdT
2211

373
391
CTCAAGTAATTTACACCAG
1985
CUCAAGUAAUUUACACCAGdTdT
2101
CUGGUGUAAAUUACUUGAGdTdT
2212

378
396
GTAATTTACACCAGAAATA
1986
GUAAUUUACACCAGAAAUAdTdT
2102
UAUUUCUGGUGUAAAUUACdTdT
2213

379
397
TAATTTACACCAGAAATAC
1987
UAAUUUACACCAGAAAUACdTdT
2103
GUAUUUCUGGUGUAAAUUAdTdT
2214

384
402
TACACCAGAAATACCAAGG
1988
UACACCAGAAAUACCAAGGdTdT
2104
CCUUGGUAUUUCUGGUGUAdTdT
2215

387
405
ACCAGAAATACCAAGGGTG
1989
ACCAGAAAUACCAAGGGUGdTdT
2105
CACCCUUGGUAUUUCUGGUdTdT
2216

390
408
AGAAATACCAAGGGTGGAG
1990
AGAAAUACCAAGGGUGGAGdTdT
2106
CUCCACCCUUGGUAUUUCUdTdT
2217

393
411
AATACCAAGGGTGGAGATG
1991
AAUACCAAGGGUGGAGAUGdTdT
2107
CAUCUCCACCCUUGGUAUUdTdT
2218

399
417
AAGGGTGGAGATGCTCCAG
1992
AAGGGUGGAGAUGCUCCAGdTdT
2108
CUGGAGCAUCUCCACCCUUdTdT
2219

402
420
GGTGGAGATGCTCCAGCTG
1993
GGUGGAGAUGCUCCAGCUGdTdT
2109
CAGCUGGAGCAUCUCCACCdTdT
2220

404
422
TGGAGATGCTCCAGCTGCT
1994
UGGAGAUGCUCCAGCUGCUdTdT
1201
AGCAGCUGGAGCAUCUCCAdTdT
1611

410
428
TGCTCCAGCTGCTGGTGAA
1995
UGCUCCAGCUGCUGGUGAAdTdT
2110
UUCACCAGCAGCUGGAGCAdTdT
2221

411
429
GCTCCAGCTGCTGGTGAAG
1996
GCUCCAGCUGCUGGUGAAGdTdT
2111
CUUCACCAGCAGCUGGAGCdTdT
2222

417
435
GCTGCTGGTGAAGATGCAT
1997
GCUGCUGGUGAAGAUGCAUdTdT
1214
AUGCAUCUUCACCAGCAGCdTdT
1624

419
437
TGCTGGTGAAGATGCATGA
1998
UGCUGGUGAAGAUGCAUGAdTdT
2112
UCAUGCAUCUUCACCAGCAdTdT
2223

423
441
GGTGAAGATGCATGAATAG
1999
GGUGAAGAUGCAUGAAUAGdTdT
2113
CUAUUCAUGCAUCUUCACCdTdT
2224

426
444
GAAGATGCATGAATAGGTC
2000
GAAGAUGCAUGAAUAGGUCdTdT
2114
GACCUAUUCAUGCAUCUUCdTdT
2225

430
448
ATGCATGAATAGGTCCAAC
2001
AUGCAUGAAUAGGUCCAACdTdT
2115
GUUGGACCUAUUCAUGCAUdTdT
2226

432
450
GCATGAATAGGTCCAACCA
2002
GCAUGAAUAGGUCCAACCAdTdT
2116
UGGUUGGACCUAUUCAUGCdTdT
2227

435
453
TGAATAGGTCCAACCAGCT
2003
UGAAUAGGUCCAACCAGCUdTdT
1232
AGCUGGUUGGACCUAUUCAdTdT
1642

441
459
GGTCCAACCAGCTGTACAT
2004
GGUCCAACCAGCUGUACAUdTdT
1238
AUGUACAGCUGGUUGGACCdTdT
1648

444
462
CCAACCAGCTGTACATTTG
2005
CCAACCAGCUGUACAUUUGdTdT
2117
CAAAUGUACAGCUGGUUGGdTdT
2228

448
466
CCAGCTGTACATTTGGAAA
2006
CCAGCUGUACAUUUGGAAAdTdT
2118
UUUCCAAAUGUACAGCUGGdTdT
2229

451
469
GCTGTACATTTGGAAAAAT
2007
GCUGUACAUUUGGAAAAAUdTdT
1248
AUUUUUCCAAAUGUACAGCdTdT
1658

454
472
GTACATTTGGAAAAATAAA
2008
GUACAUUUGGAAAAAUAAAdTdT
2119
UUUAUUUUUCCAAAUGUACdTdT
2230

456
474
ACATTTGGAAAAATAAAAC
2009
ACAUUUGGAAAAAUAAAACdTdT
2120
GUUUUAUUUUUCCAAAUGUdTdT
2231

462
480
GGAAAAATAAAACTTTATT
2010
GGAAAAAUAAAACUUUAUUdTdT
2121
AAUAAAGUUUUAUUUUUCCdTdT
2232

465
483
AAAATAAAACTTTATTAAA
2011
AAAAUAAAACUUUAUUAAAdTdT
2122
UUUAAUAAAGUUUUAUUUUdTdT
2233

TABLE 6

RPS25 Unmodified duplex Sequences

Start
End

Site in
Site in
Sense
SEQ
Antisense
SEQ

SEQ

NM_
NM_
Oligo Sequence
ID
Oligo Sequence
ID
Target Sequence
ID

001028.3
00128.3
5’ to 3’
NO:
5’ to 3’
NO:
5’ to 3’
NO:

245
263
UCCCAACUAUAAACUUAUA
1722
UAUAAGUUUAUAGUUGGGA
1833
TCCCAACTATAAACTTATA
1948

246
264
CCCAACUAUAAACUUAUAA
2234
UUAUAAGUUUAUAGUUGGG
2287
CCCAACTATAAACTTATAA
2340

188
206
GCUCAAUAACUUAGUCUUG
1710
CAAGACUAAGUUAUUGAGC
1821
GCTCAATAACTTAGTCTTG
1931

343
361
GACUUAUCAAACUGGUUUC
1744
GAAACCAGUUUGAUAAGUC
1855
GACTTATCAAACTGGTTTC
1976

244
262
UUCCCAACUAUAAACUUAU
246
AUAAGUUUAUAGUUGGGAA
656
TTCCCAACTATAAACTTAT
2341

189
207
CUCAAUAACUUAGUCUUGU
191
ACAAGACUAAGUUAUUGAG
601
CTCAATAACTTAGTCTTGT
1932

247
265
CCAACUAUAAACUUAUAAC
2235
GUUAUAAGUUUAUAGUUGG
2288
CCAACTATAAACTTATAAC
2342

182
200
GGACAAGCUCAAUAACUUA
1709
UAAGUUAUUGAGCUUGUCC
1820
GGACAAGCTCAATAACTTA
1930

181
199
GGGACAAGCUCAAUAACUU
183
AAGUUAUUGAGCUUGUCCC
593
GGGACAAGCTCAATAACTT
1929

248
266
CAACUAUAAACUUAUAACC
1723
GGUUAUAAGUUUAUAGUUG
1834
CAACTATAAACTTATAACC
1949

243
261
GUUCCCAACUAUAAACUUA
1721
UAAGUUUAUAGUUGGGAAC
1832
GTTCCCAACTATAAACTTA
1947

187
205
AGCUCAAUAACUUAGUCUU
189
AAGACUAAGUUAUUGAGCU
599
AGCTCAATAACTTAGTCTT
2343

368
386
CAGAGCUCAAGUAAUUUAC
1750
GUAAAUUACUUGAGCUCUG
1861
CAGAGCTCAAGTAATTTAC
1983

344
362
ACUUAUCAAACUGGUUUCA
2236
UGAAACCAGUUUGAUAAGU
2289
ACTTATCAAACTGGTTTCA
2344

330
348
CUCCUUAGUAAAGGACUUA
1741
UAAGUCCUUUACUAAGGAG
1852
CTCCTTAGTAAAGGACTTA
1972

342
360
GGACUUAUCAAACUGGUUU
319
AAACCAGUUUGAUAAGUCC
729
GGACTTATCAAACTGGTTT
2345

345
363
CUUAUCAAACUGGUUUCAA
1745
UUGAAACCAGUUUGAUAAG
1856
CTTATCAAACTGGTTTCAA
1977

369
387
AGAGCUCAAGUAAUUUACA
1751
UGUAAAUUACUUGAGCUCU
1862
AGAGCTCAAGTAATTTACA
1984

454
472
GUACAUUUGGAAAAAUAAA
1770
UUUAUUUUUCCAAAUGUAC
1881
GTACATTTGGAAAAATAAA
2008

378
396
GUAAUUUACACCAGAAAUA
1753
UAUUUCUGGUGUAAAUUAC
1864
GTAATTTACACCAGAAATA
1986

242
260
AGUUCCCAACUAUAAACUU
244
AAGUUUAUAGUUGGGAACU
654
AGTTCCCAACTATAAACTT
2346

346
364
UUAUCAAACUGGUUUCAAA
2237
UUUGAAACCAGUUUGAUAA
2290
TTATCAAACTGGTTTCAAA
2347

347
365
UAUCAAACUGGUUUCAAAG
2238
CUUUGAAACCAGUUUGAUA
2291
TATCAAACTGGTTTCAAAG
2348

451
469
GCUGUACAUUUGGAAAAAU
428
AUUUUUCCAAAUGUACAGC
838
GCTGTACATTTGGAAAAAT
2007

333
351
CUUAGUAAAGGACUUAUCA
1742
UGAUAAGUCCUUUACUAAG
1853
CTTAGTAAAGGACTTATCA
1973

377
395
AGUAAUUUACACCAGAAAU
354
AUUUCUGGUGUAAAUUACU
764
AGTAATTTACACCAGAAAT
2349

452
470
CUGUACAUUUGGAAAAAUA
2239
UAUUUUUCCAAAUGUACAG
2292
CTGTACATTTGGAAAAATA
2350

183
201
GACAAGCUCAAUAACUUAG
2240
CUAAGUUAUUGAGCUUGUC
2293
GACAAGCTCAATAACTTAG
2351

239
257
GGAAGUUCCCAACUAUAAA
1720
UUUAUAGUUGGGAACUUCC
1831
GGAAGTTCCCAACTATAAA
1946

372
390
GCUCAAGUAAUUUACACCA
2241
UGGUGUAAAUUACUUGAGC
2294
GCTCAAGTAATTTACACCA
2352

217
235
CUACCUAUGAUAAACUCUG
1715
CAGAGUUUAUCAUAGGUAG
1826
CTACCTATGATAAACTCTG
1940

448
466
CCAGCUGUACAUUUGGAAA
1769
UUUCCAAAUGUACAGCUGG
1880
CCAGCTGTACATTTGGAAA
2006

329
347
GCUCCUUAGUAAAGGACUU
306
AAGUCCUUUACUAAGGAGC
716
GCTCCTTAGTAAAGGACTT
2353

331
349
UCCUUAGUAAAGGACUUAU
308
AUAAGUCCUUUACUAAGGA
718
TCCTTAGTAAAGGACTTAT
2354

31
49
GUGUCUGCUGCUAUUCUCC
1669
GGAGAAUAGCAGCAGACAC
1780
GTGTCTGCTGCTATTCTCC
1894

179
197
UCGGGACAAGCUCAAUAAC
2242
GUUAUUGAGCUUGUCCCGA
2295
TCGGGACAAGCTCAATAAC
2355

6
24
UGUCCGACAUCUUGACGAG
1665
CUCGUCAAGAUGUCGGACA
1776
TGTCCGACATCTTGACGAG
1887

220
238
CCUAUGAUAAACUCUGUAA
1716
UUACAGAGUUUAUCAUAGG
1827
CCTATGATAAACTCTGTAA
1941

376
394
AAGUAAUUUACACCAGAAA
2243
UUUCUGGUGUAAAUUACUU
2296
AAGTAATTTACACCAGAAA
2356

453
471
UGUACAUUUGGAAAAAUAA
2244
UUAUUUUUCCAAAUGUACA
2297
TGTACATTTGGAAAAATAA
2357

332
350
CCUUAGUAAAGGACUUAUC
2245
GAUAAGUCCUUUACUAAGG
2298
CCTTAGTAAAGGACTTATC
2358

449
467
CAGCUGUACAUUUGGAAAA
2246
UUUUCCAAAUGUACAGCUG
2299
CAGCTGTACATTTGGAAAA
2359

278
296
CUCUGAGAGACUGAAGAUU
264
AAUCUUCAGUCUCUCAGAG
674
CTCTGAGAGACTGAAGATT
1957

279
297
UCUGAGAGACUGAAGAUUC
1730
GAAUCUUCAGUCUCUCAGA
1841
TCTGAGAGACTGAAGATTC
1958

276
294
GUCUCUGAGAGACUGAAGA
2247
UCUUCAGUCUCUCAGAGAC
2300
GTCTCTGAGAGACTGAAGA
2360

370
388
GAGCUCAAGUAAUUUACAC
2248
GUGUAAAUUACUUGAGCUC
2301
GAGCTCAAGTAATTTACAC
2361

229
247
AACUCUGUAAGGAAGUUCC
1718
GGAACUUCCUUACAGAGUU
1829
AACTCTGTAAGGAAGTTCC
1943

185
203
CAAGCUCAAUAACUUAGUC
2249
GACUAAGUUAUUGAGCUUG
2302
CAAGCTCAATAACTTAGTC
2362

221
239
CUAUGAUAAACUCUGUAAG
2250
CUUACAGAGUUUAUCAUAG
2303
CTATGATAAACTCTGTAAG
2363

33
51
GUCUGCUGCUAUUCUCCGA
1670
UCGGAGAAUAGCAGCAGAC
1781
GTCTGCTGCTATTCTCCGA
1895

163
181
GGUCCAAAGGCAAAGUUCG
1704
CGAACUUUGCCUUUGGACC
1815
GGTCCAAAGGCAAAGTTCG
1924

373
391
CUCAAGUAAUUUACACCAG
1752
CUGGUGUAAAUUACUUGAG
1863
CTCAAGTAATTTACACCAG
1985

375
393
CAAGUAAUUUACACCAGAA
2251
UUCUGGUGUAAAUUACUUG
2304
CAAGTAATTTACACCAGAA
2364

450
468
AGCUGUACAUUUGGAAAAA
2252
UUUUUCCAAAUGUACAGCU
2305
AGCTGTACATTTGGAAAAA
2365

180
198
CGGGACAAGCUCAAUAACU
182
AGUUAUUGAGCUUGUCCCG
592
CGGGACAAGCTCAATAACT
2366

190
208
UCAAUAACUUAGUCUUGUU
192
AACAAGACUAAGUUAUUGA
602
TCAATAACTTAGTCTTGTT
2367

203
221
CUUGUUUGACAAAGCUACC
1713
GGUAGCUUUGUCAAACAAG
1824
CTTGTTTGACAAAGCTACC
1936

462
480
GGAAAAAUAAAACUUUAUU
1772
AAUAAAGUUUUAUUUUUCC
1883
GGAAAAATAAAACTTTATT
2010

231
249
CUCUGUAAGGAAGUUCCCA
1719
UGGGAACUUCCUUACAGAG
1830
CTCTGTAAGGAAGTTCCCA
1944

30
48
GGUGUCUGCUGCUAUUCUC
2253
GAGAAUAGCAGCAGACACC
2306
GGTGTCTGCTGCTATTCTC
2368

200
218
AGUCUUGUUUGACAAAGCU
202
AGCUUUGUCAAACAAGACU
612
AGTCTTGTTTGACAAAGCT
1935

216
234
GCUACCUAUGAUAAACUCU
218
AGAGUUUAUCAUAGGUAGC
628
GCTACCTATGATAAACTCT
1939

341
359
AGGACUUAUCAAACUGGUU
318
AACCAGUUUGAUAAGUCCU
728
AGGACTTATCAAACTGGTT
2369

218
236
UACCUAUGAUAAACUCUGU
220
ACAGAGUUUAUCAUAGGUA
630
TACCTATGATAAACTCTGT
2370

461
479
UGGAAAAAUAAAACUUUAU
2254
AUAAAGUUUUAUUUUUCCA
2307
TGGAAAAATAAAACTTTAT
2371

162
180
UGGUCCAAAGGCAAAGUUC
2255
GAACUUUGCCUUUGGACCA
2308
TGGTCCAAAGGCAAAGTTC
2372

379
397
UAAUUUACACCAGAAAUAC
1754
GUAUUUCUGGUGUAAAUUA
1865
TAATTTACACCAGAAATAC
1987

280
298
CUGAGAGACUGAAGAUUCG
2256
CGAAUCUUCAGUCUCUCAG
2309
CTGAGAGACTGAAGATTCG
2373

191
209
CAAUAACUUAGUCUUGUUU
193
AAACAAGACUAAGUUAUUG
603
CAATAACTTAGTCTTGTTT
2374

212
230
CAAAGCUACCUAUGAUAAA
1714
UUUAUCAUAGGUAGCUUUG
1825
CAAAGCTACCTATGATAAA
1938

367
385
ACAGAGCUCAAGUAAUUUA
2257
UAAAUUACUUGAGCUCUGU
2310
ACAGAGCTCAAGTAATTTA
2375

230
248
ACUCUGUAAGGAAGUUCCC
2258
GGGAACUUCCUUACAGAGU
2311
ACTCTGTAAGGAAGTTCCC
2376

274
292
UGGUCUCUGAGAGACUGAA
1729
UUCAGUCUCUCAGAGACCA
1840
TGGTCTCTGAGAGACTGAA
1956

366
384
CACAGAGCUCAAGUAAUUU
343
AAAUUACUUGAGCUCUGUG
753
CACAGAGCTCAAGTAATTT
2377

371
389
AGCUCAAGUAAUUUACACC
2259
GGUGUAAAUUACUUGAGCU
2312
AGCTCAAGTAATTTACACC
2378

447
465
ACCAGCUGUACAUUUGGAA
2260
UUCCAAAUGUACAGCUGGU
2313
ACCAGCTGTACATTTGGAA
2379

223
241
AUGAUAAACUCUGUAAGGA
2261
UCCUUACAGAGUUUAUCAU
2314
ATGATAAACTCTGTAAGGA
2380

460
478
UUGGAAAAAUAAAACUUUA
2262
UAAAGUUUUAUUUUUCCAA
2315
TTGGAAAAATAAAACTTTA
2381

184
202
ACAAGCUCAAUAACUUAGU
186
ACUAAGUUAUUGAGCUUGU
596
ACAAGCTCAATAACTTAGT
2382

277
295
UCUCUGAGAGACUGAAGAU
263
AUCUUCAGUCUCUCAGAGA
673
TCTCTGAGAGACTGAAGAT
2383

232
250
UCUGUAAGGAAGUUCCCAA
2263
UUGGGAACUUCCUUACAGA
2316
TCTGTAAGGAAGTTCCCAA
2384

64
82
CGCCUAAGGACGACAAGAA
1678
UUCUUGUCGUCCUUAGGCG
1789
CGCCTAAGGACGACAAGAA
1904

282
300
GAGAGACUGAAGAUUCGAG
1731
CUCGAAUCUUCAGUCUCUC
1842
GAGAGACTGAAGATTCGAG
1959

224
242
UGAUAAACUCUGUAAGGAA
1717
UUCCUUACAGAGUUUAUCA
1828
TGATAAACTCTGTAAGGAA
1942

222
240
UAUGAUAAACUCUGUAAGG
2264
CCUUACAGAGUUUAUCAUA
2317
TATGATAAACTCTGTAAGG
2385

238
256
AGGAAGUUCCCAACUAUAA
2265
UUAUAGUUGGGAACUUCCU
2318
AGGAAGTTCCCAACTATAA
2386

254
272
UAAACUUAUAACCCCAGCU
1724
AGCUGGGGUUAUAAGUUUA
1835
TAAACTTATAACCCCAGCT
1950

275
293
GGUCUCUGAGAGACUGAAG
2266
CUUCAGUCUCUCAGAGACC
2319
GGTCTCTGAGAGACTGAAG
2387

219
237
ACCUAUGAUAAACUCUGUA
2267
UACAGAGUUUAUCAUAGGU
2320
ACCTATGATAAACTCTGTA
2388

186
204
AAGCUCAAUAACUUAGUCU
188
AGACUAAGUUAUUGAGCUU
598
AAGCTCAATAACTTAGTCT
2389

455
473
UACAUUUGGAAAAAUAAAA
2268
UUUUAUUUUUCCAAAUGUA
2321
TACATTTGGAAAAATAAAA
2390

197
215
CUUAGUCUUGUUUGACAAA
1712
UUUGUCAAACAAGACUAAG
1823
CTTAGTCTTGTTTGACAAA
1934

29
47
CGGUGUCUGCUGCUAUUCU
51
AGAAUAGCAGCAGACACCG
461
CGGTGTCTGCTGCTATTCT
1893

456
474
ACAUUUGGAAAAAUAAAAC
1771
GUUUUAUUUUUCCAAAUGU
1882
ACATTTGGAAAAATAAAAC
2009

34
52
UCUGCUGCUAUUCUCCGAG
2269
CUCGGAGAAUAGCAGCAGA
2322
TCTGCTGCTATTCTCCGAG
2391

423
441
GGUGAAGAUGCAUGAAUAG
1764
CUAUUCAUGCAUCUUCACC
1875
GGTGAAGATGCATGAATAG
1999

1
19
CUUUUUGUCCGACAUCUUG
1663
CAAGAUGUCGGACAAAAAG
1774
CTTTTTGTCCGACATCTTG
1885

348
366
AUCAAACUGGUUUCAAAGC
1746
GCUUUGAAACCAGUUUGAU
1857
ATCAAACTGGTTTCAAAGC
1978

240
258
GAAGUUCCCAACUAUAAAC
2270
GUUUAUAGUUGGGAACUUC
2323
GAAGTTCCCAACTATAAAC
2392

255
273
AAACUUAUAACCCCAGCUG
1725
CAGCUGGGGUUAUAAGUUU
1836
AAACTTATAACCCCAGCTG
1951

215
233
AGCUACCUAUGAUAAACUC
2271
GAGUUUAUCAUAGGUAGCU
2324
AGCTACCTATGATAAACTC
2393

382
400
UUUACACCAGAAAUACCAA
2272
UUGGUAUUUCUGGUGUAAA
2325
TTTACACCAGAAATACCAA
2394

353
371
ACUGGUUUCAAAGCACAGA
1747
UCUGUGCUUUGAAACCAGU
1858
ACTGGTTTCAAAGCACAGA
1979

326
344
GGAGCUCCUUAGUAAAGGA
1740
UCCUUUACUAAGGAGCUCC
1851
GGAGCTCCTTAGTAAAGGA
1971

202
220
UCUUGUUUGACAAAGCUAC
2273
GUAGCUUUGUCAAACAAGA
2326
TCTTGTTTGACAAAGCTAC
2395

45
63
UCUCCGAGCUUCGCAAUGC
1672
GCAUUGCGAAGCUCGGAGA
1783
TCTCCGAGCTTCGCAATGC
1898

419
437
UGCUGGUGAAGAUGCAUGA
1763
UCAUGCAUCUUCACCAGCA
1874
TGCTGGTGAAGATGCATGA
1998

178
196
UUCGGGACAAGCUCAAUAA
1708
UUAUUGAGCUUGUCCCGAA
1819
TTCGGGACAAGCTCAATAA
1928

44
62
UUCUCCGAGCUUCGCAAUG
2274
CAUUGCGAAGCUCGGAGAA
2327
TTCTCCGAGCTTCGCAATG
2396

335
353
UAGUAAAGGACUUAUCAAA
1743
UUUGAUAAGUCCUUUACUA
1854
TAGTAAAGGACTTATCAAA
1974

251
269
CUAUAAACUUAUAACCCCA
2275
UGGGGUUAUAAGUUUAUAG
2328
CTATAAACTTATAACCCCA
2397

374
392
UCAAGUAAUUUACACCAGA
2276
UCUGGUGUAAAUUACUUGA
2329
TCAAGTAATTTACACCAGA
2398

151
169
AAAAGAAGAAGUGGUCCAA
1702
UUGGACCACUUCUUCUUUU
1813
AAAAGAAGAAGTGGTCCAA
1921

164
182
GUCCAAAGGCAAAGUUCGG
2277
CCGAACUUUGCCUUUGGAC
2330
GTCCAAAGGCAAAGTTCGG
2399

253
271
AUAAACUUAUAACCCCAGC
2278
GCUGGGGUUAUAAGUUUAU
2331
ATAAACTTATAACCCCAGC
2400

32
50
UGUCUGCUGCUAUUCUCCG
2279
CGGAGAAUAGCAGCAGACA
2332
TGTCTGCTGCTATTCTCCG
2401

146
164
GGCCAAAAAGAAGAAGUGG
1700
CCACUUCUUCUUUUUGGCC
1811
GGCCAAAAAGAAGAAGTGG
1919

323
341
UCAGGAGCUCCUUAGUAAA
1739
UUUACUAAGGAGCUCCUGA
1850
TCAGGAGCTCCTTAGTAAA
1970

358
376
UUUCAAAGCACAGAGCUCA
1748
UGAGCUCUGUGCUUUGAAA
1859
TTTCAAAGCACAGAGCTCA
1980

241
259
AAGUUCCCAACUAUAAACU
243
AGUUUAUAGUUGGGAACUU
653
AAGTTCCCAACTATAAACT
2402

206
224
GUUUGACAAAGCUACCUAU
208
AUAGGUAGCUUUGUCAAAC
618
GTTTGACAAAGCTACCTAT
1937

328
346
AGCUCCUUAGUAAAGGACU
305
AGUCCUUUACUAAGGAGCU
715
AGCTCCTTAGTAAAGGACT
2403

213
231
AAAGCUACCUAUGAUAAAC
2280
GUUUAUCAUAGGUAGCUUU
2333
AAAGCTACCTATGATAAAC
2404

148
166
CCAAAAAGAAGAAGUGGUC
1701
GACCACUUCUUCUUUUUGG
1812
CCAAAAAGAAGAAGTGGTC
1920

37
55
GCUGCUAUUCUCCGAGCUU
59
AAGCUCGGAGAAUAGCAGC
469
GCTGCTATTCTCCGAGCTT
1896

349
367
UCAAACUGGUUUCAAAGCA
2281
UGCUUUGAAACCAGUUUGA
2334
TCAAACTGGTTTCAAAGCA
2405

365
383
GCACAGAGCUCAAGUAAUU
342
AAUUACUUGAGCUCUGUGC
752
GCACAGAGCTCAAGTAATT
1982

350
368
CAAACUGGUUUCAAAGCAC
2282
GUGCUUUGAAACCAGUUUG
2335
CAAACTGGTTTCAAAGCAC
2406

336
354
AGUAAAGGACUUAUCAAAC
2283
GUUUGAUAAGUCCUUUACU
2336
AGTAAAGGACTTATCAAAC
2407

337
355
GUAAAGGACUUAUCAAACU
314
AGUUUGAUAAGUCCUUUAC
724
GTAAAGGACTTATCAAACT
2408

214
232
AAGCUACCUAUGAUAAACU
216
AGUUUAUCAUAGGUAGCUU
626
AAGCTACCTATGATAAACT
2409

354
372
CUGGUUUCAAAGCACAGAG
2284
CUCUGUGCUUUGAAACCAG
2337
CTGGTTTCAAAGCACAGAG
2410

196
214
ACUUAGUCUUGUUUGACAA
2285
UUGUCAAACAAGACUAAGU
2338
ACTTAGTCTTGTTTGACAA
2411

236
254
UAAGGAAGUUCCCAACUAU
238
AUAGUUGGGAACUUCCUUA
648
TAAGGAAGTTCCCAACTAT
1945

357
375
GUUUCAAAGCACAGAGCUC
2286
GAGCUCUGUGCUUUGAAAC
2339
GTTTCAAAGCACAGAGCTC
2412

TABLE 7

RPS25 Modified duplex Sequences

Sense

Antisense

Start
End
Target
SEQ
Oligo
SEQ
Oligo
SEQ

Site in
Site in
Sequence
ID
Sequence
ID
Sequence
ID

NM_001028.3
NM_00128.3
5′ to 3′
NO:
5′ to 3′
NO:
5′ to 3′
NO:

245
263
TCCCAACTATAA
1948
UCCCAACUAUAA
2071
UAUAAGUUUAUA
2182

ACTTATA

ACUUAUAdTdT

GUUGGGAdTdT

246
264
CCCAACTATAAA
2340
CCCAACUAUAAA
2413
UUAUAAGUUUAU
2466

CTTATAA

CUUAUAAdTdT

AGUUGGGdTdT

188
206
GCTCAATAACTT
1931
GCUCAAUAACUU
2059
CAAGACUAAGUU
2170

AGTCTTG

AGUCUUGdTdT

AUUGAGCdTdT

343
361
GACTTATCAAAC
1976
GACUUAUCAAAC
2093
GAAACCAGUUUG
2204

TGGTTTC

UGGUUUCdTdT

AUAAGUCdTdT

244
262
TTCCCAACTATA
2341
UUCCCAACUAUA
1066
AUAAGUUUAUAG
1476

AACTTAT

AACUUAUdTdT

UUGGGAAdTdT

189
207
CTCAATAACTTA
1932
CUCAAUAACUUA
1011
ACAAGACUAAGU
1421

GTCTTGT

GUCUUGUdTdT

UAUUGAGdTdT

247
265
CCAACTATAAAC
2342
CCAACUAUAAAC
2414
GUUAUAAGUUUA
2467

TTATAAC

UUAUAACdTdT

UAGUUGGdTdT

182
200
GGACAAGCTCAA
1930
GGACAAGCUCAA
2058
UAAGUUAUUGAG
2169

TAACTTA

UAACUUAdTdT

CUUGUCCdTdT

181
199
GGGACAAGCTCA
1929
GGGACAAGCUCA
1003
AAGUUAUUGAGC
1413

ATAACTT

AUAACUUdTdT

UUGUCCCdTdT

248
266
CAACTATAAACT
1949
CAACUAUAAACU
2072
GGUUAUAAGUUU
2183

TATAACC

UAUAACCdTdT

AUAGUUGdTdT

243
261
GTTCCCAACTAT
1947
GUUCCCAACUAU
2070
UAAGUUUAUAGU
2181

AAACTTA

AAACUUAdTdT

UGGGAACdTdT

187
205
AGCTCAATAACT
2343
AGCUCAAUAACU
1009
AAGACUAAGUUA
1419

TAGTCTT

UAGUCUUdTdT

UUGAGCUdTdT

368
386
CAGAGCTCAAGT
1983
CAGAGCUCAAGU
2099
GUAAAUUACUUG
2210

AATTTAC

AAUUUACdTdT

AGCUCUGdTdT

344
362
ACTTATCAAACT
2344
ACUUAUCAAACU
2415
UGAAACCAGUUU
2468

GGTTTCA

GGUUUCAdTdT

GAUAAGUdTdT

330
348
CTCCTTAGTAAA
1972
CUCCUUAGUAAA
2090
UAAGUCCUUUAC
2201

GGACTTA

GGACUUAdTdT

UAAGGAGdTdT

342
360
GGACTTATCAAA
2345
GGACUUAUCAAA
1139
AAACCAGUUUGA
1549

CTGGTTT

CUGGUUUdTdT

UAAGUCCdTdT

345
363
CTTATCAAACTG
1977
CUUAUCAAACUG
2094
UUGAAACCAGUU
2205

GTTTCAA

GUUUCAAdTdT

UGAUAAGdTdT

369
387
AGAGCTCAAGTA
1984
AGAGCUCAAGUA
2100
UGUAAAUUACUU
2211

ATTTACA

AUUUACAdTdT

GAGCUCUdTdT

454
472
GTACATTTGGAA
2008
GUACAUUUGGAA
2119
UUUAUUUUUCCA
2230

AAATAAA

AAAUAAAdTdT

AAUGUACdTdT

378
396
GTAATTTACACC
1986
GUAAUUUACACC
2102
UAUUUCUGGUGU
2213

AGAAATA

AGAAAUAdTdT

AAAUUACdTdT

242
260
AGTTCCCAACTA
2346
AGUUCCCAACUA
1064
AAGUUUAUAGUU
1474

TAAACTT

UAAACUUdTdT

GGGAACUdTdT

346
364
TTATCAAACTGG
2347
UUAUCAAACUGG
2416
UUUGAAACCAGU
2469

TTTCAAA

UUUCAAAdTdT

UUGAUAAdTdT

347
365
TATCAAACTGGT
2348
UAUCAAACUGGU
2417
CUUUGAAACCAG
2470

TTCAAAG

UUCAAAGdTdT

UUUGAUAdTdT

451
469
GCTGTACATTTG
2007
GCUGUACAUUUG
1248
AUUUUUCCAAAU
1658

GAAAAAT

GAAAAAUdTdT

GUACAGCdTdT

333
351
CTTAGTAAAGGA
1973
CUUAGUAAAGGA
2091
UGAUAAGUCCUU
2202

CTTATCA

CUUAUCAdTdT

UACUAAGdTdT

377
395
AGTAATTTACAC
2349
AGUAAUUUACAC
1174
AUUUCUGGUGUA
1584

CAGAAAT

CAGAAAUdTdT

AAUUACUdTdT

452
470
CTGTACATTTGG
2350
CUGUACAUUUGG
2418
UAUUUUUCCAAA
2471

AAAAATA

AAAAAUAdTdT

UGUACAGdTdT

183
201
GACAAGCTCAAT
2351
GACAAGCUCAAU
2419
CUAAGUUAUUGA
2472

AACTTAG

AACUUAGdTdT

GCUUGUCdTdT

239
257
GGAAGTTCCCAA
1946
GGAAGUUCCCAA
2069
UUUAUAGUUGGG
2180

CTATAAA

CUAUAAAdTdT

AACUUCCdTdT

372
390
GCTCAAGTAATT
2352
GCUCAAGUAAUU
2420
UGGUGUAAAUUA
2473

TACACCA

UACACCAdTdT

CUUGAGCdTdT

217
235
CTACCTATGATA
1940
CUACCUAUGAUA
2064
CAGAGUUUAUCA
2175

AACTCTG

AACUCUGdTdT

UAGGUAGdTdT

448
466
CCAGCTGTACAT
2006
CCAGCUGUACAU
2118
UUUCCAAAUGUA
2229

TTGGAAA

UUGGAAAdTdT

CAGCUGGdTdT

329
347
GCTCCTTAGTAA
2353
GCUCCUUAGUAA
1126
AAGUCCUUUACU
1536

AGGACTT

AGGACUUdTdT

AAGGAGCdTdT

331
349
TCCTTAGTAAAG
2354
UCCUUAGUAAAG
1128
AUAAGUCCUUUA
1538

GACTTAT

GACUUAUdTdT

CUAAGGAdTdT

31
49
GTGTCTGCTGCT
1894
GUGUCUGCUGCU
2018
GGAGAAUAGCAG
2129

ATTCTCC

AUUCUCCdTdT

CAGACACdTdT

179
197
TCGGGACAAGCT
2355
UCGGGACAAGCU
2421
GUUAUUGAGCUU
2474

CAATAAC

CAAUAACdTdT

GUCCCGAdTdT

6
24
TGTCCGACATCT
1887
UGUCCGACAUCU
2014
CUCGUCAAGAUG
2125

TGACGAG

UGACGAGdTdT

UCGGACAdTdT

220
238
CCTATGATAAAC
1941
CCUAUGAUAAAC
2065
UUACAGAGUUUA
2176

TCTGTAA

UCUGUAAdTdT

UCAUAGGdTdT

376
394
AAGTAATTTACA
2356
AAGUAAUUUACA
2422
UUUCUGGUGUAA
2475

CCAGAAA

CCAGAAAdTdT

AUUACUUdTdT

453
471
TGTACATTTGGA
2357
UGUACAUUUGGA
2423
UUAUUUUUCCAA
2476

AAAATAA

AAAAUAAdTdT

AUGUACAdTdT

332
350
CCTTAGTAAAGG
2358
CCUUAGUAAAGG
2424
GAUAAGUCCUUU
2477

ACTTATC

ACUUAUCdTdT

ACUAAGGdTdT

449
467
CAGCTGTACATT
2359
CAGCUGUACAUU
2425
UUUUCCAAAUGU
2478

TGGAAAA

UGGAAAAdTdT

ACAGCUGdTdT

278
296
CTCTGAGAGACT
1957
CUCUGAGAGACU
1084
AAUCUUCAGUCU
1494

GAAGATT

GAAGAUUdTdT

CUCAGAGdTdT

279
297
TCTGAGAGACTG
1958
UCUGAGAGACUG
2079
GAAUCUUCAGUC
2190

AAGATTC

AAGAUUCdTdT

UCUCAGAdTdT

276
294
GTCTCTGAGAGA
2360
GUCUCUGAGAGA
2426
UCUUCAGUCUCU
2479

CTGAAGA

CUGAAGAdTdT

CAGAGACdTdT

370
388
GAGCTCAAGTAA
2361
GAGCUCAAGUAA
2427
GUGUAAAUUACU
2480

TTTACAC

UUUACACdTdT

UGAGCUCdTdT

229
247
AACTCTGTAAGG
1943
AACUCUGUAAGG
2067
GGAACUUCCUUA
2178

AAGTTCC

AAGUUCCdTdT

CAGAGUUdTdT

185
203
CAAGCTCAATAA
2362
CAAGCUCAAUAA
2428
GACUAAGUUAUU
2481

CTTAGTC

CUUAGUCdTdT

GAGCUUGdTdT

221
239
CTATGATAAACT
2363
CUAUGAUAAACU
2429
CUUACAGAGUUU
2482

CTGTAAG

CUGUAAGdTdT

AUCAUAGdTdT

33
51
GTCTGCTGCTAT
1895
GUCUGCUGCUAU
2019
UCGGAGAAUAGC
2130

TCTCCGA

UCUCCGAdTdT

AGCAGACdTdT

163
181
GGTCCAAAGGCA
1924
GGUCCAAAGGCA
2053
CGAACUUUGCCU
2164

AAGTTCG

AAGUUCGdTdT

UUGGACCdTdT

373
391
CTCAAGTAATTT
1985
CUCAAGUAAUUU
2101
CUGGUGUAAAUU
2212

ACACCAG

ACACCAGdTdT

ACUUGAGdTdT

375
393
CAAGTAATTTAC
2364
CAAGUAAUUUAC
2430
UUCUGGUGUAAA
2483

ACCAGAA

ACCAGAAdTdT

UUACUUGdTdT

450
468
AGCTGTACATTT
2365
AGCUGUACAUUU
2431
UUUUUCCAAAUG
2484

GGAAAAA

GGAAAAAdTdT

UACAGCUdTdT

180
198
CGGGACAAGCTC
2366
CGGGACAAGCUC
1002
AGUUAUUGAGCU
1412

AATAACT

AAUAACUdTdT

UGUCCCGdTdT

190
208
TCAATAACTTAG
2367
UCAAUAACUUAG
1012
AACAAGACUAAG
1422

TCTTGTT

UCUUGUUdTdT

UUAUUGAdTdT

203
221
CTTGTTTGACAA
1936
CUUGUUUGACAA
2062
GGUAGCUUUGUC
2173

AGCTACC

AGCUACCdTdT

AAACAAGdTdT

462
480
GGAAAAATAAAA
2010
GGAAAAAUAAAA
2121
AAUAAAGUUUUA
2232

CTTTATT

CUUUAUUdTdT

UUUUUCCdTdT

231
249
CTCTGTAAGGAA
1944
CUCUGUAAGGAA
2068
UGGGAACUUCCU
2179

GTTCCCA

GUUCCCAdTdT

UACAGAGdTdT

30
48
GGTGTCTGCTGC
2368
GGUGUCUGCUGC
2432
GAGAAUAGCAGC
2485

TATTCTC

UAUUCUCdTdT

AGACACCdTdT

200
218
AGTCTTGTTTGA
1935
AGUCUUGUUUGA
1022
AGCUUUGUCAAA
1432

CAAAGCT

CAAAGCUdTdT

CAAGACUdTdT

216
234
GCTACCTATGAT
1939
GCUACCUAUGAU
1038
AGAGUUUAUCAU
1448

AAACTCT

AAACUCUdTdT

AGGUAGCdTdT

341
359
AGGACTTATCAA
2369
AGGACUUAUCAA
1138
AACCAGUUUGAU
1548

ACTGGTT

ACUGGUUdTdT

AAGUCCUdTdT

218
236
TACCTATGATAA
2370
UACCUAUGAUAA
1040
ACAGAGUUUAUC
1450

ACTCTGT

ACUCUGUdTdT

AUAGGUAdTdT

461
479
TGGAAAAATAAA
2371
UGGAAAAAUAAA
2433
AUAAAGUUUUAU
2486

ACTTTAT

ACUUUAUdTdT

UUUUCCAdTdT

162
180
TGGTCCAAAGGC
2372
UGGUCCAAAGGC
2434
GAACUUUGCCUU
2487

AAAGTTC

AAAGUUCdTdT

UGGACCAdTdT

379
397
TAATTTACACCA
1987
UAAUUUACACCA
2103
GUAUUUCUGGUG
2214

GAAATAC

GAAAUACdTdT

UAAAUUAdTdT

280
298
CTGAGAGACTGA
2373
CUGAGAGACUGA
2435
CGAAUCUUCAGU
2488

AGATTCG

AGAUUCGdTdT

CUCUCAGdTdT

191
209
CAATAACTTAGT
2374
CAAUAACUUAGU
1013
AAACAAGACUAA
1423

CTTGTTT

CUUGUUUdTdT

GUUAUUGdTdT

212
230
CAAAGCTACCTA
1938
CAAAGCUACCUA
2063
UUUAUCAUAGGU
2174

TGATAAA

UGAUAAAdTdT

AGCUUUGdTdT

367
385
ACAGAGCTCAAG
2375
ACAGAGCUCAAG
2436
UAAAUUACUUGA
2489

TAATTTA

UAAUUUAdTdT

GCUCUGUdTdT

230
248
ACTCTGTAAGGA
2376
ACUCUGUAAGGA
2437
GGGAACUUCCUU
2490

AGTTCCC

AGUUCCCdTdT

ACAGAGUdTdT

274
292
TGGTCTCTGAGA
1956
UGGUCUCUGAGA
2078
UUCAGUCUCUCA
2189

GACTGAA

GACUGAAdTdT

GAGACCAdTdT

366
384
CACAGAGCTCAA
2377
CACAGAGCUCAA
1163
AAAUUACUUGAG
1573

GTAATTT

GUAAUUUdTdT

CUCUGUGdTdT

371
389
AGCTCAAGTAAT
2378
AGCUCAAGUAAU
2438
GGUGUAAAUUAC
2491

TTACACC

UUACACCdTdT

UUGAGCUdTdT

447
465
ACCAGCTGTACA
2379
ACCAGCUGUACA
2439
UUCCAAAUGUAC
2492

TTTGGAA

UUUGGAAdTdT

AGCUGGUdTdT

223
241
ATGATAAACTCT
2380
AUGAUAAACUCU
2440
UCCUUACAGAGU
2493

GTAAGGA

GUAAGGAdTdT

UUAUCAUdTdT

460
478
TTGGAAAAATAA
2381
UUGGAAAAAUAA
2441
UAAAGUUUUAUU
2494

AACTTTA

AACUUUAdTdT

UUUCCAAdTdT

184
202
ACAAGCTCAATA
2382
ACAAGCUCAAUA
1006
ACUAAGUUAUUG
1416

ACTTAGT

ACUUAGUdTdT

AGCUUGUdTdT

277
295
TCTCTGAGAGAC
2383
UCUCUGAGAGAC
1083
AUCUUCAGUCUC
1493

TGAAGAT

UGAAGAUdTdT

UCAGAGAdTdT

232
250
TCTGTAAGGAAG
2384
UCUGUAAGGAAG
2442
UUGGGAACUUCC
2495

TTCCCAA

UUCCCAAdTdT

UUACAGAdTdT

64
82
CGCCTAAGGACG
1904
CGCCUAAGGACG
2027
UUCUUGUCGUCC
2138

ACAAGAA

ACAAGAAdTdT

UUAGGCGdTdT

282
300
GAGAGACTGAAG
1959
GAGAGACUGAAG
2080
CUCGAAUCUUCA
2191

ATTCGAG

AUUCGAGdTdT

GUCUCUCdTdT

224
242
TGATAAACTCTG
1942
UGAUAAACUCUG
2066
UUCCUUACAGAG
2177

TAAGGAA

UAAGGAAdTdT

UUUAUCAdTdT

222
240
TATGATAAACTC
2385
UAUGAUAAACUC
2443
CCUUACAGAGUU
2496

TGTAAGG

UGUAAGGdTdT

UAUCAUAdTdT

238
256
AGGAAGTTCCCA
2386
AGGAAGUUCCCA
2444
UUAUAGUUGGGA
2497

ACTATAA

ACUAUAAdTdT

ACUUCCUdTdT

254
272
TAAACTTATAAC
1950
UAAACUUAUAAC
2073
AGCUGGGGUUAU
2184

CCCAGCT

CCCAGCUdTdT

AAGUUUAdTdT

275
293
GGTCTCTGAGAG
2387
GGUCUCUGAGAG
2445
CUUCAGUCUCUC
2498

ACTGAAG

ACUGAAGdTdT

AGAGACCdTdT

219
237
ACCTATGATAAA
2388
ACCUAUGAUAAA
2446
UACAGAGUUUAU
2499

CTCTGTA

CUCUGUAdTdT

CAUAGGUdTdT

186
204
AAGCTCAATAAC
2389
AAGCUCAAUAAC
1008
AGACUAAGUUAU
1418

TTAGTCT

UUAGUCUdTdT

UGAGCUUdTdT

455
473
TACATTTGGAAA
2390
UACAUUUGGAAA
2447
UUUUAUUUUUCC
2500

AATAAAA

AAUAAAAdTdT

AAAUGUAdTdT

197
215
CTTAGTCTTGTT
1934
CUUAGUCUUGUU
2061
UUUGUCAAACAA
2172

TGACAAA

UGACAAAdTdT

GACUAAGdTdT

29
47
CGGTGTCTGCTG
1893
CGGUGUCUGCUG
871
AGAAUAGCAGCA
1281

CTATTCT

CUAUUCUdTdT

GACACCGdTdT

456
474
ACATTTGGAAAA
2009
AGAUUUGGAAAA
2120
GUUUUAUUUUUC
2231

ATAAAAC

AUAAAACdTdT

CAAAUGUdTdT

34
52
TCTGCTGCTATT
2391
UCUGCUGCUAUU
2448
CUCGGAGAAUAG
2501

CTCCGAG

CUCCGAGdTdT

CAGCAGAdTdT

423
441
GGTGAAGATGCA
1999
GGUGAAGAUGCA
2113
CUAUUCAUGCAU
2224

TGAATAG

UGAAUAGdTdT

CUUCACCdTdT

1
19
CTTTTTGTCCGA
1885
CUUUUUGUCCGA
2012
CAAGAUGUCGGA
2123

CATCTTG

CAUCUUGdTdT

CAAAAAGdTdT

348
366
ATCAAACTGGTT
1978
AUCAAACUGGUU
2095
GCUUUGAAACCA
2206

TCAAAGC

UCAAAGCdTdT

GUUUGAUdTdT

240
258
GAAGTTCCCAAC
2392
GAAGUUCCCAAC
2449
GUUUAUAGUUGG
2502

TATAAAC

UAUAAACdTdT

GAACUUCdTdT

255
273
AAACTTATAACC
1951
AAACUUAUAACC
2074
CAGCUGGGGUUA
2185

CCAGCTG

CCAGCUGdTdT

UAAGUUUdTdT

215
233
AGCTACCTATGA
2393
AGCUACCUAUGA
2450
GAGUUUAUCAUA
2503

TAAACTC

UAAACUCdTdT

GGUAGCUdTdT

382
400
TTTACACCAGAA
2394
UUUACACCAGAA
2451
UUGGUAUUUCUG
2504

ATACCAA

AUACCAAdTdT

GUGUAAAdTdT

353
371
ACTGGTTTCAAA
1979
ACUGGUUUCAAA
2096
UCUGUGCUUUGA
2207

GCACAGA

GCACAGAdTdT

AACCAGUdTdT

326
344
GGAGCTCCTTAG
1971
GGAGCUCCUUAG
2089
UCCUUUACUAAG
2200

TAAAGGA

UAAAGGAdTdT

GAGCUCCdTdT

202
220
TCTTGTTTGACA
2395
UCUUGUUUGACA
2452
GUAGCUUUGUCA
2505

AAGCTAC

AAGCUACdTdT

AACAAGAdTdT

45
63
TCTCCGAGCTTC
1898
UCUCCGAGCUUC
2021
GCAUUGCGAAGC
2132

GCAATGC

GCAAUGCdTdT

UCGGAGAdTdT

419
437
TGCTGGTGAAGA
1998
UGCUGGUGAAGA
2112
UCAUGCAUCUUC
2223

TGCATGA

UGCAUGAdTdT

ACCAGCAdTdT

178
196
TTCGGGACAAGC
1928
UUCGGGACAAGC
2057
UUAUUGAGCUUG
2168

TCAATAA

UCAAUAAdTdT

UCCCGAAdTdT

44
62
TTCTCCGAGCTT
2396
UUCUCCGAGCUU
2453
CAUUGCGAAGCU
2506

CGCAATG

CGCAAUGdTdT

CGGAGAAdTdT

335
353
TAGTAAAGGACT
1974
UAGUAAAGGACU
2092
UUUGAUAAGUCC
2203

TATCAAA

UAUCAAAdTdT

UUUACUAdTdT

251
269
CTATAAACTTAT
2397
CUAUAAACUUAU
2454
UGGGGUUAUAAG
2507

AACCCCA

AACCCCAdTdT

UUUAUAGdTdT

374
392
TCAAGTAATTTA
2398
UCAAGUAAUUUA
2455
UCUGGUGUAAAU
2508

CACCAGA

CACCAGAdTdT

UACUUGAdTdT

151
169
AAAAGAAGAAGT
1921
AAAAGAAGAAGU
2051
UUGGACCACUUC
2162

GGTCCAA

GGUCCAAdTdT

UUCUUUUdTdT

164
182
GTCCAAAGGCAA
2399
GUCCAAAGGCAA
2456
CCGAACUUUGCC
2509

AGTTCGG

AGUUCGGdTdT

UUUGGACdTdT

253
271
ATAAACTTATAA
2400
AUAAACUUAUAA
2457
GCUGGGGUUAUA
2510

CCCCAGC

CCCCAGCdTdT

AGUUUAUdTdT

32
50
TGTCTGCTGCTA
2401
UGUCUGCUGCUA
2458
CGGAGAAUAGCA
2511

TTCTCCG

UUCUCCGdTdT

GCAGACAdTdT

146
164
GGCCAAAAAGAA
1919
GGCCAAAAAGAA
2049
CCACUUCUUCUU
2160

GAAGTGG

GAAGUGGdTdT

UUUGGCCdTdT

323
341
TCAGGAGCTCCT
1970
UCAGGAGCUCCU
2088
UUUACUAAGGAG
2199

TAGTAAA

UAGUAAAdTdT

CUCCUGAdTdT

358
376
TTTCAAAGCACA
1980
UUUCAAAGCACA
2097
UGAGCUCUGUGC
2208

GAGCTCA

GAGCUCAdTdT

UUUGAAAdTdT

241
259
AAGTTCCCAACT
2402
AAGUUCCCAACU
1063
AGUUUAUAGUUG
1473

ATAAACT

AUAAACUdTdT

GGAACUUdTdT

206
224
GTTTGACAAAGC
1937
GUUUGACAAAGC
1028
AUAGGUAGCUUU
1438

TACCTAT

UACCUAUdTdT

GUCAAACdTdT

328
346
AGCTCCTTAGTA
2403
AGCUCCUUAGUA
1125
AGUCCUUUACUA
1535

AAGGACT

AAGGACUdTdT

AGGAGCUdTdT

213
231
AAAGCTACCTAT
2404
AAAGCUACCUAU
2459
GUUUAUCAUAGG
2512

GATAAAC

GAUAAACdTdT

UAGCUUUdTdT

148
166
CCAAAAAGAAGA
1920
CCAAAAAGAAGA
2050
GACCACUUCUUC
2161

AGTGGTC

AGUGGUCdTdT

UUUUUGGdTdT

37
55
GCTGCTATTCTC
1896
GCUGCUAUUCUC
879
AAGCUCGGAGAA
1289

CGAGCTT

CGAGCUUdTdT

UAGCAGCdTdT

349
367
TCAAACTGGTTT
2405
UCAAACUGGUUU
2460
UGCUUUGAAACC
2513

CAAAGCA

CAAAGCAdTdT

AGUUUGAdTdT

365
383
GCACAGAGCTCA
1982
GCACAGAGCUCA
1162
AAUUACUUGAGC
1572

AGTAATT

AGUAAUUdTdT

UCUGUGCdTdT

350
368
CAAACTGGTTTC
2406
CAAACUGGUUUC
2461
GUGCUUUGAAAC
2514

AAAGCAC

AAAGCACdTdT

CAGUUUGdTdT

336
354
AGTAAAGGACTT
2407
AGUAAAGGACUU
2462
GUUUGAUAAGUC
2515

ATCAAAC

AUCAAACdTdT

CUUUACUdTdT

337
355
GTAAAGGACTTA
2408
GUAAAGGACUUA
1134
AGUUUGAUAAGU
1544

TCAAACT

UCAAACUdTdT

CCUUUACdTdT

214
232
AAGCTACCTATG
2409
AAGCUACCUAUG
1036
AGUUUAUCAUAG
1446

ATAAACT

AUAAACUdTdT

GUAGCUUdTdT

354
372
CTGGTTTCAAAG
2410
CUGGUUUCAAAG
2463
CUCUGUGCUUUG
2516

CACAGAG

CACAGAGdTdT

AAACCAGdTdT

196
214
ACTTAGTCTTGT
2411
ACUUAGUCUUGU
2464
UUGUCAAACAAG
2517

TTGACAA

UUGACAAdTdT

ACUAAGUdTdT

236
254
TAAGGAAGTTCC
1945
UAAGGAAGUUCC
1058
AUAGUUGGGAAC
1468

CAACTAT

CAACUAUdTdT

UUCCUUAdTdT

357
375
GTTTCAAAGCAC
2412
GUUUCAAAGCAC
2465
GAGCUCUGUGCU
2518

AGAGCTC

AGAGCUCdTdT

UUGAAACdTdT

TABLE 8

RPS25 Unmodified duplex Sequences

Start
End
Sense
SEQ
Antisense
SEQ
Target
SEQ

Site in
Site in
Oligo Sequence
ID
Oligo Sequence
ID
Sequence
ID

NM_001028.3
NM_00128.3
5′ to 3′
NO:
5′ to 3′
NO:
5′ to 3′
NO:

1
19
CUUUUUGUCCGACAUCUUG
1663
CAAGAUGUCGGACAAAAAG
1774
CTTTTTGTCCGACATCTTG
1885

3
21
UUUUGUCCGACAUCUUGAC
1664
GUCAAGAUGUCGGACAAAA
1775
TTTTGTCCGACATCTTGAC
1886

6
24
UGUCCGACAUCUUGACGAG
1665
CUCGUCAAGAUGUCGGACA
1776
TGTCCGACATCTTGACGAG
1887

9
27
CCGACAUCUUGACGAGGCU
31
AGCCUCGUCAAGAUGUCGG
441
CCGACATCTTGACGAGGCT
1888

12
30
ACAUCUUGACGAGGCUGCG
1666
CGCAGCCUCGUCAAGAUGU
1777
ACATCTTGACGAGGCTGCG
1889

16
34
CUUGACGAGGCUGCGGUGU
38
ACACCGCAGCCUCGUCAAG
448
CTTGACGAGGCTGCGGTGT
1890

22
40
GAGGCUGCGGUGUCUGCUG
1667
CAGCAGACACCGCAGCCUC
1778
GAGGCTGCGGTGTCTGCTG
1891

25
43
GCUGCGGUGUCUGCUGCUA
1668
UAGCAGCAGACACCGCAGC
1779
GCTGCGGTGTCTGCTGCTA
1892

29
47
CGGUGUCUGCUGCUAUUCU
51
AGAAUAGCAGCAGACACCG
461
CGGTGTCTGCTGCTATTCT
1893

31
49
GUGUCUGCUGCUAUUCUCC
1669
GGAGAAUAGCAGCAGACAC
1780
GTGTCTGCTGCTATTCTCC
1894

33
51
GUCUGCUGCUAUUCUCCGA
1670
UCGGAGAAUAGCAGCAGAC
1781
GTCTGCTGCTATTCTCCGA
1895

37
55
GCUGCUAUUCUCCGAGCUU
59
AAGCUCGGAGAAUAGCAGC
469
GCTGCTATTCTCCGAGCTT
1896

42
60
UAUUCUCCGAGCUUCGCAA
1671
UUGCGAAGCUCGGAGAAUA
1782
TATTCTCCGAGCTTCGCAA
1897

45
63
UCUCCGAGCUUCGCAAUGC
1672
GCAUUGCGAAGCUCGGAGA
1783
TCTCCGAGCTTCGCAATGC
1898

48
66
CCGAGCUUCGCAAUGCCGC
1673
GCGGCAUUGCGAAGCUCGG
1784
CCGAGCTTCGCAATGCCGC
1899

53
71
CUUCGCAAUGCCGCCUAAG
1674
CUUAGGCGGCAUUGCGAAG
1785
CTTCGCAATGCCGCCTAAG
1900

54
72
UUCGCAAUGCCGCCUAAGG
1675
CCUUAGGCGGCAUUGCGAA
1786
TTCGCAATGCCGCCTAAGG
1901

60
78
AUGCCGCCUAAGGACGACA
1676
UGUCGUCCUUAGGCGGCAU
1787
ATGCCGCCTAAGGACGACA
1902

62
80
GCCGCCUAAGGACGACAAG
1677
CUUGUCGUCCUUAGGCGGC
1788
GCCGCCTAAGGACGACAAG
1903

64
82
CGCCUAAGGACGACAAGAA
1678
UUCUUGUCGUCCUUAGGCG
1789
CGCCTAAGGACGACAAGAA
1904

70
88
AGGACGACAAGAAGAAGAA
1679
UUCUUCUUCUUGUCGUCCU
1790
AGGACGACAAGAAGAAGAA
2520

71
89
GGACGACAAGAAGAAGAAG
1680
CUUCUUCUUCUUGUCGUCC
1791
GGACGACAAGAAGAAGAAG
2521

76
94
ACAAGAAGAAGAAGGACGC
1681
GCGUCCUUCUUCUUCUUGU
1792
ACAAGAAGAAGAAGGACGC
2522

79
97
AGAAGAAGAAGGACGCUGG
1682
CCAGCGUCCUUCUUCUUCU
1793
AGAAGAAGAAGGACGCTGG
1905

83
101
GAAGAAGGACGCUGGAAAG
1683
CUUUCCAGCGUCCUUCUUC
1794
GAAGAAGGACGCTGGAAAG
1906

85
103
AGAAGGACGCUGGAAAGUC
1684
GACUUUCCAGCGUCCUUCU
1795
AGAAGGACGCTGGAAAGTC
1907

91
109
ACGCUGGAAAGUCGGCCAA
1685
UUGGCCGACUUUCCAGCGU
1796
ACGCTGGAAAGTCGGCCAA
1908

94
112
CUGGAAAGUCGGCCAAGAA
1686
UUCUUGGCCGACUUUCCAG
1797
CTGGAAAGTCGGCCAAGAA
1909

96
114
GGAAAGUCGGCCAAGAAAG
1687
CUUUCUUGGCCGACUUUCC
1798
GGAAAGTCGGCCAAGAAAG
1910

101
119
GUCGGCCAAGAAAGACAAA
1688
UUUGUCUUUCUUGGCCGAC
1799
GTCGGCCAAGAAAGACAAA
1911

103
121
CGGCCAAGAAAGACAAAGA
1689
UCUUUGUCUUUCUUGGCCG
1800
CGGCCAAGAAAGACAAAGA
2523

107
125
CAAGAAAGACAAAGACCCA
1690
UGGGUCUUUGUCUUUCUUG
1801
CAAGAAAGACAAAGACCCA
2524

109
127
AGAAAGACAAAGACCCAGU
130
ACUGGGUCUUUGUCUUUCU
540
AGAAAGACAAAGACCCAGT
1912

115
133
ACAAAGACCCAGUGAACAA
1691
UUGUUCACUGGGUCUUUGU
1802
ACAAAGACCCAGTGAACAA
1913

116
134
CAAAGACCCAGUGAACAAA
1692
UUUGUUCACUGGGUCUUUG
1803
CAAAGACCCAGTGAACAAA
1914

120
138
GACCCAGUGAACAAAUCCG
1693
CGGAUUUGUUCACUGGGUC
1804
GACCCAGTGAACAAATCCG
1915

125
143
AGUGAACAAAUCCGGGGGC
1694
GCCCCCGGAUUUGUUCACU
1805
AGTGAACAAATCCGGGGGC
1916

127
145
UGAACAAAUCCGGGGGCAA
1695
UUGCCCCCGGAUUUGUUCA
1806
TGAACAAATCCGGGGGCAA
1917

130
148
ACAAAUCCGGGGGCAAGGC
1696
GCCUUGCCCCCGGAUUUGU
1807
ACAAATCCGGGGGCAAGGC
1918

136
154
CCGGGGGCAAGGCCAAAAA
1697
UUUUUGGCCUUGCCCCCGG
1808
CCGGGGGCAAGGCCAAAAA
2525

140
158
GGGCAAGGCCAAAAAGAAG
1698
CUUCUUUUUGGCCUUGCCC
1809
GGGCAAGGCCAAAAAGAAG
2526

142
160
GCAAGGCCAAAAAGAAGAA
1699
UUCUUCUUUUUGGCCUUGC
1810
GCAAGGCCAAAAAGAAGAA
2527

146
164
GGCCAAAAAGAAGAAGUGG
1700
CCACUUCUUCUUUUUGGCC
1811
GGCCAAAAAGAAGAAGTGG
1919

148
166
CCAAAAAGAAGAAGUGGUC
1701
GACCACUUCUUCUUUUUGG
1812
CCAAAAAGAAGAAGTGGTC
1920

151
169
AAAAGAAGAAGUGGUCCAA
1702
UUGGACCACUUCUUCUUUU
1813
AAAAGAAGAAGTGGTCCAA
1921

154
172
AGAAGAAGUGGUCCAAAGG
1703
CCUUUGGACCACUUCUUCU
1814
AGAAGAAGTGGTCCAAAGG
1922

160
178
AGUGGUCCAAAGGCAAAGU
162
ACUUUGCCUUUGGACCACU
572
AGTGGTCCAAAGGCAAAGT
1923

163
181
GGUCCAAAGGCAAAGUUCG
1704
CGAACUUUGCCUUUGGACC
1815
GGTCCAAAGGCAAAGTTCG
1924

165
183
UCCAAAGGCAAAGUUCGGG
1705
CCCGAACUUUGCCUUUGGA
1816
TCCAAAGGCAAAGTTCGGG
1925

169
187
AAGGCAAAGUUCGGGACAA
1706
UUGUCCCGAACUUUGCCUU
1817
AAGGCAAAGTTCGGGACAA
1926

173
191
CAAAGUUCGGGACAAGCUC
1707
GAGCUUGUCCCGAACUUUG
1818
CAAAGTTCGGGACAAGCTC
1927

178
196
UUCGGGACAAGCUCAAUAA
1708
UUAUUGAGCUUGUCCCGAA
1819
TTCGGGACAAGCTCAATAA
1928

181
199
GGGACAAGCUCAAUAACUU
183
AAGUUAUUGAGCUUGUCCC
593
GGGACAAGCTCAATAACTT
1929

182
200
GGACAAGCUCAAUAACUUA
1709
UAAGUUAUUGAGCUUGUCC
1820
GGACAAGCTCAATAACTTA
1930

188
206
GCUCAAUAACUUAGUCUUG
1710
CAAGACUAAGUUAUUGAGC
1821
GCTCAATAACTTAGTCTTG
1931

189
207
CUCAAUAACUUAGUCUUGU
191
ACAAGACUAAGUUAUUGAG
601
CTCAATAACTTAGTCTTGT
1932

192
210
AAUAACUUAGUCUUGUUUG
1711
CAAACAAGACUAAGUUAUU
1822
AATAACTTAGTCTTGTTTG
1933

197
215
CUUAGUCUUGUUUGACAAA
1712
UUUGUCAAACAAGACUAAG
1823
CTTAGTCTTGTTTGACAAA
1934

200
218
AGUCUUGUUUGACAAAGCU
202
AGCUUUGUCAAACAAGACU
612
AGTCTTGTTTGACAAAGCT
1935

203
221
CUUGUUUGACAAAGCUACC
1713
GGUAGCUUUGUCAAACAAG
1824
CTTGTTTGACAAAGCTACC
1936

206
224
GUUUGACAAAGCUACCUAU
208
AUAGGUAGCUUUGUCAAAC
618
GTTTGACAAAGCTACCTAT
1937

212
230
CAAAGCUACCUAUGAUAAA
1714
UUUAUCAUAGGUAGCUUUG
1825
CAAAGCTACCTATGATAAA
1938

216
234
GCUACCUAUGAUAAACUCU
218
AGAGUUUAUCAUAGGUAGC
628
GCTACCTATGATAAACTCT
1939

217
235
CUACCUAUGAUAAACUCUG
1715
CAGAGUUUAUCAUAGGUAG
1826
CTACCTATGATAAACTCTG
1940

220
238
CCUAUGAUAAACUCUGUAA
1716
UUACAGAGUUUAUCAUAGG
1827
CCTATGATAAACTCTGTAA
1941

224
242
UGAUAAACUCUGUAAGGAA
1717
UUCCUUACAGAGUUUAUCA
1828
TGATAAACTCTGTAAGGAA
1942

229
247
AACUCUGUAAGGAAGUUCC
1718
GGAACUUCCUUACAGAGUU
1829
AACTCTGTAAGGAAGTTCC
1943

231
249
CUCUGUAAGGAAGUUCCCA
1719
UGGGAACUUCCUUACAGAG
1830
CTCTGTAAGGAAGTTCCCA
1944

236
254
UAAGGAAGUUCCCAACUAU
238
AUAGUUGGGAACUUCCUUA
648
TAAGGAAGTTCCCAACTAT
1945

239
257
GGAAGUUCCCAACUAUAAA
1720
UUUAUAGUUGGGAACUUCC
1831
GGAAGTTCCCAACTATAAA
1946

243
261
GUUCCCAACUAUAAACUUA
1721
UAAGUUUAUAGUUGGGAAC
1832
GTTCCCAACTATAAACTTA
1947

245
263
UCCCAACUAUAAACUUAUA
1722
UAUAAGUUUAUAGUUGGGA
1833
TCCCAACTATAAACTTATA
1948

248
266
CAACUAUAAACUUAUAACC
1723
GGUUAUAAGUUUAUAGUUG
1834
CAACTATAAACTTATAACC
1949

254
272
UAAACUUAUAACCCCAGCU
1724
AGCUGGGGUUAUAAGUUUA
1835
TAAACTTATAACCCCAGCT
1950

255
273
AAACUUAUAACCCCAGCUG
1725
CAGCUGGGGUUAUAAGUUU
1836
AAACTTATAACCCCAGCTG
1951

258
276
CUUAUAACCCCAGCUGUGG
1726
CCACAGCUGGGGUUAUAAG
1837
CTTATAACCCCAGCTGTGG
1952

264
282
ACCCCAGCUGUGGUCUCUG
1727
CAGAGACCACAGCUGGGGU
1838
ACCCCAGCTGTGGTCTCTG
1953

267
285
CCAGCUGUGGUCUCUGAGA
1728
UCUCAGAGACCACAGCUGG
1839
CCAGCTGTGGTCTCTGAGA
1954

271
289
CUGUGGUCUCUGAGAGACU
257
AGUCUCUCAGAGACCACAG
667
CTGTGGTCTCTGAGAGACT
1955

274
292
UGGUCUCUGAGAGACUGAA
1729
UUCAGUCUCUCAGAGACCA
1840
TGGTCTCTGAGAGACTGAA
1956

278
296
CUCUGAGAGACUGAAGAUU
264
AAUCUUCAGUCUCUCAGAG
674
CTCTGAGAGACTGAAGATT
1957

279
297
UCUGAGAGACUGAAGAUUC
1730
GAAUCUUCAGUCUCUCAGA
1841
TCTGAGAGACTGAAGATTC
1958

282
300
GAGAGACUGAAGAUUCGAG
1731
CUCGAAUCUUCAGUCUCUC
1842
GAGAGACTGAAGATTCGAG
1959

287
305
ACUGAAGAUUCGAGGCUCC
1732
GGAGCCUCGAAUCUUCAGU
1843
ACTGAAGATTCGAGGCTCC
1960

289
307
UGAAGAUUCGAGGCUCCCU
275
AGGGAGCCUCGAAUCUUCA
685
TGAAGATTCGAGGCTCCCT
1961

293
311
GAUUCGAGGCUCCCUGGCC
1733
GGCCAGGGAGCCUCGAAUC
1844
GATTCGAGGCTCCCTGGCC
1962

298
316
GAGGCUCCCUGGCCAGGGC
1734
GCCCUGGCCAGGGAGCCUC
1845
GAGGCTCCCTGGCCAGGGC
1963

302
320
CUCCCUGGCCAGGGCAGCC
1735
GGCUGCCCUGGCCAGGGAG
1846
CTCCCTGGCCAGGGCAGCC
1964

306
324
CUGGCCAGGGCAGCCCUUC
1736
GAAGGGCUGCCCUGGCCAG
1847
CTGGCCAGGGCAGCCCTTC
1965

308
326
GGCCAGGGCAGCCCUUCAG
1737
CUGAAGGGCUGCCCUGGCC
1848
GGCCAGGGCAGCCCTTCAG
1966

313
331
GGGCAGCCCUUCAGGAGCU
290
AGCUCCUGAAGGGCUGCCC
700
GGGCAGCCCTTCAGGAGCT
1967

316
334
CAGCCCUUCAGGAGCUCCU
293
AGGAGCUCCUGAAGGGCUG
703
CAGCCCTTCAGGAGCTCCT
1968

318
336
GCCCUUCAGGAGCUCCUUA
1738
UAAGGAGCUCCUGAAGGGC
1849
GCCCTTCAGGAGCTCCTTA
1969

323
341
UCAGGAGCUCCUUAGUAAA
1739
UUUACUAAGGAGCUCCUGA
1850
TCAGGAGCTCCTTAGTAAA
1970

326
344
GGAGCUCCUUAGUAAAGGA
1740
UCCUUUACUAAGGAGCUCC
1851
GGAGCTCCTTAGTAAAGGA
1971

330
348
CUCCUUAGUAAAGGACUUA
1741
UAAGUCCUUUACUAAGGAG
1852
CTCCTTAGTAAAGGACTTA
1972

333
351
CUUAGUAAAGGACUUAUCA
1742
UGAUAAGUCCUUUACUAAG
1853
CTTAGTAAAGGACTTATCA
1973

335
353
UAGUAAAGGACUUAUCAAA
1743
UUUGAUAAGUCCUUUACUA
1854
TAGTAAAGGACTTATCAAA
1974

340
358
AAGGACUUAUCAAACUGGU
317
ACCAGUUUGAUAAGUCCUU
727
AAGGACTTATCAAACTGGT
1975

343
361
GACUUAUCAAACUGGUUUC
1744
GAAACCAGUUUGAUAAGUC
1855
GACTTATCAAACTGGTTTC
1976

345
363
CUUAUCAAACUGGUUUCAA
1745
UUGAAACCAGUUUGAUAAG
1856
CTTATCAAACTGGTTTCAA
1977

348
366
AUCAAACUGGUUUCAAAGC
1746
GCUUUGAAACCAGUUUGAU
1857
ATCAAACTGGTTTCAAAGC
1978

353
371
ACUGGUUUCAAAGCACAGA
1747
UCUGUGCUUUGAAACCAGU
1858
ACTGGTTTCAAAGCACAGA
1979

358
376
UUUCAAAGCACAGAGCUCA
1748
UGAGCUCUGUGCUUUGAAA
1859
TTTCAAAGCACAGAGCTCA
1980

359
377
UUCAAAGCACAGAGCUCAA
1749
UUGAGCUCUGUGCUUUGAA
1860
TTCAAAGCACAGAGCTCAA
1981

365
383
GCACAGAGCUCAAGUAAUU
342
AAUUACUUGAGCUCUGUGC
752
GCACAGAGCTCAAGTAATT
1982

368
386
CAGAGCUCAAGUAAUUUAC
1750
GUAAAUUACUUGAGCUCUG
1861
CAGAGCTCAAGTAATTTAC
1983

369
387
AGAGCUCAAGUAAUUUACA
1751
UGUAAAUUACUUGAGCUCU
1862
AGAGCTCAAGTAATTTACA
1984

373
391
CUCAAGUAAUUUACACCAG
1752
CUGGUGUAAAUUACUUGAG
1863
CTCAAGTAATTTACACCAG
1985

378
396
GUAAUUUACACCAGAAAUA
1753
UAUUUCUGGUGUAAAUUAC
1864
GTAATTTACACCAGAAATA
1986

379
397
UAAUUUACACCAGAAAUAC
1754
GUAUUUCUGGUGUAAAUUA
1865
TAATTTACACCAGAAATAC
1987

384
402
UACACCAGAAAUACCAAGG
1755
CCUUGGUAUUUCUGGUGUA
1866
TACACCAGAAATACCAAGG
1988

387
405
ACCAGAAAUACCAAGGGUG
1756
CACCCUUGGUAUUUCUGGU
1867
ACCAGAAATACCAAGGGTG
1989

390
408
AGAAAUACCAAGGGUGGAG
1757
CUCCACCCUUGGUAUUUCU
1868
AGAAATACCAAGGGTGGAG
1990

393
411
AAUACCAAGGGUGGAGAUG
1758
CAUCUCCACCCUUGGUAUU
1869
AATACCAAGGGTGGAGATG
1991

399
417
AAGGGUGGAGAUGCUCCAG
1759
CUGGAGCAUCUCCACCCUU
1870
AAGGGTGGAGATGCTCCAG
1992

402
420
GGUGGAGAUGCUCCAGCUG
1760
CAGCUGGAGCAUCUCCACC
1871
GGTGGAGATGCTCCAGCTG
1993

404
422
UGGAGAUGCUCCAGCUGCU
381
AGCAGCUGGAGCAUCUCCA
791
TGGAGATGCTCCAGCTGCT
1994

410
428
UGCUCCAGCUGCUGGUGAA
1761
UUCACCAGCAGCUGGAGCA
1872
TGCTCCAGCTGCTGGTGAA
1995

411
429
GCUCCAGCUGCUGGUGAAG
1762
CUUCACCAGCAGCUGGAGC
1873
GCTCCAGCTGCTGGTGAAG
1996

417
435
GCUGCUGGUGAAGAUGCAU
394
AUGCAUCUUCACCAGCAGC
804
GCTGCTGGTGAAGATGCAT
1997

419
437
UGCUGGUGAAGAUGCAUGA
1763
UCAUGCAUCUUCACCAGCA
1874
TGCTGGTGAAGATGCATGA
1998

423
441
GGUGAAGAUGCAUGAAUAG
1764
CUAUUCAUGCAUCUUCACC
1875
GGTGAAGATGCATGAATAG
1999

426
444
GAAGAUGCAUGAAUAGGUC
1765
GACCUAUUCAUGCAUCUUC
1876
GAAGATGCATGAATAGGTC
2000

430
448
AUGCAUGAAUAGGUCCAAC
1766
GUUGGACCUAUUCAUGCAU
1877
ATGCATGAATAGGTCCAAC
2001

432
450
GCAUGAAUAGGUCCAACCA
1767
UGGUUGGACCUAUUCAUGC
1878
GCATGAATAGGTCCAACCA
2002

435
453
UGAAUAGGUCCAACCAGCU
412
AGCUGGUUGGACCUAUUCA
822
TGAATAGGTCCAACCAGCT
2003

441
459
GGUCCAACCAGCUGUACAU
418
AUGUACAGCUGGUUGGACC
828
GGTCCAACCAGCTGTACAT
2004

444
462
CCAACCAGCUGUACAUUUG
1768
CAAAUGUACAGCUGGUUGG
1879
CCAACCAGCTGTACATTTG
2005

448
466
CCAGCUGUACAUUUGGAAA
1769
UUUCCAAAUGUACAGCUGG
1880
CCAGCTGTACATTTGGAAA
2006

451
469
GCUGUACAUUUGGAAAAAU
428
AUUUUUCCAAAUGUACAGC
838
GCTGTACATTTGGAAAAAT
2007

454
472
GUACAUUUGGAAAAAUAAA
1770
UUUAUUUUUCCAAAUGUAC
1881
GTACATTTGGAAAAATAAA
2008

456
474
ACAUUUGGAAAAAUAAAAC
1771
GUUUUAUUUUUCCAAAUGU
1882
ACATTTGGAAAAATAAAAC
2009

462
480
GGAAAAAUAAAACUUUAUU
1772
AAUAAAGUUUUAUUUUUCC
1883
GGAAAAATAAAACTTTATT
2010

465
483
AAAAUAAAACUUUAUUAAA
1773
UUUAAUAAAGUUUUAUUUU
1884
AAAATAAAACTTTATTAAA
2011

TABLE 9

RPS25 Modified duplex Sequences

Start
End

SEQ
Sense
SEQ
Antisense
SEQ

Site in
Site in
Target Sequence
ID
Oligo Sequence
ID
Oligo Sequence
ID

NM_001028.3
NM_00128.3
5′ to 3′
NO:
5′ to 3′
NO:
5′ to 3′
NO:

1
19
CTTTTTGTCCGACATCTTG
1885
CUUUUUGUCCGACAUCUUGdTdT
2012
CAAGAUGUCGGACAAAAAGdTdT
2123

3
21
TTTTGTCCGACATCTTGAC
1886
UUUUGUCCGACAUCUUGACdTdT
2013
GUCAAGAUGUCGGACAAAAdTdT
2124

6
24
TGTCCGACATCTTGACGAG
1887
UGUCCGACAUCUUGACGAGdTdT
2014
CUCGUCAAGAUGUCGGACAdTdT
2125

9
27
CCGACATCTTGACGAGGCT
1888
CCGACAUCUUGACGAGGCUdTdT
851
AGCCUCGUCAAGAUGUCGGdTdT
1261

12
30
ACATCTTGACGAGGCTGCG
1889
ACAUCUUGACGAGGCUGCGdTdT
2015
CGCAGCCUCGUCAAGAUGUdTdT
2126

16
34
CTTGACGAGGCTGCGGTGT
1890
CUUGACGAGGCUGCGGUGUdTdT
858
ACACCGCAGCCUCGUCAAGdTdT
1268

22
40
GAGGCTGCGGTGTCTGCTG
1891
GAGGCUGCGGUGUCUGCUGdTdT
2016
CAGCAGACACCGCAGCCUCdTdT
2127

25
43
GCTGCGGTGTCTGCTGCTA
1892
GCUGCGGUGUCUGCUGCUAdTdT
2017
UAGCAGCAGACACCGCAGCdTdT
2128

29
47
CGGTGTCTGCTGCTATTCT
1893
CGGUGUCUGCUGCUAUUCUdTdT
871
AGAAUAGCAGCAGACACCGdTdT
1281

31
49
GTGTCTGCTGCTATTCTCC
1894
GUGUCUGCUGCUAUUCUCCdTdT
2018
GGAGAAUAGCAGCAGACACdTdT
2129

33
51
GTCTGCTGCTATTCTCCGA
1895
GUCUGCUGCUAUUCUCCGAdTdT
2019
UCGGAGAAUAGCAGCAGACdTdT
2130

37
55
GCTGCTATTCTCCGAGCTT
1896
GCUGCUAUUCUCCGAGCUUdTdT
879
AAGCUCGGAGAAUAGCAGCdTdT
1289

42
60
TATTCTCCGAGCTTCGCAA
1897
UAUUCUCCGAGCUUCGCAAdTdT
2020
UUGCGAAGCUCGGAGAAUAdTdT
2131

45
63
TCTCCGAGCTTCGCAATGC
1898
UCUCCGAGCUUCGCAAUGCdTdT
2021
GCAUUGCGAAGCUCGGAGAdTdT
2132

48
66
CCGAGCTTCGCAATGCCGC
1899
CCGAGCUUCGCAAUGCCGCdTdT
2022
GCGGCAUUGCGAAGCUCGGdTdT
2133

53
71
CTTCGCAATGCCGCCTAAG
1900
CUUCGCAAUGCCGCCUAAGdTdT
2023
CUUAGGCGGCAUUGCGAAGdTdT
2134

54
72
TTCGCAATGCCGCCTAAGG
1901
UUCGCAAUGCCGCCUAAGGdTdT
2024
CCUUAGGCGGCAUUGCGAAdTdT
2135

60
78
ATGCCGCCTAAGGACGACA
1902
AUGCCGCCUAAGGACGACAdTdT
2025
UGUCGUCCUUAGGCGGCAUdTdT
2136

62
80
GCCGCCTAAGGACGACAAG
1903
GCCGCCUAAGGACGACAAGdTdT
2026
CUUGUCGUCCUUAGGCGGCdTdT
2137

64
82
CGCCTAAGGACGACAAGAA
1904
CGCCUAAGGACGACAAGAAdTdT
2027
UUCUUGUCGUCCUUAGGCGdTdT
2138

70
88
AGGACGACAAGAAGAAGAA
2520
AGGACGACAAGAAGAAGAAdTdT
2028
UUCUUCUUCUUGUCGUCCUdTdT
2139

71
89
GGACGACAAGAAGAAGAAG
2521
GGACGACAAGAAGAAGAAGdTdT
2029
CUUCUUCUUCUUGUCGUCCdTdT
2140

76
94
ACAAGAAGAAGAAGGACGC
2522
ACAAGAAGAAGAAGGACGCdTdT
2030
GCGUCCUUCUUCUUCUUGUdTdT
2141

79
97
AGAAGAAGAAGGACGCTGG
1905
AGAAGAAGAAGGACGCUGGdTdT
2031
CCAGCGUCCUUCUUCUUCUdTdT
2142

83
101
GAAGAAGGACGCTGGAAAG
1906
GAAGAAGGACGCUGGAAAGdTdT
2032
CUUUCCAGCGUCCUUCUUCdTdT
2143

85
103
AGAAGGACGCTGGAAAGTC
1907
AGAAGGACGCUGGAAAGUCdTdT
2033
GACUUUCCAGCGUCCUUCUdTdT
2144

91
109
ACGCTGGAAAGTCGGCCAA
1908
ACGCUGGAAAGUCGGCCAAdTdT
2034
UUGGCCGACUUUCCAGCGUdTdT
2145

94
112
CTGGAAAGTCGGCCAAGAA
1909
CUGGAAAGUCGGCCAAGAAdTdT
2035
UUCUUGGCCGACUUUCCAGdTdT
2146

96
114
GGAAAGTCGGCCAAGAAAG
1910
GGAAAGUCGGCCAAGAAAGdTdT
2036
CUUUCUUGGCCGACUUUCCdTdT
2147

101
119
GTCGGCCAAGAAAGACAAA
1911
GUCGGCCAAGAAAGACAAAdTdT
2037
UUUGUCUUUCUUGGCCGACdTdT
2148

103
121
CGGCCAAGAAAGACAAAGA
2523
CGGCCAAGAAAGACAAAGAdTdT
2038
UCUUUGUCUUUCUUGGCCGdTdT
2149

107
125
CAAGAAAGACAAAGACCCA
2524
CAAGAAAGACAAAGACCCAdTdT
2039
UGGGUCUUUGUCUUUCUUGdTdT
2150

109
127
AGAAAGACAAAGACCCAGT
1912
AGAAAGACAAAGACCCAGUdTdT
950
ACUGGGUCUUUGUCUUUCUdTdT
1360

115
133
ACAAAGACCCAGTGAACAA
1913
ACAAAGACCCAGUGAACAAdTdT
2040
UUGUUCACUGGGUCUUUGUdTdT
2151

116
134
CAAAGACCCAGTGAACAAA
1914
CAAAGACCCAGUGAACAAAdTdT
2041
UUUGUUCACUGGGUCUUUGdTdT
2152

120
138
GACCCAGTGAACAAATCCG
1915
GACCCAGUGAACAAAUCCGdTdT
2042
CGGAUUUGUUCACUGGGUCdTdT
2153

125
143
AGTGAACAAATCCGGGGGC
1916
AGUGAACAAAUCCGGGGGCdTdT
2043
GCCCCCGGAUUUGUUCACUdTdT
2154

127
145
TGAACAAATCCGGGGGCAA
1917
UGAACAAAUCCGGGGGCAAdTdT
2044
UUGCCCCCGGAUUUGUUCAdTdT
2155

130
148
ACAAATCCGGGGGCAAGGC
1918
ACAAAUCCGGGGGCAAGGCdTdT
2045
GCCUUGCCCCCGGAUUUGUdTdT
2156

136
154
CCGGGGGCAAGGCCAAAAA
2525
CCGGGGGCAAGGCCAAAAAdTdT
2046
UUUUUGGCCUUGCCCCCGGdTdT
2157

140
158
GGGCAAGGCCAAAAAGAAG
2526
GGGCAAGGCCAAAAAGAAGdTdT
2047
CUUCUUUUUGGCCUUGCCCdTdT
2158

142
160
GCAAGGCCAAAAAGAAGAA
2527
GCAAGGCCAAAAAGAAGAAdTdT
2048
UUCUUCUUUUUGGCCUUGCdTdT
2159

146
164
GGCCAAAAAGAAGAAGTGG
1919
GGCCAAAAAGAAGAAGUGGdTdT
2049
CCACUUCUUCUUUUUGGCCdTdT
2160

148
166
CCAAAAAGAAGAAGTGGTC
1920
CCAAAAAGAAGAAGUGGUCdTdT
2050
GACCACUUCUUCUUUUUGGdTdT
2161

151
169
AAAAGAAGAAGTGGTCCAA
1921
AAAAGAAGAAGUGGUCCAAdTdT
2051
UUGGACCACUUCUUCUUUUdTdT
2162

154
172
AGAAGAAGTGGTCCAAAGG
1922
AGAAGAAGUGGUCCAAAGGdTdT
2052
CCUUUGGACCACUUCUUCUdTdT
2163

160
178
AGTGGTCCAAAGGCAAAGT
1923
AGUGGUCCAAAGGCAAAGUdTdT
982
ACUUUGCCUUUGGACCACUdTdT
1392

163
181
GGTCCAAAGGCAAAGTTCG
1924
GGUCCAAAGGCAAAGUUCGdTdT
2053
CGAACUUUGCCUUUGGACCdTdT
2164

165
183
TCCAAAGGCAAAGTTCGGG
1925
UCCAAAGGCAAAGUUCGGGdTdT
2054
CCCGAACUUUGCCUUUGGAdTdT
2165

169
187
AAGGCAAAGTTCGGGACAA
1926
AAGGCAAAGUUCGGGACAAdTdT
2055
UUGUCCCGAACUUUGCCUUdTdT
2166

173
191
CAAAGTTCGGGACAAGCTC
1927
CAAAGUUCGGGACAAGCUCdTdT
2056
GAGCUUGUCCCGAACUUUGdTdT
2167

178
196
TTCGGGACAAGCTCAATAA
1928
UUCGGGACAAGCUCAAUAAdTdT
2057
UUAUUGAGCUUGUCCCGAAdTdT
2168

181
199
GGGACAAGCTCAATAACTT
1929
GGGACAAGCUCAAUAACUUdTdT
1003
AAGUUAUUGAGCUUGUCCCdTdT
1413

182
200
GGACAAGCTCAATAACTTA
1930
GGACAAGCUCAAUAACUUAdTdT
2058
UAAGUUAUUGAGCUUGUCCdTdT
2169

188
206
GCTCAATAACTTAGTCTTG
1931
GCUCAAUAACUUAGUCUUGdTdT
2059
CAAGACUAAGUUAUUGAGCdTdT
2170

189
207
CTCAATAACTTAGTCTTGT
1932
CUCAAUAACUUAGUCUUGUdTdT
1011
ACAAGACUAAGUUAUUGAGdTdT
1421

192
210
AATAACTTAGTCTTGTTTG
1933
AAUAACUUAGUCUUGUUUGdTdT
2060
CAAACAAGACUAAGUUAUUdTdT
2171

197
215
CTTAGTCTTGTTTGACAAA
1934
CUUAGUCUUGUUUGACAAAdTdT
2061
UUUGUCAAACAAGACUAAGdTdT
2172

200
218
AGTCTTGTTTGACAAAGCT
1935
AGUCUUGUUUGACAAAGCUdTdT
1022
AGCUUUGUCAAACAAGACUdTdT
1432

203
221
CTTGTTTGACAAAGCTACC
1936
CUUGUUUGACAAAGCUACCdTdT
2062
GGUAGCUUUGUCAAACAAGdTdT
2173

206
224
GTTTGACAAAGCTACCTAT
1937
GUUUGACAAAGCUACCUAUdTdT
1028
AUAGGUAGCUUUGUCAAACdTdT
1438

212
230
CAAAGCTACCTATGATAAA
1938
CAAAGCUACCUAUGAUAAAdTdT
2063
UUUAUCAUAGGUAGCUUUGdTdT
2174

216
234
GCTACCTATGATAAACTCT
1939
GCUACCUAUGAUAAACUCUdTdT
1038
AGAGUUUAUCAUAGGUAGCdTdT
1448

217
235
CTACCTATGATAAACTCTG
1940
CUACCUAUGAUAAACUCUGdTdT
2064
CAGAGUUUAUCAUAGGUAGdTdT
2175

220
238
CCTATGATAAACTCTGTAA
1941
CCUAUGAUAAACUCUGUAAdTdT
2065
UUACAGAGUUUAUCAUAGGdTdT
2176

224
242
TGATAAACTCTGTAAGGAA
1942
UGAUAAACUCUGUAAGGAAdTdT
2066
UUCCUUACAGAGUUUAUCAdTdT
2177

229
247
AACTCTGTAAGGAAGTTCC
1943
AACUCUGUAAGGAAGUUCCdTdT
2067
GGAACUUCCUUACAGAGUUdTdT
2178

231
249
CTCTGTAAGGAAGTTCCCA
1944
CUCUGUAAGGAAGUUCCCAdTdT
2068
UGGGAACUUCCUUACAGAGdTdT
2179

236
254
TAAGGAAGTTCCCAACTAT
1945
UAAGGAAGUUCCCAACUAUdTdT
1058
AUAGUUGGGAACUUCCUUAdTdT
1468

239
257
GGAAGTTCCCAACTATAAA
1946
GGAAGUUCCCAACUAUAAAdTdT
2069
UUUAUAGUUGGGAACUUCCdTdT
2180

243
261
GTTCCCAACTATAAACTTA
1947
GUUCCCAACUAUAAACUUAdTdT
2070
UAAGUUUAUAGUUGGGAACdTdT
2181

245
263
TCCCAACTATAAACTTATA
1948
UCCCAACUAUAAACUUAUAdTdT
2071
UAUAAGUUUAUAGUUGGGAdTdT
2182

248
266
CAACTATAAACTTATAACC
1949
CAACUAUAAACUUAUAACCdTdT
2072
GGUUAUAAGUUUAUAGUUGdTdT
2183

254
272
TAAACTTATAACCCCAGCT
1950
UAAACUUAUAACCCCAGCUdTdT
2073
AGCUGGGGUUAUAAGUUUAdTdT
2184

255
273
AAACTTATAACCCCAGCTG
1951
AAACUUAUAACCCCAGCUGdTdT
2074
CAGCUGGGGUUAUAAGUUUdTdT
2185

258
276
CTTATAACCCCAGCTGTGG
1952
CUUAUAACCCCAGCUGUGGdTdT
2075
CCACAGCUGGGGUUAUAAGdTdT
2186

264
282
ACCCCAGCTGTGGTCTCTG
1953
ACCCCAGCUGUGGUCUCUGdTdT
2076
CAGAGACCACAGCUGGGGUdTdT
2187

267
285
CCAGCTGTGGTCTCTGAGA
1954
CCAGCUGUGGUCUCUGAGAdTdT
2077
UCUCAGAGACCACAGCUGGdTdT
2188

271
289
CTGTGGTCTCTGAGAGACT
1955
CUGUGGUCUCUGAGAGACUdTdT
1077
AGUCUCUCAGAGACCACAGdTdT
1487

274
292
TGGTCTCTGAGAGACTGAA
1956
UGGUCUCUGAGAGACUGAAdTdT
2078
UUCAGUCUCUCAGAGACCAdTdT
2189

278
296
CTCTGAGAGACTGAAGATT
1957
CUCUGAGAGACUGAAGAUUdTdT
1084
AAUCUUCAGUCUCUCAGAGdTdT
1494

279
297
TCTGAGAGACTGAAGATTC
1958
UCUGAGAGACUGAAGAUUCdTdT
2079
GAAUCUUCAGUCUCUCAGAdTdT
2190

282
300
GAGAGACTGAAGATTCGAG
1959
GAGAGACUGAAGAUUCGAGdTdT
2080
CUCGAAUCUUCAGUCUCUCdTdT
2191

287
305
ACTGAAGATTCGAGGCTCC
1960
ACUGAAGAUUCGAGGCUCCdTdT
2081
GGAGCCUCGAAUCUUCAGUdTdT
2192

289
307
TGAAGATTCGAGGCTCCCT
1961
UGAAGAUUCGAGGCUCCCUdTdT
1095
AGGGAGCCUCGAAUCUUCAdTdT
1505

293
311
GATTCGAGGCTCCCTGGCC
1962
GAUUCGAGGCUCCCUGGCCdTdT
2082
GGCCAGGGAGCCUCGAAUCdTdT
2193

298
316
GAGGCTCCCTGGCCAGGGC
1963
GAGGCUCCCUGGCCAGGGCdTdT
2083
GCCCUGGCCAGGGAGCCUCdTdT
2194

302
320
CTCCCTGGCCAGGGCAGCC
1964
CUCCCUGGCCAGGGCAGCCdTdT
2084
GGCUGCCCUGGCCAGGGAGdTdT
2195

306
324
CTGGCCAGGGCAGCCCTTC
1965
CUGGCCAGGGCAGCCCUUCdTdT
2085
GAAGGGCUGCCCUGGCCAGdTdT
2196

308
326
GGCCAGGGCAGCCCTTCAG
1966
GGCCAGGGCAGCCCUUCAGdTdT
2086
CUGAAGGGCUGCCCUGGCCdTdT
2197

313
331
GGGCAGCCCTTCAGGAGCT
1967
GGGCAGCCCUUCAGGAGCUdTdT
1110
AGCUCCUGAAGGGCUGCCCdTdT
1520

316
334
CAGCCCTTCAGGAGCTCCT
1968
CAGCCCUUCAGGAGCUCCUdTdT
1113
AGGAGCUCCUGAAGGGCUGdTdT
1523

318
336
GCCCTTCAGGAGCTCCTTA
1969
GCCCUUCAGGAGCUCCUUAdTdT
2087
UAAGGAGCUCCUGAAGGGCdTdT
2198

323
341
TCAGGAGCTCCTTAGTAAA
1970
UCAGGAGCUCCUUAGUAAAdTdT
2088
UUUACUAAGGAGCUCCUGAdTdT
2199

326
344
GGAGCTCCTTAGTAAAGGA
1971
GGAGCUCCUUAGUAAAGGAdTdT
2089
UCCUUUACUAAGGAGCUCCdTdT
2200

330
348
CTCCTTAGTAAAGGACTTA
1972
CUCCUUAGUAAAGGACUUAdTdT
2090
UAAGUCCUUUACUAAGGAGdTdT
2201

333
351
CTTAGTAAAGGACTTATCA
1973
CUUAGUAAAGGACUUAUCAdTdT
2091
UGAUAAGUCCUUUACUAAGdTdT
2202

335
353
TAGTAAAGGACTTATCAAA
1974
UAGUAAAGGACUUAUCAAAdTdT
2092
UUUGAUAAGUCCUUUACUAdTdT
2203

340
358
AAGGACTTATCAAACTGGT
1975
AAGGACUUAUCAAACUGGUdTdT
1137
ACCAGUUUGAUAAGUCCUUdTdT
1547

343
361
GACTTATCAAACTGGTTTC
1976
GACUUAUCAAACUGGUUUCdTdT
2093
GAAACCAGUUUGAUAAGUCdTdT
2204

345
363
CTTATCAAACTGGTTTCAA
1977
CUUAUCAAACUGGUUUCAAdTdT
2094
UUGAAACCAGUUUGAUAAGdTdT
2205

348
366
ATCAAACTGGTTTCAAAGC
1978
AUCAAACUGGUUUCAAAGCdTdT
2095
GCUUUGAAACCAGUUUGAUdTdT
2206

353
371
ACTGGTTTCAAAGCACAGA
1979
ACUGGUUUCAAAGCACAGAdTdT
2096
UCUGUGCUUUGAAACCAGUdTdT
2207

358
376
TTTCAAAGCACAGAGCTCA
1980
UUUCAAAGCACAGAGCUCAdTdT
2097
UGAGCUCUGUGCUUUGAAAdTdT
2208

359
377
TTCAAAGCACAGAGCTCAA
1981
UUCAAAGCACAGAGCUCAAdTdT
2098
UUGAGCUCUGUGCUUUGAAdTdT
2209

365
383
GCACAGAGCTCAAGTAATT
1982
GCACAGAGCUCAAGUAAUUdTdT
1162
AAUUACUUGAGCUCUGUGCdTdT
1572

368
386
CAGAGCTCAAGTAATTTAC
1983
CAGAGCUCAAGUAAUUUACdTdT
2099
GUAAAUUACUUGAGCUCUGdTdT
2210

369
387
AGAGCTCAAGTAATTTACA
1984
AGAGCUCAAGUAAUUUACAdTdT
2100
UGUAAAUUACUUGAGCUCUdTdT
2211

373
391
CTCAAGTAATTTACACCAG
1985
CUCAAGUAAUUUACACCAGdTdT
2101
CUGGUGUAAAUUACUUGAGdTdT
2212

378
396
GTAATTTACACCAGAAATA
1986
GUAAUUUACACCAGAAAUAdTdT
2102
UAUUUCUGGUGUAAAUUACdTdT
2213

379
397
TAATTTACACCAGAAATAC
1987
UAAUUUACACCAGAAAUACdTdT
2103
GUAUUUCUGGUGUAAAUUAdTdT
2214

384
402
TACACCAGAAATACCAAGG
1988
UACACCAGAAAUACCAAGGdTdT
2104
CCUUGGUAUUUCUGGUGUAdTdT
2215

387
405
ACCAGAAATACCAAGGGTG
1989
ACCAGAAAUACCAAGGGUGdTdT
2105
CACCCUUGGUAUUUCUGGUdTdT
2216

390
408
AGAAATACCAAGGGTGGAG
1990
AGAAAUACCAAGGGUGGAGdTdT
2106
CUCCACCCUUGGUAUUUCUdTdT
2217

393
411
AATACCAAGGGTGGAGATG
1991
AAUACCAAGGGUGGAGAUGdTdT
2107
CAUCUCCACCCUUGGUAUUdTdT
2218

399
417
AAGGGTGGAGATGCTCCAG
1992
AAGGGUGGAGAUGCUCCAGdTdT
2108
CUGGAGCAUCUCCACCCUUdTdT
2219

402
420
GGTGGAGATGCTCCAGCTG
1993
GGUGGAGAUGCUCCAGCUGdTdT
2109
CAGCUGGAGCAUCUCCACCdTdT
2220

404
422
TGGAGATGCTCCAGCTGCT
1994
UGGAGAUGCUCCAGCUGCUdTdT
1201
AGCAGCUGGAGCAUCUCCAdTdT
1611

410
428
TGCTCCAGCTGCTGGTGAA
1995
UGCUCCAGCUGCUGGUGAAdTdT
2110
UUCACCAGCAGCUGGAGCAdTdT
2221

411
429
GCTCCAGCTGCTGGTGAAG
1996
GCUCCAGCUGCUGGUGAAGdTdT
2111
CUUCACCAGCAGCUGGAGCdTdT
2222

417
435
GCTGCTGGTGAAGATGCAT
1997
GCUGCUGGUGAAGAUGCAUdTdT
1214
AUGCAUCUUCACCAGCAGCdTdT
1624

419
437
TGCTGGTGAAGATGCATGA
1998
UGCUGGUGAAGAUGCAUGAdTdT
2112
UCAUGCAUCUUCACCAGCAdTdT
2223

423
441
GGTGAAGATGCATGAATAG
1999
GGUGAAGAUGCAUGAAUAGdTdT
2113
CUAUUCAUGCAUCUUCACCdTdT
2224

426
444
GAAGATGCATGAATAGGTC
2000
GAAGAUGCAUGAAUAGGUCdTdT
2114
GACCUAUUCAUGCAUCUUCdTdT
2225

430
448
ATGCATGAATAGGTCCAAC
2001
AUGCAUGAAUAGGUCCAACdTdT
2115
GUUGGACCUAUUCAUGCAUdTdT
2226

432
450
GCATGAATAGGTCCAACCA
2002
GCAUGAAUAGGUCCAACCAdTdT
2116
UGGUUGGACCUAUUCAUGCdTdT
2227

435
453
TGAATAGGTCCAACCAGCT
2003
UGAAUAGGUCCAACCAGCUdTdT
1232
AGCUGGUUGGACCUAUUCAdTdT
1642

441
459
GGTCCAACCAGCTGTACAT
2004
GGUCCAACCAGCUGUACAUdTdT
1238
AUGUACAGCUGGUUGGACCdTdT
1648

444
462
CCAACCAGCTGTACATTTG
2005
CCAACCAGCUGUACAUUUGdTdT
2117
CAAAUGUACAGCUGGUUGGdTdT
2228

448
466
CCAGCTGTACATTTGGAAA
2006
CCAGCUGUACAUUUGGAAAdTdT
2118
UUUCCAAAUGUACAGCUGGdTdT
2229

451
469
GCTGTACATTTGGAAAAAT
2007
GCUGUACAUUUGGAAAAAUdTdT
1248
AUUUUUCCAAAUGUACAGCdTdT
1658

454
472
GTACATTTGGAAAAATAAA
2008
GUACAUUUGGAAAAAUAAAdTdT
2119
UUUAUUUUUCCAAAUGUACdTdT
2230

456
474
ACATTTGGAAAAATAAAAC
2009
ACAUUUGGAAAAAUAAAACdTdT
2120
GUUUUAUUUUUCCAAAUGUdTdT
2231

462
480
GGAAAAATAAAACTTTATT
2010
GGAAAAAUAAAACUUUAUUdTdT
2121
AAUAAAGUUUUAUUUUUCCdTdT
2232

465
483
AAAATAAAACTTTATTAAA
2011
AAAAUAAAACUUUAUUAAAdTdT
2122
UUUAAUAAAGUUUUAUUUUdTdT
2233

TABLE 10

RPS25 Unmodified duplex Sequences

Start
End
Sense
SEQ
Antisense
SEQ

SEQ

Site in
Site in
Oligo Sequence
ID
Oligo Sequence
ID
Target Sequence
ID

NM_001028.3
NM_00128.3
5′ to 3′
NO:
5′ to 3′
NO:
5′ to 3′
NO:

245
263
UCCCAACUAUAAACUUAUA
1722
UAUAAGUUUAUAGUUGGGA
1833
TCCCAACTATAAACTTATA
1948

246
264
CCCAACUAUAAACUUAUAA
2234
UUAUAAGUUUAUAGUUGGG
2287
CCCAACTATAAACTTATAA
2340

188
206
GCUCAAUAACUUAGUCUUG
1710
CAAGACUAAGUUAUUGAGC
1821
GCTCAATAACTTAGTCTTG
1931

343
361
GACUUAUCAAACUGGUUUC
1744
GAAACCAGUUUGAUAAGUC
1855
GACTTATCAAACTGGTTTC
1976

244
262
UUCCCAACUAUAAACUUAU
246
AUAAGUUUAUAGUUGGGAA
656
TTCCCAACTATAAACTTAT
2341

189
207
CUCAAUAACUUAGUCUUGU
191
ACAAGACUAAGUUAUUGAG
601
CTCAATAACTTAGTCTTGT
1932

247
265
CCAACUAUAAACUUAUAAC
2235
GUUAUAAGUUUAUAGUUGG
2288
CCAACTATAAACTTATAAC
2342

182
200
GGACAAGCUCAAUAACUUA
1709
UAAGUUAUUGAGCUUGUCC
1820
GGACAAGCTCAATAACTTA
1930

181
199
GGGACAAGCUCAAUAACUU
183
AAGUUAUUGAGCUUGUCCC
593
GGGACAAGCTCAATAACTT
1929

248
266
CAACUAUAAACUUAUAACC
1723
GGUUAUAAGUUUAUAGUUG
1834
CAACTATAAACTTATAACC
1949

243
261
GUUCCCAACUAUAAACUUA
1721
UAAGUUUAUAGUUGGGAAC
1832
GTTCCCAACTATAAACTTA
1947

187
205
AGCUCAAUAACUUAGUCUU
189
AAGACUAAGUUAUUGAGCU
599
AGCTCAATAACTTAGTCTT
2343

368
386
CAGAGCUCAAGUAAUUUAC
1750
GUAAAUUACUUGAGCUCUG
1861
CAGAGCTCAAGTAATTTAC
1983

344
362
ACUUAUCAAACUGGUUUCA
2236
UGAAACCAGUUUGAUAAGU
2289
ACTTATCAAACTGGTTTCA
2344

330
348
CUCCUUAGUAAAGGACUUA
1741
UAAGUCCUUUACUAAGGAG
1852
CTCCTTAGTAAAGGACTTA
1972

342
360
GGACUUAUCAAACUGGUUU
319
AAACCAGUUUGAUAAGUCC
729
GGACTTATCAAACTGGTTT
2345

345
363
CUUAUCAAACUGGUUUCAA
1745
UUGAAACCAGUUUGAUAAG
1856
CTTATCAAACTGGTTTCAA
1977

369
387
AGAGCUCAAGUAAUUUACA
1751
UGUAAAUUACUUGAGCUCU
1862
AGAGCTCAAGTAATTTACA
1984

454
472
GUACAUUUGGAAAAAUAAA
1770
UUUAUUUUUCCAAAUGUAC
1881
GTACATTTGGAAAAATAAA
2008

378
396
GUAAUUUACACCAGAAAUA
1753
UAUUUCUGGUGUAAAUUAC
1864
GTAATTTACACCAGAAATA
1986

242
260
AGUUCCCAACUAUAAACUU
244
AAGUUUAUAGUUGGGAACU
654
AGTTCCCAACTATAAACTT
2346

346
364
UUAUCAAACUGGUUUCAAA
2237
UUUGAAACCAGUUUGAUAA
2290
TTATCAAACTGGTTTCAAA
2347

347
365
UAUCAAACUGGUUUCAAAG
2238
CUUUGAAACCAGUUUGAUA
2291
TATCAAACTGGTTTCAAAG
2348

451
469
GCUGUACAUUUGGAAAAAU
428
AUUUUUCCAAAUGUACAGC
838
GCTGTACATTTGGAAAAAT
2007

333
351
CUUAGUAAAGGACUUAUCA
1742
UGAUAAGUCCUUUACUAAG
1853
CTTAGTAAAGGACTTATCA
1973

377
395
AGUAAUUUACACCAGAAAU
354
AUUUCUGGUGUAAAUUACU
764
AGTAATTTACACCAGAAAT
2349

452
470
CUGUACAUUUGGAAAAAUA
2239
UAUUUUUCCAAAUGUACAG
2292
CTGTACATTTGGAAAAATA
2350

183
201
GACAAGCUCAAUAACUUAG
2240
CUAAGUUAUUGAGCUUGUC
2293
GACAAGCTCAATAACTTAG
2351

239
257
GGAAGUUCCCAACUAUAAA
1720
UUUAUAGUUGGGAACUUCC
1831
GGAAGTTCCCAACTATAAA
1946

372
390
GCUCAAGUAAUUUACACCA
2241
UGGUGUAAAUUACUUGAGC
2294
GCTCAAGTAATTTACACCA
2352

217
235
CUACCUAUGAUAAACUCUG
1715
CAGAGUUUAUCAUAGGUAG
1826
CTACCTATGATAAACTCTG
1940

448
466
CCAGCUGUACAUUUGGAAA
1769
UUUCCAAAUGUACAGCUGG
1880
CCAGCTGTACATTTGGAAA
2006

329
347
GCUCCUUAGUAAAGGACUU
306
AAGUCCUUUACUAAGGAGC
716
GCTCCTTAGTAAAGGACTT
2353

331
349
UCCUUAGUAAAGGACUUAU
308
AUAAGUCCUUUACUAAGGA
718
TCCTTAGTAAAGGACTTAT
2354

31
49
GUGUCUGCUGCUAUUCUCC
1669
GGAGAAUAGCAGCAGACAC
1780
GTGTCTGCTGCTATTCTCC
1894

179
197
UCGGGACAAGCUCAAUAAC
2242
GUUAUUGAGCUUGUCCCGA
2295
TCGGGACAAGCTCAATAAC
2355

6
24
UGUCCGACAUCUUGACGAG
1665
CUCGUCAAGAUGUCGGACA
1776
TGTCCGACATCTTGACGAG
1887

220
238
CCUAUGAUAAACUCUGUAA
1716
UUACAGAGUUUAUCAUAGG
1827
CCTATGATAAACTCTGTAA
1941

376
394
AAGUAAUUUACACCAGAAA
2243
UUUCUGGUGUAAAUUACUU
2296
AAGTAATTTACACCAGAAA
2356

453
471
UGUACAUUUGGAAAAAUAA
2244
UUAUUUUUCCAAAUGUACA
2297
TGTACATTTGGAAAAATAA
2357

332
350
CCUUAGUAAAGGACUUAUC
2245
GAUAAGUCCUUUACUAAGG
2298
CCTTAGTAAAGGACTTATC
2358

449
467
CAGCUGUACAUUUGGAAAA
2246
UUUUCCAAAUGUACAGCUG
2299
CAGCTGTACATTTGGAAAA
2359

278
296
CUCUGAGAGACUGAAGAUU
264
AAUCUUCAGUCUCUCAGAG
674
CTCTGAGAGACTGAAGATT
1957

279
297
UCUGAGAGACUGAAGAUUC
1730
GAAUCUUCAGUCUCUCAGA
1841
TCTGAGAGACTGAAGATTC
1958

276
294
GUCUCUGAGAGACUGAAGA
2247
UCUUCAGUCUCUCAGAGAC
2300
GTCTCTGAGAGACTGAAGA
2360

370
388
GAGCUCAAGUAAUUUACAC
2248
GUGUAAAUUACUUGAGCUC
2301
GAGCTCAAGTAATTTACAC
2361

229
247
AACUCUGUAAGGAAGUUCC
1718
GGAACUUCCUUACAGAGUU
1829
AACTCTGTAAGGAAGTTCC
1943

185
203
CAAGCUCAAUAACUUAGUC
2249
GACUAAGUUAUUGAGCUUG
2302
CAAGCTCAATAACTTAGTC
2362

221
239
CUAUGAUAAACUCUGUAAG
2250
CUUACAGAGUUUAUCAUAG
2303
CTATGATAAACTCTGTAAG
2363

33
51
GUCUGCUGCUAUUCUCCGA
1670
UCGGAGAAUAGCAGCAGAC
1781
GTCTGCTGCTATTCTCCGA
1895

163
181
GGUCCAAAGGCAAAGUUCG
1704
CGAACUUUGCCUUUGGACC
1815
GGTCCAAAGGCAAAGTTCG
1924

373
391
CUCAAGUAAUUUACACCAG
1752
CUGGUGUAAAUUACUUGAG
1863
CTCAAGTAATTTACACCAG
1985

375
393
CAAGUAAUUUACACCAGAA
2251
UUCUGGUGUAAAUUACUUG
2304
CAAGTAATTTACACCAGAA
2364

450
468
AGCUGUACAUUUGGAAAAA
2252
UUUUUCCAAAUGUACAGCU
2305
AGCTGTACATTTGGAAAAA
2365

180
198
CGGGACAAGCUCAAUAACU
182
AGUUAUUGAGCUUGUCCCG
592
CGGGACAAGCTCAATAACT
2366

190
208
UCAAUAACUUAGUCUUGUU
192
AACAAGACUAAGUUAUUGA
602
TCAATAACTTAGTCTTGTT
2367

203
221
CUUGUUUGACAAAGCUACC
1713
GGUAGCUUUGUCAAACAAG
1824
CTTGTTTGACAAAGCTACC
1936

462
480
GGAAAAAUAAAACUUUAUU
1772
AAUAAAGUUUUAUUUUUCC
1883
GGAAAAATAAAACTTTATT
2010

231
249
CUCUGUAAGGAAGUUCCCA
1719
UGGGAACUUCCUUACAGAG
1830
CTCTGTAAGGAAGTTCCCA
1944

30
48
GGUGUCUGCUGCUAUUCUC
2253
GAGAAUAGCAGCAGACACC
2306
GGTGTCTGCTGCTATTCTC
2368

200
218
AGUCUUGUUUGACAAAGCU
202
AGCUUUGUCAAACAAGACU
612
AGTCTTGTTTGACAAAGCT
1935

216
234
GCUACCUAUGAUAAACUCU
218
AGAGUUUAUCAUAGGUAGC
628
GCTACCTATGATAAACTCT
1939

341
359
AGGACUUAUCAAACUGGUU
318
AACCAGUUUGAUAAGUCCU
728
AGGACTTATCAAACTGGTT
2369

218
236
UACCUAUGAUAAACUCUGU
220
ACAGAGUUUAUCAUAGGUA
630
TACCTATGATAAACTCTGT
2370

461
479
UGGAAAAAUAAAACUUUAU
2254
AUAAAGUUUUAUUUUUCCA
2307
TGGAAAAATAAAACTTTAT
2371

162
180
UGGUCCAAAGGCAAAGUUC
2255
GAACUUUGCCUUUGGACCA
2308
TGGTCCAAAGGCAAAGTTC
2372

379
397
UAAUUUACACCAGAAAUAC
1754
GUAUUUCUGGUGUAAAUUA
1865
TAATTTACACCAGAAATAC
1987

280
298
CUGAGAGACUGAAGAUUCG
2256
CGAAUCUUCAGUCUCUCAG
2309
CTGAGAGACTGAAGATTCG
2373

191
209
CAAUAACUUAGUCUUGUUU
193
AAACAAGACUAAGUUAUUG
603
CAATAACTTAGTCTTGTTT
2374

212
230
CAAAGCUACCUAUGAUAAA
1714
UUUAUCAUAGGUAGCUUUG
1825
CAAAGCTACCTATGATAAA
1938

367
385
ACAGAGCUCAAGUAAUUUA
2257
UAAAUUACUUGAGCUCUGU
2310
ACAGAGCTCAAGTAATTTA
2375

230
248
ACUCUGUAAGGAAGUUCCC
2258
GGGAACUUCCUUACAGAGU
2311
ACTCTGTAAGGAAGTTCCC
2376

274
292
UGGUCUCUGAGAGACUGAA
1729
UUCAGUCUCUCAGAGACCA
1840
TGGTCTCTGAGAGACTGAA
1956

366
384
CACAGAGCUCAAGUAAUUU
343
AAAUUACUUGAGCUCUGUG
753
CACAGAGCTCAAGTAATTT
2377

371
389
AGCUCAAGUAAUUUACACC
2259
GGUGUAAAUUACUUGAGCU
2312
AGCTCAAGTAATTTACACC
2378

447
465
ACCAGCUGUACAUUUGGAA
2260
UUCCAAAUGUACAGCUGGU
2313
ACCAGCTGTACATTTGGAA
2379

223
241
AUGAUAAACUCUGUAAGGA
2261
UCCUUACAGAGUUUAUCAU
2314
ATGATAAACTCTGTAAGGA
2380

460
478
UUGGAAAAAUAAAACUUUA
2262
UAAAGUUUUAUUUUUCCAA
2315
TTGGAAAAATAAAACTTTA
2381

184
202
ACAAGCUCAAUAACUUAGU
186
ACUAAGUUAUUGAGCUUGU
596
ACAAGCTCAATAACTTAGT
2382

277
295
UCUCUGAGAGACUGAAGAU
263
AUCUUCAGUCUCUCAGAGA
673
TCTCTGAGAGACTGAAGAT
2383

232
250
UCUGUAAGGAAGUUCCCAA
2263
UUGGGAACUUCCUUACAGA
2316
TCTGTAAGGAAGTTCCCAA
2384

64
82
CGCCUAAGGACGACAAGAA
1678
UUCUUGUCGUCCUUAGGCG
1789
CGCCTAAGGACGACAAGAA
1904

282
300
GAGAGACUGAAGAUUCGAG
1731
CUCGAAUCUUCAGUCUCUC
1842
GAGAGACTGAAGATTCGAG
1959

224
242
UGAUAAACUCUGUAAGGAA
1717
UUCCUUACAGAGUUUAUCA
1828
TGATAAACTCTGTAAGGAA
1942

222
240
UAUGAUAAACUCUGUAAGG
2264
CCUUACAGAGUUUAUCAUA
2317
TATGATAAACTCTGTAAGG
2385

238
256
AGGAAGUUCCCAACUAUAA
2265
UUAUAGUUGGGAACUUCCU
2318
AGGAAGTTCCCAACTATAA
2386

254
272
UAAACUUAUAACCCCAGCU
1724
AGCUGGGGUUAUAAGUUUA
1835
TAAACTTATAACCCCAGCT
1950

275
293
GGUCUCUGAGAGACUGAAG
2266
CUUCAGUCUCUCAGAGACC
2319
GGTCTCTGAGAGACTGAAG
2387

219
237
ACCUAUGAUAAACUCUGUA
2267
UACAGAGUUUAUCAUAGGU
2320
ACCTATGATAAACTCTGTA
2388

186
204
AAGCUCAAUAACUUAGUCU
188
AGACUAAGUUAUUGAGCUU
598
AAGCTCAATAACTTAGTCT
2389

455
473
UACAUUUGGAAAAAUAAAA
2268
UUUUAUUUUUCCAAAUGUA
2321
TACATTTGGAAAAATAAAA
2390

197
215
CUUAGUCUUGUUUGACAAA
1712
UUUGUCAAACAAGACUAAG
1823
CTTAGTCTTGTTTGACAAA
1934

29
47
CGGUGUCUGCUGCUAUUCU
51
AGAAUAGCAGCAGACACCG
461
CGGTGTCTGCTGCTATTCT
1893

456
474
ACAUUUGGAAAAAUAAAAC
1771
GUUUUAUUUUUCCAAAUGU
1882
ACATTTGGAAAAATAAAAC
2009

34
52
UCUGCUGCUAUUCUCCGAG
2269
CUCGGAGAAUAGCAGCAGA
2322
TCTGCTGCTATTCTCCGAG
2391

423
441
GGUGAAGAUGCAUGAAUAG
1764
CUAUUCAUGCAUCUUCACC
1875
GGTGAAGATGCATGAATAG
1999

1
19
CUUUUUGUCCGACAUCUUG
1663
CAAGAUGUCGGACAAAAAG
1774
CTTTTTGTCCGACATCTTG
1885

348
366
AUCAAACUGGUUUCAAAGC
1746
GCUUUGAAACCAGUUUGAU
1857
ATCAAACTGGTTTCAAAGC
1978

240
258
GAAGUUCCCAACUAUAAAC
2270
GUUUAUAGUUGGGAACUUC
2323
GAAGTTCCCAACTATAAAC
2392

255
273
AAACUUAUAACCCCAGCUG
1725
CAGCUGGGGUUAUAAGUUU
1836
AAACTTATAACCCCAGCTG
1951

215
233
AGCUACCUAUGAUAAACUC
2271
GAGUUUAUCAUAGGUAGCU
2324
AGCTACCTATGATAAACTC
2393

382
400
UUUACACCAGAAAUACCAA
2272
UUGGUAUUUCUGGUGUAAA
2325
TTTACACCAGAAATACCAA
2394

353
371
ACUGGUUUCAAAGCACAGA
1747
UCUGUGCUUUGAAACCAGU
1858
ACTGGTTTCAAAGCACAGA
1979

326
344
GGAGCUCCUUAGUAAAGGA
1740
UCCUUUACUAAGGAGCUCC
1851
GGAGCTCCTTAGTAAAGGA
1971

202
220
UCUUGUUUGACAAAGCUAC
2273
GUAGCUUUGUCAAACAAGA
2326
TCTTGTTTGACAAAGCTAC
2395

45
63
UCUCCGAGCUUCGCAAUGC
1672
GCAUUGCGAAGCUCGGAGA
1783
TCTCCGAGCTTCGCAATGC
1898

419
437
UGCUGGUGAAGAUGCAUGA
1763
UCAUGCAUCUUCACCAGCA
1874
TGCTGGTGAAGATGCATGA
1998

178
196
UUCGGGACAAGCUCAAUAA
1708
UUAUUGAGCUUGUCCCGAA
1819
TTCGGGACAAGCTCAATAA
1928

44
62
UUCUCCGAGCUUCGCAAUG
2274
CAUUGCGAAGCUCGGAGAA
2327
TTCTCCGAGCTTCGCAATG
2396

335
353
UAGUAAAGGACUUAUCAAA
1743
UUUGAUAAGUCCUUUACUA
1854
TAGTAAAGGACTTATCAAA
1974

251
269
CUAUAAACUUAUAACCCCA
2275
UGGGGUUAUAAGUUUAUAG
2328
CTATAAACTTATAACCCCA
2397

374
392
UCAAGUAAUUUACACCAGA
2276
UCUGGUGUAAAUUACUUGA
2329
TCAAGTAATTTACACCAGA
2398

151
169
AAAAGAAGAAGUGGUCCAA
1702
UUGGACCACUUCUUCUUUU
1813
AAAAGAAGAAGTGGTCCAA
1921

164
182
GUCCAAAGGCAAAGUUCGG
2277
CCGAACUUUGCCUUUGGAC
2330
GTCCAAAGGCAAAGTTCGG
2399

253
271
AUAAACUUAUAACCCCAGC
2278
GCUGGGGUUAUAAGUUUAU
2331
ATAAACTTATAACCCCAGC
2400

32
50
UGUCUGCUGCUAUUCUCCG
2279
CGGAGAAUAGCAGCAGACA
2332
TGTCTGCTGCTATTCTCCG
2401

146
164
GGCCAAAAAGAAGAAGUGG
1700
CCACUUCUUCUUUUUGGCC
1811
GGCCAAAAAGAAGAAGTGG
1919

323
341
UCAGGAGCUCCUUAGUAAA
1739
UUUACUAAGGAGCUCCUGA
1850
TCAGGAGCTCCTTAGTAAA
1970

358
376
UUUCAAAGCACAGAGCUCA
1748
UGAGCUCUGUGCUUUGAAA
1859
TTTCAAAGCACAGAGCTCA
1980

241
259
AAGUUCCCAACUAUAAACU
243
AGUUUAUAGUUGGGAACUU
653
AAGTTCCCAACTATAAACT
2402

206
224
GUUUGACAAAGCUACCUAU
208
AUAGGUAGCUUUGUCAAAC
618
GTTTGACAAAGCTACCTAT
1937

328
346
AGCUCCUUAGUAAAGGACU
305
AGUCCUUUACUAAGGAGCU
715
AGCTCCTTAGTAAAGGACT
2403

213
231
AAAGCUACCUAUGAUAAAC
2280
GUUUAUCAUAGGUAGCUUU
2333
AAAGCTACCTATGATAAAC
2404

148
166
CCAAAAAGAAGAAGUGGUC
1701
GACCACUUCUUCUUUUUGG
1812
CCAAAAAGAAGAAGTGGTC
1920

37
55
GCUGCUAUUCUCCGAGCUU
59
AAGCUCGGAGAAUAGCAGC
469
GCTGCTATTCTCCGAGCTT
1896

349
367
UCAAACUGGUUUCAAAGCA
2281
UGCUUUGAAACCAGUUUGA
2334
TCAAACTGGTTTCAAAGCA
2405

365
383
GCACAGAGCUCAAGUAAUU
342
AAUUACUUGAGCUCUGUGC
752
GCACAGAGCTCAAGTAATT
1982

350
368
CAAACUGGUUUCAAAGCAC
2282
GUGCUUUGAAACCAGUUUG
2335
CAAACTGGTTTCAAAGCAC
2406

336
354
AGUAAAGGACUUAUCAAAC
2283
GUUUGAUAAGUCCUUUACU
2336
AGTAAAGGACTTATCAAAC
2407

337
355
GUAAAGGACUUAUCAAACU
314
AGUUUGAUAAGUCCUUUAC
724
GTAAAGGACTTATCAAACT
2408

214
232
AAGCUACCUAUGAUAAACU
216
AGUUUAUCAUAGGUAGCUU
626
AAGCTACCTATGATAAACT
2409

354
372
CUGGUUUCAAAGCACAGAG
2284
CUCUGUGCUUUGAAACCAG
2337
CTGGTTTCAAAGCACAGAG
2410

196
214
ACUUAGUCUUGUUUGACAA
2285
UUGUCAAACAAGACUAAGU
2338
ACTTAGTCTTGTTTGACAA
2411

236
254
UAAGGAAGUUCCCAACUAU
238
AUAGUUGGGAACUUCCUUA
648
TAAGGAAGTTCCCAACTAT
1945

357
375
GUUUCAAAGCACAGAGCUC
2286
GAGCUCUGUGCUUUGAAAC
2339
GTTTCAAAGCACAGAGCTC
2412

TABLE 11

RPS25 Modified duplex Sequences

Start
End

Site in
Site in

SEQ

SEQ

SEQ

NM_
NM_
Target Sequence
ID
Sense Oligo Sequence
ID
Antisense Oligo Sequence
ID

001028.3
00128.3
5′ to 3′
NO:
5′ to 3′
NO:
5′ to 3′
NO:

245
263
TCCCAACTATAAACTTATA
1948
UCCCAACUAUAAACUUAUAdTdT
2071
UAUAAGUUUAUAGUUGGGAdTdT
2182

246
264
CCCAACTATAAACTTATAA
2340
CCCAACUAUAAACUUAUAAdTdT
2413
UUAUAAGUUUAUAGUUGGGdTdT
2466

188
206
GCTCAATAACTTAGTCTTG
1931
GCUCAAUAACUUAGUCUUGdTdT
2059
CAAGACUAAGUUAUUGAGCdTdT
2170

343
361
GACTTATCAAACTGGTTTC
1976
GACUUAUCAAACUGGUUUCdTdT
2093
GAAACCAGUUUGAUAAGUCdTdT
2204

244
262
TTCCCAACTATAAACTTAT
2341
UUCCCAACUAUAAACUUAUdTdT
1066
AUAAGUUUAUAGUUGGGAAdTdT
1476

189
207
CTCAATAACTTAGTCTTGT
1932
CUCAAUAACUUAGUCUUGUdTdT
1011
ACAAGACUAAGUUAUUGAGdTdT
1421

247
265
CCAACTATAAACTTATAAC
2342
CCAACUAUAAACUUAUAACdTdT
2414
GUUAUAAGUUUAUAGUUGGdTdT
2467

182
200
GGACAAGCTCAATAACTTA
1930
GGACAAGCUCAAUAACUUAdTdT
2058
UAAGUUAUUGAGCUUGUCCdTdT
2169

181
199
GGGACAAGCTCAATAACTT
1929
GGGACAAGCUCAAUAACUUdTdT
1003
AAGUUAUUGAGCUUGUCCCdTdT
1413

248
266
CAACTATAAACTTATAACC
1949
CAACUAUAAACUUAUAACCdTdT
2072
GGUUAUAAGUUUAUAGUUGdTdT
2183

243
261
GTTCCCAACTATAAACTTA
1947
GUUCCCAACUAUAAACUUAdTdT
2070
UAAGUUUAUAGUUGGGAACdTdT
2181

187
205
AGCTCAATAACTTAGTCTT
2343
AGCUCAAUAACUUAGUCUUdTdT
1009
AAGACUAAGUUAUUGAGCUdTdT
1419

368
386
CAGAGCTCAAGTAATTTAC
1983
CAGAGCUCAAGUAAUUUACdTdT
2099
GUAAAUUACUUGAGCUCUGdTdT
2210

344
362
ACTTATCAAACTGGTTTCA
2344
ACUUAUCAAACUGGUUUCAdTdT
2415
UGAAACCAGUUUGAUAAGUdTdT
2468

330
348
CTCCTTAGTAAAGGACTTA
1972
CUCCUUAGUAAAGGACUUAdTdT
2090
UAAGUCCUUUACUAAGGAGdTdT
2201

342
360
GGACTTATCAAACTGGTTT
2345
GGACUUAUCAAACUGGUUUdTdT
1139
AAACCAGUUUGAUAAGUCCdTdT
1549

345
363
CTTATCAAACTGGTTTCAA
1977
CUUAUCAAACUGGUUUCAAdTdT
2094
UUGAAACCAGUUUGAUAAGdTdT
2205

369
387
AGAGCTCAAGTAATTTACA
1984
AGAGCUCAAGUAAUUUACAdTdT
2100
UGUAAAUUACUUGAGCUCUdTdT
2211

454
472
GTACATTTGGAAAAATAAA
2008
GUACAUUUGGAAAAAUAAAdTdT
2119
UUUAUUUUUCCAAAUGUACdTdT
2230

378
396
GTAATTTACACCAGAAATA
1986
GUAAUUUACACCAGAAAUAdTdT
2102
UAUUUCUGGUGUAAAUUACdTdT
2213

242
260
AGTTCCCAACTATAAACTT
2346
AGUUCCCAACUAUAAACUUdTdT
1064
AAGUUUAUAGUUGGGAACUdTdT
1474

346
364
TTATCAAACTGGTTTCAAA
2347
UUAUCAAACUGGUUUCAAAdTdT
2416
UUUGAAACCAGUUUGAUAAdTdT
2469

347
365
TATCAAACTGGTTTCAAAG
2348
UAUCAAACUGGUUUCAAAGdTdT
2417
CUUUGAAACCAGUUUGAUAdTdT
2470

451
469
GCTGTACATTTGGAAAAAT
2007
GCUGUACAUUUGGAAAAAUdTdT
1248
AUUUUUCCAAAUGUACAGCdTdT
1658

333
351
CTTAGTAAAGGACTTATCA
1973
CUUAGUAAAGGACUUAUCAdTdT
2091
UGAUAAGUCCUUUACUAAGdTdT
2202

377
395
AGTAATTTACACCAGAAAT
2349
AGUAAUUUACACCAGAAAUdTdT
1174
AUUUCUGGUGUAAAUUACUdTdT
1584

452
470
CTGTACATTTGGAAAAATA
2350
CUGUACAUUUGGAAAAAUAdTdT
2418
UAUUUUUCCAAAUGUACAGdTdT
2471

183
201
GACAAGCTCAATAACTTAG
2351
GACAAGCUCAAUAACUUAGdTdT
2419
CUAAGUUAUUGAGCUUGUCdTdT
2472

239
257
GGAAGTTCCCAACTATAAA
1946
GGAAGUUCCCAACUAUAAAdTdT
2069
UUUAUAGUUGGGAACUUCCdTdT
2180

372
390
GCTCAAGTAATTTACACCA
2352
GCUCAAGUAAUUUACACCAdTdT
2420
UGGUGUAAAUUACUUGAGCdTdT
2473

217
235
CTACCTATGATAAACTCTG
1940
CUACCUAUGAUAAACUCUGdTdT
2064
CAGAGUUUAUCAUAGGUAGdTdT
2175

448
466
CCAGCTGTACATTTGGAAA
2006
CCAGCUGUACAUUUGGAAAdTdT
2118
UUUCCAAAUGUACAGCUGGdTdT
2229

329
347
GCTCCTTAGTAAAGGACTT
2353
GCUCCUUAGUAAAGGACUUdTdT
1126
AAGUCCUUUACUAAGGAGCdTdT
1536

331
349
TCCTTAGTAAAGGACTTAT
2354
UCCUUAGUAAAGGACUUAUdTdT
1128
AUAAGUCCUUUACUAAGGAdTdT
1538

31
49
GTGTCTGCTGCTATTCTCC
1894
GUGUCUGCUGCUAUUCUCCdTdT
2018
GGAGAAUAGCAGCAGACACdTdT
2129

179
197
TCGGGACAAGCTCAATAAC
2355
UCGGGACAAGCUCAAUAACdTdT
2421
GUUAUUGAGCUUGUCCCGAdTdT
2474

6
24
TGTCCGACATCTTGACGAG
1887
UGUCCGACAUCUUGACGAGdTdT
2014
CUCGUCAAGAUGUCGGACAdTdT
2125

220
238
CCTATGATAAACTCTGTAA
1941
CCUAUGAUAAACUCUGUAAdTdT
2065
UUACAGAGUUUAUCAUAGGdTdT
2176

376
394
AAGTAATTTACACCAGAAA
2356
AAGUAAUUUACACCAGAAAdTdT
2422
UUUCUGGUGUAAAUUACUUdTdT
2475

453
471
TGTACATTTGGAAAAATAA
2357
UGUACAUUUGGAAAAAUAAdTdT
2423
UUAUUUUUCCAAAUGUACAdTdT
2476

332
350
CCTTAGTAAAGGACTTATC
2358
CCUUAGUAAAGGACUUAUCdTdT
2424
GAUAAGUCCUUUACUAAGGdTdT
2477

449
467
CAGCTGTACATTTGGAAAA
2359
CAGCUGUACAUUUGGAAAAdTdT
2425
UUUUCCAAAUGUACAGCUGdTdT
2478

278
296
CTCTGAGAGACTGAAGATT
1957
CUCUGAGAGACUGAAGAUUdTdT
1084
AAUCUUCAGUCUCUCAGAGdTdT
1494

279
297
TCTGAGAGACTGAAGATTC
1958
UCUGAGAGACUGAAGAUUCdTdT
2079
GAAUCUUCAGUCUCUCAGAdTdT
2190

276
294
GTCTCTGAGAGACTGAAGA
2360
GUCUCUGAGAGACUGAAGAdTdT
2426
UCUUCAGUCUCUCAGAGACdTdT
2479

370
388
GAGCTCAAGTAATTTACAC
2361
GAGCUCAAGUAAUUUACACdTdT
2427
GUGUAAAUUACUUGAGCUCdTdT
2480

229
247
AACTCTGTAAGGAAGTTCC
1943
AACUCUGUAAGGAAGUUCCdTdT
2067
GGAACUUCCUUACAGAGUUdTdT
2178

185
203
CAAGCTCAATAACTTAGTC
2362
CAAGCUCAAUAACUUAGUCdTdT
2428
GACUAAGUUAUUGAGCUUGdTdT
2481

221
239
CTATGATAAACTCTGTAAG
2363
CUAUGAUAAACUCUGUAAGdTdT
2429
CUUACAGAGUUUAUCAUAGdTdT
2482

33
51
GTCTGCTGCTATTCTCCGA
1895
GUCUGCUGCUAUUCUCCGAdTdT
2019
UCGGAGAAUAGCAGCAGACdTdT
2130

163
181
GGTCCAAAGGCAAAGTTCG
1924
GGUCCAAAGGCAAAGUUCGdTdT
2053
CGAACUUUGCCUUUGGACCdTdT
2164

373
391
CTCAAGTAATTTACACCAG
1985
CUCAAGUAAUUUACACCAGdTdT
2101
CUGGUGUAAAUUACUUGAGdTdT
2212

375
393
CAAGTAATTTACACCAGAA
2364
CAAGUAAUUUACACCAGAAdTdT
2430
UUCUGGUGUAAAUUACUUGdTdT
2483

450
468
AGCTGTACATTTGGAAAAA
2365
AGCUGUACAUUUGGAAAAAdTdT
2431
UUUUUCCAAAUGUACAGCUdTdT
2484

180
198
CGGGACAAGCTCAATAACT
2366
CGGGACAAGCUCAAUAACUdTdT
1002
AGUUAUUGAGCUUGUCCCGdTdT
1412

190
208
TCAATAACTTAGTCTTGTT
2367
UCAAUAACUUAGUCUUGUUdTdT
1012
AACAAGACUAAGUUAUUGAdTdT
1422

203
221
CTTGTTTGACAAAGCTACC
1936
CUUGUUUGACAAAGCUACCdTdT
2062
GGUAGCUUUGUCAAACAAGdTdT
2173

462
480
GGAAAAATAAAACTTTATT
2010
GGAAAAAUAAAACUUUAUUdTdT
2121
AAUAAAGUUUUAUUUUUCCdTdT
2232

231
249
CTCTGTAAGGAAGTTCCCA
1944
CUCUGUAAGGAAGUUCCCAdTdT
2068
UGGGAACUUCCUUACAGAGdTdT
2179

30
48
GGTGTCTGCTGCTATTCTC
2368
GGUGUCUGCUGCUAUUCUCdTdT
2432
GAGAAUAGCAGCAGACACCdTdT
2485

200
218
AGTCTTGTTTGACAAAGCT
1935
AGUCUUGUUUGACAAAGCUdTdT
1022
AGCUUUGUCAAACAAGACUdTdT
1432

216
234
GCTACCTATGATAAACTCT
1939
GCUACCUAUGAUAAACUCUdTdT
1038
AGAGUUUAUCAUAGGUAGCdTdT
1448

341
359
AGGACTTATCAAACTGGTT
2369
AGGACUUAUCAAACUGGUUdTdT
1138
AACCAGUUUGAUAAGUCCUdTdT
1548

218
236
TACCTATGATAAACTCTGT
2370
UACCUAUGAUAAACUCUGUdTdT
1040
ACAGAGUUUAUCAUAGGUAdTdT
1450

461
479
TGGAAAAATAAAACTTTAT
2371
UGGAAAAAUAAAACUUUAUdTdT
2433
AUAAAGUUUUAUUUUUCCAdTdT
2486

162
180
TGGTCCAAAGGCAAAGTTC
2372
UGGUCCAAAGGCAAAGUUCdTdT
2434
GAACUUUGCCUUUGGACCAdTdT
2487

379
397
TAATTTACACCAGAAATAC
1987
UAAUUUACACCAGAAAUACdTdT
2103
GUAUUUCUGGUGUAAAUUAdTdT
2214

280
298
CTGAGAGACTGAAGATTCG
2373
CUGAGAGACUGAAGAUUCGdTdT
2435
CGAAUCUUCAGUCUCUCAGdTdT
2488

191
209
CAATAACTTAGTCTTGTTT
2374
CAAUAACUUAGUCUUGUUUdTdT
1013
AAACAAGACUAAGUUAUUGdTdT
1423

212
230
CAAAGCTACCTATGATAAA
1938
CAAAGCUACCUAUGAUAAAdTdT
2063
UUUAUCAUAGGUAGCUUUGdTdT
2174

367
385
ACAGAGCTCAAGTAATTTA
2375
ACAGAGCUCAAGUAAUUUAdTdT
2436
UAAAUUACUUGAGCUCUGUdTdT
2489

230
248
ACTCTGTAAGGAAGTTCCC
2376
ACUCUGUAAGGAAGUUCCCdTdT
2437
GGGAACUUCCUUACAGAGUdTdT
2490

274
292
TGGTCTCTGAGAGACTGAA
1956
UGGUCUCUGAGAGACUGAAdTdT
2078
UUCAGUCUCUCAGAGACCAdTdT
2189

366
384
CACAGAGCTCAAGTAATTT
2377
CACAGAGCUCAAGUAAUUUdTdT
1163
AAAUUACUUGAGCUCUGUGdTdT
1573

371
389
AGCTCAAGTAATTTACACC
2378
AGCUCAAGUAAUUUACACCdTdT
2438
GGUGUAAAUUACUUGAGCUdTdT
2491

447
465
ACCAGCTGTACATTTGGAA
2379
ACCAGCUGUACAUUUGGAAdTdT
2439
UUCCAAAUGUACAGCUGGUdTdT
2492

223
241
ATGATAAACTCTGTAAGGA
2380
AUGAUAAACUCUGUAAGGAdTdT
2440
UCCUUACAGAGUUUAUCAUdTdT
2493

460
478
TTGGAAAAATAAAACTTTA
2381
UUGGAAAAAUAAAACUUUAdTdT
2441
UAAAGUUUUAUUUUUCCAAdTdT
2494

184
202
ACAAGCTCAATAACTTAGT
2382
AGAAGCUCAAUAACUUAGUdTdT
1006
ACUAAGUUAUUGAGCUUGUdTdT
1416

277
295
TCTCTGAGAGACTGAAGAT
2383
UCUCUGAGAGACUGAAGAUdTdT
1083
AUCUUCAGUCUCUCAGAGAdTdT
1493

232
250
TCTGTAAGGAAGTTCCCAA
2384
UCUGUAAGGAAGUUCCCAAdTdT
2442
UUGGGAACUUCCUUACAGAdTdT
2495

64
82
CGCCTAAGGACGACAAGAA
1904
CGCCUAAGGACGACAAGAAdTdT
2027
UUCUUGUCGUCCUUAGGCGdTdT
2138

282
300
GAGAGACTGAAGATTCGAG
1959
GAGAGACUGAAGAUUCGAGdTdT
2080
CUCGAAUCUUCAGUCUCUCdTdT
2191

224
242
TGATAAACTCTGTAAGGAA
1942
UGAUAAACUCUGUAAGGAAdTdT
2066
UUCCUUACAGAGUUUAUCAdTdT
2177

222
240
TATGATAAACTCTGTAAGG
2385
UAUGAUAAACUCUGUAAGGdTdT
2443
CCUUACAGAGUUUAUCAUAdTdT
2496

238
256
AGGAAGTTCCCAACTATAA
2386
AGGAAGUUCCCAACUAUAAdTdT
2444
UUAUAGUUGGGAACUUCCUdTdT
2497

254
272
TAAACTTATAACCCCAGCT
1950
UAAACUUAUAACCCCAGCUdTdT
2073
AGCUGGGGUUAUAAGUUUAdTdT
2184

275
293
GGTCTCTGAGAGACTGAAG
2387
GGUCUCUGAGAGACUGAAGdTdT
2445
CUUCAGUCUCUCAGAGACCdTdT
2498

219
237
ACCTATGATAAACTCTGTA
2388
ACCUAUGAUAAACUCUGUAdTdT
2446
UACAGAGUUUAUCAUAGGUdTdT
2499

186
204
AAGCTCAATAACTTAGTCT
2389
AAGCUCAAUAACUUAGUCUdTdT
1008
AGACUAAGUUAUUGAGCUUdTdT
1418

455
473
TACATTTGGAAAAATAAAA
2390
UACAUUUGGAAAAAUAAAAdTdT
2447
UUUUAUUUUUCCAAAUGUAdTdT
2500

197
215
CTTAGTCTTGTTTGACAAA
1934
CUUAGUCUUGUUUGACAAAdTdT
2061
UUUGUCAAACAAGACUAAGdTdT
2172

29
47
CGGTGTCTGCTGCTATTCT
1893
CGGUGUCUGCUGCUAUUCUdTdT
871
AGAAUAGCAGCAGACACCGdTdT
1281

456
474
ACATTTGGAAAAATAAAAC
2009
AGAUUUGGAAAAAUAAAACdTdT
2120
GUUUUAUUUUUCCAAAUGUdTdT
2231

34
52
TCTGCTGCTATTCTCCGAG
2391
UCUGCUGCUAUUCUCCGAGdTdT
2448
CUCGGAGAAUAGCAGCAGAdTdT
2501

423
441
GGTGAAGATGCATGAATAG
1999
GGUGAAGAUGCAUGAAUAGdTdT
2113
CUAUUCAUGCAUCUUCACCdTdT
2224

1
19
CTTTTTGTCCGACATCTTG
1885
CUUUUUGUCCGACAUCUUGdTdT
2012
CAAGAUGUCGGACAAAAAGdTdT
2123

348
366
ATCAAACTGGTTTCAAAGC
1978
AUCAAACUGGUUUCAAAGCdTdT
2095
GCUUUGAAACCAGUUUGAUdTdT
2206

240
258
GAAGTTCCCAACTATAAAC
2392
GAAGUUCCCAACUAUAAACdTdT
2449
GUUUAUAGUUGGGAACUUCdTdT
2502

255
273
AAACTTATAACCCCAGCTG
1951
AAACUUAUAACCCCAGCUGdTdT
2074
CAGCUGGGGUUAUAAGUUUdTdT
2185

215
233
AGCTACCTATGATAAACTC
2393
AGCUACCUAUGAUAAACUCdTdT
2450
GAGUUUAUCAUAGGUAGCUdTdT
2503

382
400
TTTACACCAGAAATACCAA
2394
UUUACACCAGAAAUACCAAdTdT
2451
UUGGUAUUUCUGGUGUAAAdTdT
2504

353
371
ACTGGTTTCAAAGCACAGA
1979
ACUGGUUUCAAAGCACAGAdTdT
2096
UCUGUGCUUUGAAACCAGUdTdT
2207

326
344
GGAGCTCCTTAGTAAAGGA
1971
GGAGCUCCUUAGUAAAGGAdTdT
2089
UCCUUUACUAAGGAGCUCCdTdT
2200

202
220
TCTTGTTTGACAAAGCTAC
2395
UCUUGUUUGACAAAGCUACdTdT
2452
GUAGCUUUGUCAAACAAGAdTdT
2505

45
63
TCTCCGAGCTTCGCAATGC
1898
UCUCCGAGCUUCGCAAUGCdTdT
2021
GCAUUGCGAAGCUCGGAGAdTdT
2132

419
437
TGCTGGTGAAGATGCATGA
1998
UGCUGGUGAAGAUGCAUGAdTdT
2112
UCAUGCAUCUUCACCAGCAdTdT
2223

178
196
TTCGGGACAAGCTCAATAA
1928
UUCGGGACAAGCUCAAUAAdTdT
2057
UUAUUGAGCUUGUCCCGAAdTdT
2168

44
62
TTCTCCGAGCTTCGCAATG
2396
UUCUCCGAGCUUCGCAAUGdTdT
2453
CAUUGCGAAGCUCGGAGAAdTdT
2506

335
353
TAGTAAAGGACTTATCAAA
1974
UAGUAAAGGACUUAUCAAAdTdT
2092
UUUGAUAAGUCCUUUACUAdTdT
2203

251
269
CTATAAACTTATAACCCCA
2397
CUAUAAACUUAUAACCCCAdTdT
2454
UGGGGUUAUAAGUUUAUAGdTdT
2507

374
392
TCAAGTAATTTACACCAGA
2398
UCAAGUAAUUUACACCAGAdTdT
2455
UCUGGUGUAAAUUACUUGAdTdT
2508

151
169
AAAAGAAGAAGTGGTCCAA
1921
AAAAGAAGAAGUGGUCCAAdTdT
2051
UUGGACCACUUCUUCUUUUdTdT
2162

164
182
GTCCAAAGGCAAAGTTCGG
2399
GUCCAAAGGCAAAGUUCGGdTdT
2456
CCGAACUUUGCCUUUGGACdTdT
2509

253
271
ATAAACTTATAACCCCAGC
2400
AUAAACUUAUAACCCCAGCdTdT
2457
GCUGGGGUUAUAAGUUUAUdTdT
2510

32
50
TGTCTGCTGCTATTCTCCG
2401
UGUCUGCUGCUAUUCUCCGdTdT
2458
CGGAGAAUAGCAGCAGACAdTdT
2511

146
164
GGCCAAAAAGAAGAAGTGG
1919
GGCCAAAAAGAAGAAGUGGdTdT
2049
CCACUUCUUCUUUUUGGCCdTdT
2160

323
341
TCAGGAGCTCCTTAGTAAA
1970
UCAGGAGCUCCUUAGUAAAdTdT
2088
UUUACUAAGGAGCUCCUGAdTdT
2199

358
376
TTTCAAAGCACAGAGCTCA
1980
UUUCAAAGCACAGAGCUCAdTdT
2097
UGAGCUCUGUGCUUUGAAAdTdT
2208

241
259
AAGTTCCCAACTATAAACT
2402
AAGUUCCCAACUAUAAACUdTdT
1063
AGUUUAUAGUUGGGAACUUdTdT
1473

206
224
GTTTGACAAAGCTACCTAT
1937
GUUUGACAAAGCUACCUAUdTdT
1028
AUAGGUAGCUUUGUCAAACdTdT
1438

328
346
AGCTCCTTAGTAAAGGACT
2403
AGCUCCUUAGUAAAGGACUdTdT
1125
AGUCCUUUACUAAGGAGCUdTdT
1535

213
231
AAAGCTACCTATGATAAAC
2404
AAAGCUACCUAUGAUAAACdTdT
2459
GUUUAUCAUAGGUAGCUUUdTdT
2512

148
166
CCAAAAAGAAGAAGTGGTC
1920
CCAAAAAGAAGAAGUGGUCdTdT
2050
GACCACUUCUUCUUUUUGGdTdT
2161

37
55
GCTGCTATTCTCCGAGCTT
1896
GCUGCUAUUCUCCGAGCUUdTdT
879
AAGCUCGGAGAAUAGCAGCdTdT
1289

349
367
TCAAACTGGTTTCAAAGCA
2405
UCAAACUGGUUUCAAAGCAdTdT
2460
UGCUUUGAAACCAGUUUGAdTdT
2513

365
383
GCACAGAGCTCAAGTAATT
1982
GCACAGAGCUCAAGUAAUUdTdT
1162
AAUUACUUGAGCUCUGUGCdTdT
1572

350
368
CAAACTGGTTTCAAAGCAC
2406
CAAACUGGUUUCAAAGCACdTdT
2461
GUGCUUUGAAACCAGUUUGdTdT
2514

336
354
AGTAAAGGACTTATCAAAC
2407
AGUAAAGGACUUAUCAAACdTdT
2462
GUUUGAUAAGUCCUUUACUdTdT
2515

337
355
GTAAAGGACTTATCAAACT
2408
GUAAAGGACUUAUCAAACUdTdT
1134
AGUUUGAUAAGUCCUUUACdTdT
1544

214
232
AAGCTACCTATGATAAACT
2409
AAGCUACCUAUGAUAAACUdTdT
1036
AGUUUAUCAUAGGUAGCUUdTdT
1446

354
372
CTGGTTTCAAAGCACAGAG
2410
CUGGUUUCAAAGCACAGAGdTdT
2463
CUCUGUGCUUUGAAACCAGdTdT
2516

196
214
ACTTAGTCTTGTTTGACAA
2411
ACUUAGUCUUGUUUGACAAdTdT
2464
UUGUCAAACAAGACUAAGUdTdT
2517

236
254
TAAGGAAGTTCCCAACTAT
1945
UAAGGAAGUUCCCAACUAUdTdT
1058
AUAGUUGGGAACUUCCUUAdTdT
1468

357
375
GTTTCAAAGCACAGAGCTC
2412
GUUUCAAAGCACAGAGCUCdTdT
2465
GAGCUCUGUGCUUUGAAACdTdT
2518

TABLE 12

RPS25 Modified duplex Sequences

Alnylam

SEQ

SEQ

Duplex

ID
Antisense

ID

Designation
Duplex ID
Sense ID
Sense Sequence 5′ to 3′
NO:
ID
Antisense Sequence 5′ to 3′
NO:

AD-
XD-18245
X61218
AUGCCGCCUAAGGACGACUdTdT
902
X61219
AGUCGUCCUUAGGCGGCAUdTdT
1312

960560.1

AD-
XD-18246
X61220
UGCCGCCUAAGGACGACAUdTdT
903
X61221
AUGUCGUCCUUAGGCGGCAdTdT
1313

960561.1

AD-
XD-18247
X61222
CCGCCUAAGGACGACAAGUdTdT
905
X61223
ACUUGUCGUCCUUAGGCGGdTdT
1315

960563.1

AD-
XD-18248
X61224
CGCCUAAGGACGACAAGAUdTdT
906
X61225
AUCUUGUCGUCCUUAGGCGdTdT
1316

960564.1

AD-
XD-18249
X61226
GCCUAAGGACGACAAGAAUdTdT
907
X61227
AUUCUUGUCGUCCUUAGGCdTdT
1317

960565.1

AD-
XD-18250
X61228
CUAAGGACGACAAGAAGAUdTdT
909
X61229
AUCUUCUUGUCGUCCUUAGdTdT
1319

960567.1

AD-
XD-18251
X61230
UAAGGACGACAAGAAGAAUdTdT
910
X61231
AUUCUUCUUGUCGUCCUUAdTdT
1320

960568.1

AD-
XD-18252
X61232
AAGGACGACAAGAAGAAGUdTdT
911
X61233
ACUUCUUCUUGUCGUCCUUdTdT
1321

960569.1

AD-
XD-18253
X61234
GGACGACAAGAAGAAGAAUdTdT
913
X61235
AUUCUUCUUCUUGUCGUCCdTdT
1323

960571.1

AD-
XD-18254
X61236
GACGACAAGAAGAAGAAGUdTdT
914
X61237
ACUUCUUCUUCUUGUCGUCdTdT
1324

960572.1

AD-
XD-18255
X61238
ACGACAAGAAGAAGAAGGUdTdT
915
X61239
ACCUUCUUCUUCUUGUCGUdTdT
1325

960573.1

AD-
XD-18256
X61240
GACAAGAAGAAGAAGGACUdTdT
917
X61241
AGUCCUUCUUCUUCUUGUCdTdT
1327

960575.1

AD-
XD-18257
X61242
ACAAGAAGAAGAAGGACGUdTdT
918
X61243
ACGUCCUUCUUCUUCUUGUdTdT
1328

960576.1

AD-
XD-18258
X61244
CAAGAAGAAGAAGGACGCUdTdT
919
X61245
AGCGUCCUUCUUCUUCUUGdTdT
1329

960577.1

AD-
XD-18259
X61246
AGAAGAAGAAGGACGCUGUdTdT
921
X61247
ACAGCGUCCUUCUUCUUCUdTdT
1331

960579.1

AD-
XD-18260
X61248
GAAGAAGAAGGACGCUGGUdTdT
922
X61249
ACCAGCGUCCUUCUUCUUCdTdT
1332

960580.1

AD-
XD-18261
X61250
AAGAAGAAGGACGCUGGAUdTdT
923
X61251
AUCCAGCGUCCUUCUUCUUdTdT
1333

960581.1

AD-
XD-18262
X61252
AGAAGAAGGACGCUGGAAUdTdT
924
X61253
AUUCCAGCGUCCUUCUUCUdTdT
1334

960582.1

AD-
XD-18263
X61254
AAGAAGGACGCUGGAAAGUdTdT
926
X61255
ACUUUCCAGCGUCCUUCUUdTdT
1336

960584.1

AD-
XD-18264
X61256
AGAAGGACGCUGGAAAGUUdTdT
927
X61257
AACUUUCCAGCGUCCUUCUdTdT
1337

960585.1

AD-
XD-18265
X61258
GAAGGACGCUGGAAAGUCUdTdT
928
X61259
AGACUUUCCAGCGUCCUUCdTdT
1338

960586.1

AD-
XD-18266
X61260
AGGACGCUGGAAAGUCGGUdTdT
930
X61261
ACCGACUUUCCAGCGUCCUdTdT
1340

960588.1

AD-
XD-18267
X61262
GGACGCUGGAAAGUCGGCUdTdT
931
X61263
AGCCGACUUUCCAGCGUCCdTdT
1341

960589.1

AD-
XD-18268
X61264
GACGCUGGAAAGUCGGCCUdTdT
932
X61265
AGGCCGACUUUCCAGCGUCdTdT
1342

960590.1

AD-
XD-18269
X61266
CGCUGGAAAGUCGGCCAAUdTdT
934
X61267
AUUGGCCGACUUUCCAGCGdTdT
1344

960592.1

AD-
XD-18270
X61268
GCUGGAAAGUCGGCCAAGUdTdT
935
X61269
ACUUGGCCGACUUUCCAGCdTdT
1345

960593.1

AD-
XD-18271
X61270
CUGGAAAGUCGGCCAAGAUdTdT
936
X61271
AUCUUGGCCGACUUUCCAGdTdT
1346

960594.1

AD-
XD-18272
X61272
GGAAAGUCGGCCAAGAAAUdTdT
938
X61273
AUUUCUUGGCCGACUUUCCdTdT
1348

960596.1

AD-
XD-18273
X61274
GAAAGUCGGCCAAGAAAGUdTdT
939
X61275
ACUUUCUUGGCCGACUUUCdTdT
1349

960597.1

AD-
XD-18274
X61276
AAAGUCGGCCAAGAAAGAUdTdT
940
X61277
AUCUUUCUUGGCCGACUUUdTdT
1350

960598.1

AD-
XD-18275
X61278
AGUCGGCCAAGAAAGACAUdTdT
942
X61279
AUGUCUUUCUUGGCCGACUdTdT
1352

960600.1

AD-
XD-18276
X61280
GUCGGCCAAGAAAGACAAUdTdT
943
X61281
AUUGUCUUUCUUGGCCGACdTdT
1353

960601.1

AD-
XD-18277
X61282
UCGGCCAAGAAAGACAAAUdTdT
944
X61283
AUUUGUCUUUCUUGGCCGAdTdT
1354

960602.1

AD-
XD-18278
X61284
GGCCAAGAAAGACAAAGAUdTdT
946
X61285
AUCUUUGUCUUUCUUGGCCdTdT
1356

960604.1

AD-
XD-18279
X61286
GCCAAGAAAGACAAAGACUdTdT
947
X61287
AGUCUUUGUCUUUCUUGGCdTdT
1357

960605.1

AD-
XD-18280
X61288
CCAAGAAAGACAAAGACCUdTdT
948
X61289
AGGUCUUUGUCUUUCUUGGdTdT
1358

960606.1

AD-
XD-18281
X61290
AGAAAGACAAAGACCCAGUdTdT
950
X61291
ACUGGGUCUUUGUCUUUCUdTdT
1360

960608.1

AD-
XD-18282
X61292
GAAAGACAAAGACCCAGUUdTdT
951
X61293
AACUGGGUCUUUGUCUUUCdTdT
1361

960609.1

AD-
XD-18283
X61294
AAAGACAAAGACCCAGUGUdTdT
952
X61295
ACACUGGGUCUUUGUCUUUdTdT
1362

960610.1

AD-
XD-18284
X61296
AGACAAAGACCCAGUGAAUdTdT
954
X61297
AUUCACUGGGUCUUUGUCUdTdT
1364

960612.1

AD-
XD-18285
X61298
GACAAAGACCCAGUGAACUdTdT
955
X61299
AGUUCACUGGGUCUUUGUCdTdT
1365

960613.1

AD-
XD-18286
X61300
ACAAAGACCCAGUGAACAUdTdT
956
X61301
AUGUUCACUGGGUCUUUGUdTdT
1366

960614.1

AD-
XD-18287
X61302
CAAAGACCCAGUGAACAAUdTdT
957
X61303
AUUGUUCACUGGGUCUUUGdTdT
1367

960615.1

AD-
XD-18288
X61304
AAGACCCAGUGAACAAAUUdTdT
959
X61305
AAUUUGUUCACUGGGUCUUdTdT
1369

960617.1

AD-
XD-18289
X61306
AGACCCAGUGAACAAAUCUdTdT
960
X61307
AGAUUUGUUCACUGGGUCUdTdT
1370

960618.1

AD-
XD-18290
X61308
GACCCAGUGAACAAAUCCUdTdT
961
X61309
AGGAUUUGUUCACUGGGUCdTdT
1371

960619.1

AD-
XD-18291
X61310
CCCAGUGAACAAAUCCGGUdTdT
963
X61311
ACCGGAUUUGUUCACUGGGdTdT
1373

960621.1

AD-
XD-18292
X61312
GGGCAAGGCCAAAAAGAAUdTdT
964
X61313
AUUCUUUUUGGCCUUGCCCdTdT
1374

960622.1

AD-
XD-18293
X61314
GGCAAGGCCAAAAAGAAGUdTdT
965
X61315
ACUUCUUUUUGGCCUUGCCdTdT
1375

960623.1

AD-
XD-18294
X61316
AGGCCAAAAAGAAGAAGUUdTdT
967
X61317
AACUUCUUCUUUUUGGCCUdTdT
1377

960625.1

AD-
XD-18295
X61318
GGCCAAAAAGAAGAAGUGUdTdT
968
X61319
ACACUUCUUCUUUUUGGCCdTdT
1378

960626.1

AD-
XD-18296
X61320
GCCAAAAAGAAGAAGUGGUdTdT
969
X61321
ACCACUUCUUCUUUUUGGCdTdT
1379

960627.1

AD-
XD-18297
X61322
CAAAAAGAAGAAGUGGUCUdTdT
971
X61323
AGACCACUUCUUCUUUUUGdTdT
1381

960629.1

AD-
XD-18298
X61324
AAAAAGAAGAAGUGGUCCUdTdT
972
X61325
AGGACCACUUCUUCUUUUUdTdT
1382

960630.1

AD-
XD-18299
X61326
AAAAGAAGAAGUGGUCCAUdTdT
973
X61327
AUGGACCACUUCUUCUUUUdTdT
1383

960631.1

AD-
XD-18300
X61328
AAGAAGAAGUGGUCCAAAUdTdT
975
X61329
AUUUGGACCACUUCUUCUUdTdT
1385

960633.1

AD-
XD-18301
X61330
AGAAGAAGUGGUCCAAAGUdTdT
976
X61331
ACUUUGGACCACUUCUUCUdTdT
1386

960634.1

AD-
XD-18302
X61332
GAAGAAGUGGUCCAAAGGUdTdT
977
X61333
ACCUUUGGACCACUUCUUCdTdT
1387

960635.1

AD-
XD-18303
X61334
AGAAGUGGUCCAAAGGCAUdTdT
979
X61335
AUGCCUUUGGACCACUUCUdTdT
1389

960637.1

AD-
XD-18304
X61336
GAAGUGGUCCAAAGGCAAUdTdT
980
X61337
AUUGCCUUUGGACCACUUCdTdT
1390

960638.1

AD-
XD-18305
X61338
AAGUGGUCCAAAGGCAAAUdTdT
981
X61339
AUUUGCCUUUGGACCACUUdTdT
1391

960639.1

AD-
XD-18306
X61340
GUGGUCCAAAGGCAAAGUUdTdT
983
X61341
AACUUUGCCUUUGGACCACdTdT
1393

960641.1

AD-
XD-18307
X61342
UGGUCCAAAGGCAAAGUUUdTdT
984
X61343
AAACUUUGCCUUUGGACCAdTdT
1394

960642.1

AD-
XD-18308
X61344
GGUCCAAAGGCAAAGUUCUdTdT
985
X61345
AGAACUUUGCCUUUGGACCdTdT
1395

960643.1

AD-
XD-18309
X61346
UCCAAAGGCAAAGUUCGGUdTdT
987
X61347
ACCGAACUUUGCCUUUGGAdTdT
1397

960645.1

AD-
XD-18310
X61348
CCAAAGGCAAAGUUCGGGUdTdT
988
X61349
ACCCGAACUUUGCCUUUGGdTdT
1398

960646.1

AD-
XD-18311
X61350
CAAAGGCAAAGUUCGGGAUdTdT
989
X61351
AUCCCGAACUUUGCCUUUGdTdT
1399

960647.1

AD-
XD-18312
X61352
AAGGCAAAGUUCGGGACAUdTdT
991
X61353
AUGUCCCGAACUUUGCCUUdTdT
1401

960649.1

AD-
XD-18313
X61354
AGGCAAAGUUCGGGACAAUdTdT
992
X61355
AUUGUCCCGAACUUUGCCUdTdT
1402

960650.1

AD-
XD-18314
X61356
GGCAAAGUUCGGGACAAGUdTdT
993
X61357
ACUUGUCCCGAACUUUGCCdTdT
1403

960651.1

AD-
XD-18315
X61358
CAAAGUUCGGGACAAGCUUdTdT
995
X61359
AAGCUUGUCCCGAACUUUGdTdT
1405

960653.1

AD-
XD-18316
X61360
AAAGUUCGGGACAAGCUCUdTdT
996
X61361
AGAGCUUGUCCCGAACUUUdTdT
1406

960654.1

AD-
XD-18317
X61362
AAGUUCGGGACAAGCUCAUdTdT
997
X61363
AUGAGCUUGUCCCGAACUUdTdT
1407

960655.1

AD-
XD-18318
X61364
AGUUCGGGACAAGCUCAAUdTdT
998
X61365
AUUGAGCUUGUCCCGAACUdTdT
1408

960656.1

AD-
XD-18319
X61366
UUCGGGACAAGCUCAAUAUdTdT
1000
X61367
AUAUUGAGCUUGUCCCGAAdTdT
1410

960658.1

AD-
XD-18320
X61368
UCGGGACAAGCUCAAUAAUdTdT
1001
X61369
AUUAUUGAGCUUGUCCCGAdTdT
1411

960659.1

AD-
XD-18321
X61370
CGGGACAAGCUCAAUAACUdTdT
1002
X61371
AGUUAUUGAGCUUGUCCCGdTdT
1412

960660.1

AD-
XD-18322
X61372
GGACAAGCUCAAUAACUUUdTdT
1004
X61373
AAAGUUAUUGAGCUUGUCCdTdT
1414

960662.1

AD-
XD-18323
X61374
GACAAGCUCAAUAACUUAUdTdT
1005
X61375
AUAAGUUAUUGAGCUUGUCdTdT
1415

960663.1

AD-
XD-18324
X61376
ACAAGCUCAAUAACUUAGUdTdT
1006
X61377
ACUAAGUUAUUGAGCUUGUdTdT
1416

960664.1

AD-
XD-18325
X61378
AAGCUCAAUAACUUAGUCUdTdT
1008
X61379
AGACUAAGUUAUUGAGCUUdTdT
1418

960666.1

AD-
XD-18326
X61380
AGCUCAAUAACUUAGUCUUdTdT
1009
X61381
AAGACUAAGUUAUUGAGCUdTdT
1419

960667.1

AD-
XD-18327
X61382
GCUCAAUAACUUAGUCUUUdTdT
1010
X61383
AAAGACUAAGUUAUUGAGCdTdT
1420

960668.1

AD-
XD-18328
X61384
UCAAUAACUUAGUCUUGUUdTdT
1012
X61385
AACAAGACUAAGUUAUUGAdTdT
1422

960670.1

AD-
XD-18329
X61386
CAAUAACUUAGUCUUGUUUdTdT
1013
X61387
AAACAAGACUAAGUUAUUGdTdT
1423

960671.1

AD-
XD-18330
X61388
AAUAACUUAGUCUUGUUUUdTdT
1014
X61389
AAAACAAGACUAAGUUAUUdTdT
1424

960672.1

AD-
XD-18331
X61390
UAACUUAGUCUUGUUUGAUdTdT
1016
X61391
AUCAAACAAGACUAAGUUAdTdT
1426

960674.1

AD-
XD-18332
X61392
AACUUAGUCUUGUUUGACUdTdT
1017
X61393
AGUCAAACAAGACUAAGUUdTdT
1427

960675.1

AD-
XD-18333
X61394
ACUUAGUCUUGUUUGACAUdTdT
1018
X61395
AUGUCAAACAAGACUAAGUdTdT
1428

960676.1

AD-
XD-18334
X61396
UUAGUCUUGUUUGACAAAUdTdT
1020
X61397
AUUUGUCAAACAAGACUAAdTdT
1430

960678.1

AD-
XD-18335
X61398
UAGUCUUGUUUGACAAAGUdTdT
1021
X61399
ACUUUGUCAAACAAGACUAdTdT
1431

960679.1

AD-
XD-18336
X61400
AGUCUUGUUUGACAAAGCUdTdT
1022
X61401
AGCUUUGUCAAACAAGACUdTdT
1432

960680.1

AD-
XD-18337
X61402
UCUUGUUUGACAAAGCUAUdTdT
1024
X61403
AUAGCUUUGUCAAACAAGAdTdT
1434

960682.1

AD-
XD-18338
X61404
CUUGUUUGACAAAGCUACUdTdT
1025
X61405
AGUAGCUUUGUCAAACAAGdTdT
1435

960683.1

AD-
XD-18339
X61406
UUGUUUGACAAAGCUACCUdTdT
1026
X61407
AGGUAGCUUUGUCAAACAAdTdT
1436

960684.1

AD-
XD-18340
X61408
GUUUGACAAAGCUACCUAUdTdT
1028
X61409
AUAGGUAGCUUUGUCAAACdTdT
1438

960686.1

AD-
XD-18341
X61410
UUUGACAAAGCUACCUAUUdTdT
1029
X61411
AAUAGGUAGCUUUGUCAAAdTdT
1439

960687.1

AD-
XD-18342
X61412
UUGACAAAGCUACCUAUGUdTdT
1030
X61413
ACAUAGGUAGCUUUGUCAAdTdT
1440

960688.1

AD-
XD-18343
X61414
UGACAAAGCUACCUAUGAUdTdT
1031
X61415
AUCAUAGGUAGCUUUGUCAdTdT
1441

960689.1

AD-
XD-18344
X61416
ACAAAGCUACCUAUGAUAUdTdT
1033
X61417
AUAUCAUAGGUAGCUUUGUdTdT
1443

960691.1

AD-
XD-18345
X61418
CAAAGCUACCUAUGAUAAUdTdT
1034
X61419
AUUAUCAUAGGUAGCUUUGdTdT
1444

960692.1

AD-
XD-18346
X61420
AAAGCUACCUAUGAUAAAUdTdT
1035
X61421
AUUUAUCAUAGGUAGCUUUdTdT
1445

960693.1

AD-
XD-18347
X61422
AGCUACCUAUGAUAAACUUdTdT
1037
X61423
AAGUUUAUCAUAGGUAGCUdTdT
1447

960695.1

AD-
XD-18348
X61424
GCUACCUAUGAUAAACUCUdTdT
1038
X61425
AGAGUUUAUCAUAGGUAGCdTdT
1448

960696.1

AD-
XD-18349
X61426
CUACCUAUGAUAAACUCUUdTdT
1039
X61427
AAGAGUUUAUCAUAGGUAGdTdT
1449

960697.1

AD-
XD-18350
X61428
ACCUAUGAUAAACUCUGUUdTdT
1041
X61429
AACAGAGUUUAUCAUAGGUdTdT
1451

960699.1

AD-
XD-18351
X61430
CCUAUGAUAAACUCUGUAUdTdT
1042
X61431
AUACAGAGUUUAUCAUAGGdTdT
1452

960700.1

AD-
XD-18352
X61432
CUAUGAUAAACUCUGUAAUdTdT
1043
X61433
AUUACAGAGUUUAUCAUAGdTdT
1453

960701.1

AD-
XD-18353
X61434
AUGAUAAACUCUGUAAGGUdTdT
1045
X61435
ACCUUACAGAGUUUAUCAUdTdT
1455

960703.1

AD-
XD-18354
X61436
UGAUAAACUCUGUAAGGAUdTdT
1046
X61437
AUCCUUACAGAGUUUAUCAdTdT
1456

960704.1

AD-
XD-18355
X61438
GAUAAACUCUGUAAGGAAUdTdT
1047
X61439
AUUCCUUACAGAGUUUAUCdTdT
1457

960705.1

AD-
XD-18356
X61440
UAAACUCUGUAAGGAAGUUdTdT
1049
X61441
AACUUCCUUACAGAGUUUAdTdT
1459

960707.1

AD-
XD-18357
X61442
AAACUCUGUAAGGAAGUUUdTdT
1050
X61443
AAACUUCCUUACAGAGUUUdTdT
1460

960708.1

AD-
XD-18358
X61444
AACUCUGUAAGGAAGUUCUdTdT
1051
X61445
AGAACUUCCUUACAGAGUUdTdT
1461

960709.1

AD-
XD-18359
X61446
CUCUGUAAGGAAGUUCCCUdTdT
1053
X61447
AGGGAACUUCCUUACAGAGdTdT
1463

960711.1

AD-
XD-18360
X61448
UCUGUAAGGAAGUUCCCAUdTdT
1054
X61449
AUGGGAACUUCCUUACAGAdTdT
1464

960712.1

AD-
XD-18361
X61450
CUGUAAGGAAGUUCCCAAUdTdT
1055
X61451
AUUGGGAACUUCCUUACAGdTdT
1465

960713.1

AD-
XD-18362
X61452
GUAAGGAAGUUCCCAACUUdTdT
1057
X61453
AAGUUGGGAACUUCCUUACdTdT
1467

960715.1

AD-
XD-18363
X61454
UAAGGAAGUUCCCAACUAUdTdT
1058
X61455
AUAGUUGGGAACUUCCUUAdTdT
1468

960716.1

AD-
XD-18364
X61456
AAGGAAGUUCCCAACUAUUdTdT
1059
X61457
AAUAGUUGGGAACUUCCUUdTdT
1469

960717.1

AD-
XD-18365
X61458
GGAAGUUCCCAACUAUAAUdTdT
1061
X61459
AUUAUAGUUGGGAACUUCCdTdT
1471

960719.1

AD-
XD-18366
X61460
GAAGUUCCCAACUAUAAAUdTdT
1062
X61461
AUUUAUAGUUGGGAACUUCdTdT
1472

960720.1

AD-
XD-18367
X61462
AAGUUCCCAACUAUAAACUdTdT
1063
X61463
AGUUUAUAGUUGGGAACUUdTdT
1473

960721.1

AD-
XD-18368
X61464
GUUCCCAACUAUAAACUUUdTdT
1065
X61465
AAAGUUUAUAGUUGGGAACdTdT
1475

960723.1

AD-
XD-18369
X61466
UUCCCAACUAUAAACUUAUdTdT
1066
X61467
AUAAGUUUAUAGUUGGGAAdTdT
1476

960724.1

AD-
XD-18370
X61468
UCCCAACUAUAAACUUAUUdTdT
1067
X61469
AAUAAGUUUAUAGUUGGGAdTdT
1477

960725.1

AD-
XD-18371
X61470
CCAACUAUAAACUUAUAAUdTdT
1069
X61471
AUUAUAAGUUUAUAGUUGGdTdT
1479

960727.1

AD-
XD-18372
X61472
CAACUAUAAACUUAUAACUdTdT
1070
X61473
AGUUAUAAGUUUAUAGUUGdTdT
1480

960728.1

AD-
XD-18373
X61474
AACUAUAAACUUAUAACCUdTdT
1071
X61475
AGGUUAUAAGUUUAUAGUUdTdT
1481

960729.1

AD-
XD-18374
X61476
CCCAGCUGUGGUCUCUGAUdTdT
1072
X61477
AUCAGAGACCACAGCUGGGdTdT
1482

960730.1

AD-
XD-18375
X61478
CAGCUGUGGUCUCUGAGAUdTdT
1074
X61479
AUCUCAGAGACCACAGCUGdTdT
1484

960732.1

AD-
XD-18376
X61480
AGCUGUGGUCUCUGAGAGUdTdT
1075
X61481
ACUCUCAGAGACCACAGCUdTdT
1485

960733.1

AD-
XD-18377
X61482
GCUGUGGUCUCUGAGAGAUdTdT
1076
X61483
AUCUCUCAGAGACCACAGCdTdT
1486

960734.1

AD-
XD-18378
X61484
UGUGGUCUCUGAGAGACUUdTdT
1078
X61485
AAGUCUCUCAGAGACCACAdTdT
1488

960736.1

AD-
XD-18379
X61486
GUGGUCUCUGAGAGACUGUdTdT
1079
X61487
ACAGUCUCUCAGAGACCACdTdT
1489

960737.1

AD-
XD-18380
X61488
UGGUCUCUGAGAGACUGAUdTdT
1080
X61489
AUCAGUCUCUCAGAGACCAdTdT
1490

960738.1

AD-
XD-18381
X61490
GUCUCUGAGAGACUGAAGUdTdT
1082
X61491
ACUUCAGUCUCUCAGAGACdTdT
1492

960740.1

AD-
XD-18382
X61492
UCUCUGAGAGACUGAAGAUdTdT
1083
X61493
AUCUUCAGUCUCUCAGAGAdTdT
1493

960741.1

AD-
XD-18383
X61494
CUCUGAGAGACUGAAGAUUdTdT
1084
X61495
AAUCUUCAGUCUCUCAGAGdTdT
1494

960742.1

AD-
XD-18384
X61496
CUGAGAGACUGAAGAUUCUdTdT
1086
X61497
AGAAUCUUCAGUCUCUCAGdTdT
1496

960744.1

AD-
XD-18385
X61498
UGAGAGACUGAAGAUUCGUdTdT
1087
X61499
ACGAAUCUUCAGUCUCUCAdTdT
1497

960745.1

AD-
XD-18386
X61500
GAGAGACUGAAGAUUCGAUdTdT
1088
X61501
AUCGAAUCUUCAGUCUCUCdTdT
1498

960746.1

AD-
XD-18387
X61502
GAGACUGAAGAUUCGAGGUdTdT
1090
X61503
ACCUCGAAUCUUCAGUCUCdTdT
1500

960748.1

AD-
XD-18388
X61504
AGACUGAAGAUUCGAGGCUdTdT
1091
X61505
AGCCUCGAAUCUUCAGUCUdTdT
1501

960749.1

AD-
XD-18389
X61506
GACUGAAGAUUCGAGGCUUdTdT
1092
X61507
AAGCCUCGAAUCUUCAGUCdTdT
1502

960750.1

AD-
XD-18390
X61508
CUGAAGAUUCGAGGCUCCUdTdT
1094
X61509
AGGAGCCUCGAAUCUUCAGdTdT
1504

960752.1

AD-
XD-18391
X61510
UGAAGAUUCGAGGCUCCCUdTdT
1095
X61511
AGGGAGCCUCGAAUCUUCAdTdT
1505

960753.1

AD-
XD-18392
X61512
GAAGAUUCGAGGCUCCCUUdTdT
1096
X61513
AAGGGAGCCUCGAAUCUUCdTdT
1506

960754.1

AD-
XD-18393
X61514
AGAUUCGAGGCUCCCUGGUdTdT
1098
X61515
ACCAGGGAGCCUCGAAUCUdTdT
1508

960756.1

AD-
XD-18394
X61516
GAUUCGAGGCUCCCUGGCUdTdT
1099
X61517
AGCCAGGGAGCCUCGAAUCdTdT
1509

960757.1

AD-
XD-18395
X61518
AUUCGAGGCUCCCUGGCCUdTdT
1100
X61519
AGGCCAGGGAGCCUCGAAUdTdT
1510

960758.1

AD-
XD-18396
X61520
UCGAGGCUCCCUGGCCAGUdTdT
1102
X61521
ACUGGCCAGGGAGCCUCGAdTdT
1512

960760.1

AD-
XD-18397
X61522
CUGGCCAGGGCAGCCCUUUdTdT
1103
X61523
AAAGGGCUGCCCUGGCCAGdTdT
1513

960761.1

AD-
XD-18398
X61524
UGGCCAGGGCAGCCCUUCUdTdT
1104
X61525
AGAAGGGCUGCCCUGGCCAdTdT
1514

960762.1

AD-
XD-18399
X61526
GGCCAGGGCAGCCCUUCAUdTdT
1105
X61527
AUGAAGGGCUGCCCUGGCCdTdT
1515

960763.1

AD-
XD-18400
X61528
CCAGGGCAGCCCUUCAGGUdTdT
1107
X61529
ACCUGAAGGGCUGCCCUGGdTdT
1517

960765.1

AD-
XD-18401
X61530
CAGGGCAGCCCUUCAGGAUdTdT
1108
X61531
AUCCUGAAGGGCUGCCCUGdTdT
1518

960766.1

AD-
XD-18402
X61532
AGGGCAGCCCUUCAGGAGUdTdT
1109
X61533
ACUCCUGAAGGGCUGCCCUdTdT
1519

960767.1

AD-
XD-18403
X61534
GGCAGCCCUUCAGGAGCUUdTdT
1111
X61535
AAGCUCCUGAAGGGCUGCCdTdT
1521

960769.1

AD-
XD-18404
X61536
GCAGCCCUUCAGGAGCUCUdTdT
1112
X61537
AGAGCUCCUGAAGGGCUGCdTdT
1522

960770.1

AD-
XD-18405
X61538
CAGCCCUUCAGGAGCUCCUdTdT
1113
X61539
AGGAGCUCCUGAAGGGCUGdTdT
1523

960771.1

AD-
XD-18406
X61540
GCCCUUCAGGAGCUCCUUUdTdT
1115
X61541
AAAGGAGCUCCUGAAGGGCdTdT
1525

960773.1

AD-
XD-18407
X61542
CCCUUCAGGAGCUCCUUAUdTdT
1116
X61543
AUAAGGAGCUCCUGAAGGGdTdT
1526

960774.1

AD-
XD-18408
X61544
CCUUCAGGAGCUCCUUAGUdTdT
1117
X61545
ACUAAGGAGCUCCUGAAGGdTdT
1527

960775.1

AD-
XD-18409
X61546
UUCAGGAGCUCCUUAGUAUdTdT
1119
X61547
AUACUAAGGAGCUCCUGAAdTdT
1529

960777.1

AD-
XD-18410
X61548
UCAGGAGCUCCUUAGUAAUdTdT
1120
X61549
AUUACUAAGGAGCUCCUGAdTdT
1530

960778.1

AD-
XD-18411
X61550
CAGGAGCUCCUUAGUAAAUdTdT
1121
X61551
AUUUACUAAGGAGCUCCUGdTdT
1531

960779.1

AD-
XD-18412
X61552
GGAGCUCCUUAGUAAAGGUdTdT
1123
X61553
ACCUUUACUAAGGAGCUCCdTdT
1533

960781.1

AD-
XD-18413
X61554
GAGCUCCUUAGUAAAGGAUdTdT
1124
X61555
AUCCUUUACUAAGGAGCUCdTdT
1534

960782.1

AD-
XD-18414
X61556
AGCUCCUUAGUAAAGGACUdTdT
1125
X61557
AGUCCUUUACUAAGGAGCUdTdT
1535

960783.1

AD-
XD-18415
X61558
CUCCUUAGUAAAGGACUUUdTdT
1127
X61559
AAAGUCCUUUACUAAGGAGdTdT
1537

960785.1

AD-
XD-18416
X61560
UCCUUAGUAAAGGACUUAUdTdT
1128
X61561
AUAAGUCCUUUACUAAGGAdTdT
1538

960786.1

AD-
XD-18417
X61562
CCUUAGUAAAGGACUUAUUdTdT
1129
X61563
AAUAAGUCCUUUACUAAGGdTdT
1539

960787.1

AD-
XD-18418
X61564
UUAGUAAAGGACUUAUCAUdTdT
1131
X61565
AUGAUAAGUCCUUUACUAAdTdT
1541

960789.1

AD-
XD-18419
X61566
UAGUAAAGGACUUAUCAAUdTdT
1132
X61567
AUUGAUAAGUCCUUUACUAdTdT
1542

960790.1

AD-
XD-18420
X61568
AGUAAAGGACUUAUCAAAUdTdT
1133
X61569
AUUUGAUAAGUCCUUUACUdTdT
1543

960791.1

AD-
XD-18421
X61570
UAAAGGACUUAUCAAACUUdTdT
1135
X61571
AAGUUUGAUAAGUCCUUUAdTdT
1545

960793.1

AD-
XD-18422
X61572
AAAGGACUUAUCAAACUGUdTdT
1136
X61573
ACAGUUUGAUAAGUCCUUUdTdT
1546

960794.1

AD-
XD-18423
X61574
AAGGACUUAUCAAACUGGUdTdT
1137
X61575
ACCAGUUUGAUAAGUCCUUdTdT
1547

960795.1

AD-
XD-18424
X61576
GGACUUAUCAAACUGGUUUdTdT
1139
X61577
AAACCAGUUUGAUAAGUCCdTdT
1549

960797.1

AD-
XD-18425
X61578
GACUUAUCAAACUGGUUUUdTdT
1140
X61579
AAAACCAGUUUGAUAAGUCdTdT
1550

960798.1

AD-
XD-18426
X61580
ACUUAUCAAACUGGUUUCUdTdT
1141
X61581
AGAAACCAGUUUGAUAAGUdTdT
1551

960799.1

AD-
XD-18427
X61582
UUAUCAAACUGGUUUCAAUdTdT
1143
X61583
AUUGAAACCAGUUUGAUAAdTdT
1553

960801.1

AD-
XD-18428
X61584
UAUCAAACUGGUUUCAAAUdTdT
1144
X61585
AUUUGAAACCAGUUUGAUAdTdT
1554

960802.1

AD-
XD-18429
X61586
AUCAAACUGGUUUCAAAGUdTdT
1145
X61587
ACUUUGAAACCAGUUUGAUdTdT
1555

960803.1

AD-
XD-18430
X61588
UCAAACUGGUUUCAAAGCUdTdT
1146
X61589
AGCUUUGAAACCAGUUUGAdTdT
1556

960804.1

AD-
XD-18431
X61590
AAACUGGUUUCAAAGCACUdTdT
1148
X61591
AGUGCUUUGAAACCAGUUUdTdT
1558

960806.1

AD-
XD-18432
X61592
AACUGGUUUCAAAGCACAUdTdT
1149
X61593
AUGUGCUUUGAAACCAGUUdTdT
1559

960807.1

AD-
XD-18433
X61594
ACUGGUUUCAAAGCACAGUdTdT
1150
X61595
ACUGUGCUUUGAAACCAGUdTdT
1560

960808.1

AD-
XD-18434
X61596
UGGUUUCAAAGCACAGAGUdTdT
1152
X61597
ACUCUGUGCUUUGAAACCAdTdT
1562

960810.1

AD-
XD-18435
X61598
GGUUUCAAAGCACAGAGCUdTdT
1153
X61599
AGCUCUGUGCUUUGAAACCdTdT
1563

960811.1

AD-
XD-18436
X61600
GUUUCAAAGCACAGAGCUUdTdT
1154
X61601
AAGCUCUGUGCUUUGAAACdTdT
1564

960812.1

AD-
XD-18437
X61602
UUCAAAGCACAGAGCUCAUdTdT
1156
X61603
AUGAGCUCUGUGCUUUGAAdTdT
1566

960814.1

AD-
XD-18438
X61604
UCAAAGCACAGAGCUCAAUdTdT
1157
X61605
AUUGAGCUCUGUGCUUUGAdTdT
1567

960815.1

AD-
XD-18439
X61606
CAAAGCACAGAGCUCAAGUdTdT
1158
X61607
ACUUGAGCUCUGUGCUUUGdTdT
1568

960816.1

AD-
XD-18440
X61608
AAGCACAGAGCUCAAGUAUdTdT
1160
X61609
AUACUUGAGCUCUGUGCUUdTdT
1570

960818.1

AD-
XD-18441
X61610
AGCACAGAGCUCAAGUAAUdTdT
1161
X61611
AUUACUUGAGCUCUGUGCUdTdT
1571

960819.1

AD-
XD-18442
X61612
GCACAGAGCUCAAGUAAUUdTdT
1162
X61613
AAUUACUUGAGCUCUGUGCdTdT
1572

960820.1

AD-
XD-18443
X61614
ACAGAGCUCAAGUAAUUUUdTdT
1164
X61615
AAAAUUACUUGAGCUCUGUdTdT
1574

960822.1

AD-
XD-18444
X61616
CAGAGCUCAAGUAAUUUAUdTdT
1165
X61617
AUAAAUUACUUGAGCUCUGdTdT
1575

960823.1

AD-
XD-18445
X61618
AGAGCUCAAGUAAUUUACUdTdT
1166
X61619
AGUAAAUUACUUGAGCUCUdTdT
1576

960824.1

AD-
XD-18446
X61620
AGCUCAAGUAAUUUACACUdTdT
1168
X61621
AGUGUAAAUUACUUGAGCUdTdT
1578

960826.1

AD-
XD-18447
X61622
GCUCAAGUAAUUUACACCUdTdT
1169
X61623
AGGUGUAAAUUACUUGAGCdTdT
1579

960827.1

AD-
XD-18448
X61624
CUCAAGUAAUUUACACCAUdTdT
1170
X61625
AUGGUGUAAAUUACUUGAGdTdT
1580

960828.1

AD-
XD-18449
X61626
CAAGUAAUUUACACCAGAUdTdT
1172
X61627
AUCUGGUGUAAAUUACUUGdTdT
1582

960830.1

AD-
XD-18450
X61628
AAGUAAUUUACACCAGAAUdTdT
1173
X61629
AUUCUGGUGUAAAUUACUUdTdT
1583

960831.1

AD-
XD-18451
X61630
AGUAAUUUACACCAGAAAUdTdT
1174
X61631
AUUUCUGGUGUAAAUUACUdTdT
1584

960832.1

AD-
XD-18452
X61632
UAAUUUACACCAGAAAUAUdTdT
1176
X61633
AUAUUUCUGGUGUAAAUUAdTdT
1586

960834.1

AD-
XD-18453
X61634
AAUUUACACCAGAAAUACUdTdT
1177
X61635
AGUAUUUCUGGUGUAAAUUdTdT
1587

960835.1

AD-
XD-18454
X61636
AUUUACACCAGAAAUACCUdTdT
1178
X61637
AGGUAUUUCUGGUGUAAAUdTdT
1588

960836.1

AD-
XD-18455
X61638
UUUACACCAGAAAUACCAUdTdT
1179
X61639
AUGGUAUUUCUGGUGUAAAdTdT
1589

960837.1

AD-
XD-18456
X61640
UACACCAGAAAUACCAAGUdTdT
1181
X61641
ACUUGGUAUUUCUGGUGUAdTdT
1591

960839.1

AD-
XD-18457
X61642
ACACCAGAAAUACCAAGGUdTdT
1182
X61643
ACCUUGGUAUUUCUGGUGUdTdT
1592

960840.1

AD-
XD-18458
X61644
CACCAGAAAUACCAAGGGUdTdT
1183
X61645
ACCCUUGGUAUUUCUGGUGdTdT
1593

960841.1

AD-
XD-18459
X61646
CCAGAAAUACCAAGGGUGUdTdT
1185
X61647
ACACCCUUGGUAUUUCUGGdTdT
1595

960843.1

AD-
XD-18460
X61648
CAGAAAUACCAAGGGUGGUdTdT
1186
X61649
ACCACCCUUGGUAUUUCUGdTdT
1596

960844.1

AD-
XD-18461
X61650
AGAAAUACCAAGGGUGGAUdTdT
1187
X61651
AUCCACCCUUGGUAUUUCUdTdT
1597

960845.1

AD-
XD-18462
X61652
AAAUACCAAGGGUGGAGAUdTdT
1189
X61653
AUCUCCACCCUUGGUAUUUdTdT
1599

960847.1

AD-
XD-18463
X61654
AAUACCAAGGGUGGAGAUUdTdT
1190
X61655
AAUCUCCACCCUUGGUAUUdTdT
1600

960848.1

AD-
XD-18464
X61656
AUACCAAGGGUGGAGAUGUdTdT
1191
X61657
ACAUCUCCACCCUUGGUAUdTdT
1601

960849.1

AD-
XD-18465
X61658
ACCAAGGGUGGAGAUGCUUdTdT
1193
X61659
AAGCAUCUCCACCCUUGGUdTdT
1603

960851.1

AD-
XD-18466
X61660
CCAAGGGUGGAGAUGCUCUdTdT
1194
X61661
AGAGCAUCUCCACCCUUGGdTdT
1604

960852.1

AD-
XD-18467
X61662
CAAGGGUGGAGAUGCUCCUdTdT
1195
X61663
AGGAGCAUCUCCACCCUUGdTdT
1605

960853.1

AD-
XD-18468
X61664
AGGGUGGAGAUGCUCCAGUdTdT
1197
X61665
ACUGGAGCAUCUCCACCCUdTdT
1607

960855.1

AD-
XD-18469
X61666
GGGUGGAGAUGCUCCAGCUdTdT
1198
X61667
AGCUGGAGCAUCUCCACCCdTdT
1608

960856.1

AD-
XD-18470
X61668
GGUGGAGAUGCUCCAGCUUdTdT
1199
X61669
AAGCUGGAGCAUCUCCACCdTdT
1609

960857.1

AD-
XD-18471
X61670
UGGAGAUGCUCCAGCUGCUdTdT
1201
X61671
AGCAGCUGGAGCAUCUCCAdTdT
1611

960859.1

AD-
XD-18472
X61672
GGAGAUGCUCCAGCUGCUUdTdT
1202
X61673
AAGCAGCUGGAGCAUCUCCdTdT
1612

960860.1

AD-
XD-18473
X61674
GAGAUGCUCCAGCUGCUGUdTdT
1203
X61675
ACAGCAGCUGGAGCAUCUCdTdT
1613

960861.1

AD-
XD-18474
X61676
GAUGCUCCAGCUGCUGGUUdTdT
1205
X61677
AACCAGCAGCUGGAGCAUCdTdT
1615

960863.1

AD-
XD-18475
X61678
AUGCUCCAGCUGCUGGUGUdTdT
1206
X61679
ACACCAGCAGCUGGAGCAUdTdT
1616

960864.1

AD-
XD-18476
X61680
UGCUCCAGCUGCUGGUGAUdTdT
1207
X61681
AUCACCAGCAGCUGGAGCAdTdT
1617

960865.1

AD-
XD-18477
X61682
CUCCAGCUGCUGGUGAAGUdTdT
1209
X61683
ACUUCACCAGCAGCUGGAGdTdT
1619

960867.1

AD-
XD-18478
X61684
UCCAGCUGCUGGUGAAGAUdTdT
1210
X61685
AUCUUCACCAGCAGCUGGAdTdT
1620

960868.1

AD-
XD-18479
X61686
CCAGCUGCUGGUGAAGAUUdTdT
1211
X61687
AAUCUUCACCAGCAGCUGGdTdT
1621

960869.1

AD-
XD-18480
X61688
AGCUGCUGGUGAAGAUGCUdTdT
1213
X61689
AGCAUCUUCACCAGCAGCUdTdT
1623

960871.1

AD-
XD-18481
X61690
GCUGCUGGUGAAGAUGCAUdTdT
1214
X61691
AUGCAUCUUCACCAGCAGCdTdT
1624

960872.1

AD-
XD-18482
X61692
CUGCUGGUGAAGAUGCAUUdTdT
1215
X61693
AAUGCAUCUUCACCAGCAGdTdT
1625

960873.1

AD-
XD-18483
X61694
GCUGGUGAAGAUGCAUGAUdTdT
1217
X61695
AUCAUGCAUCUUCACCAGCdTdT
1627

960875.1

AD-
XD-18484
X61696
CUGGUGAAGAUGCAUGAAUdTdT
1218
X61697
AUUCAUGCAUCUUCACCAGdTdT
1628

960876.1

AD-
XD-18485
X61698
UGGUGAAGAUGCAUGAAUUdTdT
1219
X61699
AAUUCAUGCAUCUUCACCAdTdT
1629

960877.1

AD-
XD-18486
X61700
GUGAAGAUGCAUGAAUAGUdTdT
1221
X61701
ACUAUUCAUGCAUCUUCACdTdT
1631

960879.1

AD-
XD-18487
X61702
UGAAGAUGCAUGAAUAGGUdTdT
1222
X61703
ACCUAUUCAUGCAUCUUCAdTdT
1632

960880.1

AD-
XD-18488
X61704
GAAGAUGCAUGAAUAGGUUdTdT
1223
X61705
AACCUAUUCAUGCAUCUUCdTdT
1633

960881.1

AD-
XD-18489
X61706
AAGAUGCAUGAAUAGGUCUdTdT
1224
X61707
AGACCUAUUCAUGCAUCUUdTdT
1634

960882.1

AD-
XD-18490
X61708
GAUGCAUGAAUAGGUCCAUdTdT
1226
X61709
AUGGACCUAUUCAUGCAUCdTdT
1636

960884.1

AD-
XD-18491
X61710
AUGCAUGAAUAGGUCCAAUdTdT
1227
X61711
AUUGGACCUAUUCAUGCAUdTdT
1637

960885.1

AD-
XD-18492
X61712
UGCAUGAAUAGGUCCAACUdTdT
1228
X61713
AGUUGGACCUAUUCAUGCAdTdT
1638

960886.1

AD-
XD-18493
X61714
CAUGAAUAGGUCCAACCAUdTdT
1230
X61715
AUGGUUGGACCUAUUCAUGdTdT
1640

960888.1

AD-
XD-18494
X61716
AUGAAUAGGUCCAACCAGUdTdT
1231
X61717
ACUGGUUGGACCUAUUCAUdTdT
1641

960889.1

AD-
XD-18495
X61718
UGAAUAGGUCCAACCAGCUdTdT
1232
X61719
AGCUGGUUGGACCUAUUCAdTdT
1642

960890.1

AD-
XD-18496
X61720
AAUAGGUCCAACCAGCUGUdTdT
1234
X61721
ACAGCUGGUUGGACCUAUUdTdT
1644

960892.1

AD-
XD-18497
X61722
AUAGGUCCAACCAGCUGUUdTdT
1235
X61723
AACAGCUGGUUGGACCUAUdTdT
1645

960893.1

AD-
XD-18498
X61724
UAGGUCCAACCAGCUGUAUdTdT
1236
X61725
AUACAGCUGGUUGGACCUAdTdT
1646

960894.1

AD-
XD-18499
X61726
AGGUCCAACCAGCUGUACUdTdT
1237
X61727
AGUACAGCUGGUUGGACCUdTdT
1647

960895.1

AD-
XD-18500
X61728
GGUCCAACCAGCUGUACAUdTdT
1238
X61729
AUGUACAGCUGGUUGGACCdTdT
1648

960896.1

AD-
XD-18501
X61730
GUCCAACCAGCUGUACAUUdTdT
1239
X61731
AAUGUACAGCUGGUUGGACdTdT
1649

960897.1

AD-
XD-18502
X61732
UCCAACCAGCUGUACAUUUdTdT
1240
X61733
AAAUGUACAGCUGGUUGGAdTdT
1650

960898.1

AD-
XD-18503
X61734
CCAACCAGCUGUACAUUUUdTdT
1241
X61735
AAAAUGUACAGCUGGUUGGdTdT
1651

960899.1

AD-
XD-18504
X61736
CAACCAGCUGUACAUUUGUdTdT
1242
X61737
ACAAAUGUACAGCUGGUUGdTdT
1652

960900.1

AD-
XD-18505
X61738
AACCAGCUGUACAUUUGGUdTdT
1243
X61739
ACCAAAUGUACAGCUGGUUdTdT
1653

960901.1

AD-
XD-18506
X61740
ACCAGCUGUACAUUUGGAUdTdT
1244
X61741
AUCCAAAUGUACAGCUGGUdTdT
1654

960902.1

AD-
XD-18507
X61742
CCAGCUGUACAUUUGGAAUdTdT
1245
X61743
AUUCCAAAUGUACAGCUGGdTdT
1655

960903.1

AD-
XD-18508
X61744
CAGCUGUACAUUUGGAAAUdTdT
1246
X61745
AUUUCCAAAUGUACAGCUGdTdT
1656

960904.1

AD-
XD-18509
X61746
AGCUGUACAUUUGGAAAAUdTdT
1247
X61747
AUUUUCCAAAUGUACAGCUdTdT
1657

960905.1

AD-
XD-18510
X61748
GCUGUACAUUUGGAAAAAUdTdT
1248
X61749
AUUUUUCCAAAUGUACAGCdTdT
1658

960906.1

AD-
XD-18511
X61750
CUGUACAUUUGGAAAAAUUdTdT
1249
X61751
AAUUUUUCCAAAUGUACAGdTdT
1659

960907.1

AD-
XD-18512
X61752
UGUACAUUUGGAAAAAUAUdTdT
1250
X61753
AUAUUUUUCCAAAUGUACAdTdT
1660

960908.1

AD-
XD-18513
X61754
GUACAUUUGGAAAAAUAAUdTdT
1251
X61755
AUUAUUUUUCCAAAUGUACdTdT
1661

960909.1

AD-
XD-18514
X61756
CAUUUGGAAAAAUAAAACUdTdT
1252
X61757
AGUUUUAUUUUUCCAAAUGdTdT
1662

960910.1

TABLE 13

RPS25 Unmodified Duplex Sequences

Start Site
End Site

Duplex
in
in
Sense Sequence
Antisense Sequence
Target Sequence

Name
NM_001028.3
NM_00128.3
5′ to 3′
5′ to 3′
5′ to 3′

AD-
56
78
CAAUGCCGCCUAAGGACGACA
UGUCGUCCUUAGGCGGCAUUGCG
CGCAATGCCGCCTAAGGACGACA

1381680

AD-
57
79
AAUGCCGCCUAAGGACGACAA
UUGUCGUCCUUAGGCGGCAUUGC
GCAATGCCGCCTAAGGACGACAA

1381681

AD-
59
81
UGCCGCCUAAGGACGACAAGA
UCUUGUCGUCCUUAGGCGGCAUU
AATGCCGCCTAAGGACGACAAGA

1381682

AD-
60
82
GCCGCCUAAGGACGACAAGAA
UUCUUGUCGUCCUUAGGCGGCAU
ATGCCGCCTAAGGACGACAAGAA

1381683

AD-
61
83
CCGCCUAAGGACGACAAGAAG
CUUCUUGUCGUCCUUAGGCGGCA
TGCCGCCTAAGGACGACAAGAAG

1381684

AD-
63
85
GCCUAAGGACGACAAGAAGAA
UUCUUCUUGUCGUCCUUAGGCGG
CCGCCTAAGGACGACAAGAAGAA

1381685

AD-
64
86
CCUAAGGACGACAAGAAGAAG
CUUCUUCUUGUCGUCCUUAGGCG
CGCCTAAGGACGACAAGAAGAAG

1381686

AD-
65
87
CUAAGGACGACAAGAAGAAGA
UCUUCUUCUUGUCGUCCUUAGGC
GCCTAAGGACGACAAGAAGAAGA

1381687

AD-
67
89
AAGGACGACAAGAAGAAGAAG
CUUCUUCUUCUUGUCGUCCUUAG
CTAAGGACGACAAGAAGAAGAAG

1381688

AD-
68
90
AGGACGACAAGAAGAAGAAGG
CCUUCUUCUUCUUGUCGUCCUUA
TAAGGACGACAAGAAGAAGAAGG

1381689

AD-
69
91
GGACGACAAGAAGAAGAAGGA
UCCUUCUUCUUCUUGUCGUCCUU
AAGGACGACAAGAAGAAGAAGGA

1381690

AD-
71
93
ACGACAAGAAGAAGAAGGACG
CGUCCUUCUUCUUCUUGUCGUCC
GGACGACAAGAAGAAGAAGGACG

1381691

AD-
72
94
CGACAAGAAGAAGAAGGACGC
GCGUCCUUCUUCUUCUUGUCGUC
GACGACAAGAAGAAGAAGGACGC

1381692

AD-
73
95
GACAAGAAGAAGAAGGACGCU
AGCGUCCUUCUUCUUCUUGUCGU
ACGACAAGAAGAAGAAGGACGCT

1381693

AD-
75
97
CAAGAAGAAGAAGGACGCUGG
CCAGCGUCCUUCUUCUUCUUGUC
GACAAGAAGAAGAAGGACGCTGG

1381694

AD-
76
98
AAGAAGAAGAAGGACGCUGGA
UCCAGCGUCCUUCUUCUUCUUGU
ACAAGAAGAAGAAGGACGCTGGA

1381695

AD-
77
99
AGAAGAAGAAGGACGCUGGAA
UUCCAGCGUCCUUCUUCUUCUUG
CAAGAAGAAGAAGGACGCTGGAA

1381696

AD-
78
100
GAAGAAGAAGGACGCUGGAAA
UUUCCAGCGUCCUUCUUCUUCUU
AAGAAGAAGAAGGACGCTGGAAA

1381697

AD-
80
102
AGAAGAAGGACGCUGGAAAGU
ACUUUCCAGCGUCCUUCUUCUUC
GAAGAAGAAGGACGCTGGAAAGT

1381698

AD-
81
103
GAAGAAGGACGCUGGAAAGUC
GACUUUCCAGCGUCCUUCUUCUU
AAGAAGAAGGACGCTGGAAAGTC

1381699

AD-
82
104
AAGAAGGACGCUGGAAAGUCG
CGACUUUCCAGCGUCCUUCUUCU
AGAAGAAGGACGCTGGAAAGTCG

1381700

AD-
84
106
GAAGGACGCUGGAAAGUCGGC
GCCGACUUUCCAGCGUCCUUCUU
AAGAAGGACGCTGGAAAGTCGGC

1381701

AD-
85
107
AAGGACGCUGGAAAGUCGGCC
GGCCGACUUUCCAGCGUCCUUCU
AGAAGGACGCTGGAAAGTCGGCC

1381702

AD-
86
108
AGGACGCUGGAAAGUCGGCCA
UGGCCGACUUUCCAGCGUCCUUC
GAAGGACGCTGGAAAGTCGGCCA

1381703

AD-
88
110
GACGCUGGAAAGUCGGCCAAG
CUUGGCCGACUUUCCAGCGUCCU
AGGACGCTGGAAAGTCGGCCAAG

1381704

AD-
89
111
ACGCUGGAAAGUCGGCCAAGA
UCUUGGCCGACUUUCCAGCGUCC
GGACGCTGGAAAGTCGGCCAAGA

1381705

AD-
90
112
CGCUGGAAAGUCGGCCAAGAA
UUCUUGGCCGACUUUCCAGCGUC
GACGCTGGAAAGTCGGCCAAGAA

1381706

AD-
92
114
CUGGAAAGUCGGCCAAGAAAG
CUUUCUUGGCCGACUUUCCAGCG
CGCTGGAAAGTCGGCCAAGAAAG

1381707

AD-
93
115
UGGAAAGUCGGCCAAGAAAGA
UCUUUCUUGGCCGACUUUCCAGC
GCTGGAAAGTCGGCCAAGAAAGA

1381708

AD-
94
116
GGAAAGUCGGCCAAGAAAGAC
GUCUUUCUUGGCCGACUUUCCAG
CTGGAAAGTCGGCCAAGAAAGAC

1381709

AD-
96
118
AAAGUCGGCCAAGAAAGACAA
UUGUCUUUCUUGGCCGACUUUCC
GGAAAGTCGGCCAAGAAAGACAA

1381710

AD-
97
119
AAGUCGGCCAAGAAAGACAAA
UUUGUCUUUCUUGGCCGACUUUC
GAAAGTCGGCCAAGAAAGACAAA

1381711

AD-
98
120
AGUCGGCCAAGAAAGACAAAG
CUUUGUCUUUCUUGGCCGACUUU
AAAGTCGGCCAAGAAAGACAAAG

1381712

AD-
100
122
UCGGCCAAGAAAGACAAAGAC
GUCUUUGUCUUUCUUGGCCGACU
AGTCGGCCAAGAAAGACAAAGAC

1381713

AD-
101
123
CGGCCAAGAAAGACAAAGACC
GGUCUUUGUCUUUCUUGGCCGAC
GTCGGCCAAGAAAGACAAAGACC

1381714

AD-
102
124
GGCCAAGAAAGACAAAGACCC
GGGUCUUUGUCUUUCUUGGCCGA
TCGGCCAAGAAAGACAAAGACCC

1381715

AD-
105
127
CAAGAAAGACAAAGACCCAGU
ACUGGGUCUUUGUCUUUCUUGGC
GCCAAGAAAGACAAAGACCCAGT

1381716

AD-
106
128
AAGAAAGACAAAGACCCAGUG
CACUGGGUCUUUGUCUUUCUUGG
CCAAGAAAGACAAAGACCCAGTG

1381717

AD-
107
129
AGAAAGACAAAGACCCAGUGA
UCACUGGGUCUUUGUCUUUCUUG
CAAGAAAGACAAAGACCCAGTGA

1381718

AD-
109
131
AAAGACAAAGACCCAGUGAAC
GUUCACUGGGUCUUUGUCUUUCU
AGAAAGACAAAGACCCAGTGAAC

1381719

AD-
110
132
AAGACAAAGACCCAGUGAACA
UGUUCACUGGGUCUUUGUCUUUC
GAAAGACAAAGACCCAGTGAACA

1381720

AD-
111
133
AGACAAAGACCCAGUGAACAA
UUGUUCACUGGGUCUUUGUCUUU
AAAGACAAAGACCCAGTGAACAA

1381721

AD-
112
134
GACAAAGACCCAGUGAACAAA
UUUGUUCACUGGGUCUUUGUCUU
AAGACAAAGACCCAGTGAACAAA

1381722

AD-
114
136
CAAAGACCCAGUGAACAAAUC
GAUUUGUUCACUGGGUCUUUGUC
GACAAAGACCCAGTGAACAAATC

1381723

AD-
115
137
AAAGACCCAGUGAACAAAUCC
GGAUUUGUUCACUGGGUCUUUGU
ACAAAGACCCAGTGAACAAATCC

1381724

AD-
116
138
AAGACCCAGUGAACAAAUCCG
CGGAUUUGUUCACUGGGUCUUUG
CAAAGACCCAGTGAACAAATCCG

1381725

AD-
118
140
GACCCAGUGAACAAAUCCGGG
CCCGGAUUUGUUCACUGGGUCUU
AAGACCCAGTGAACAAATCCGGG

1381726

AD-
136
158
GGGGGCAAGGCCAAAAAGAAG
CUUCUUUUUGGCCUUGCCCCCGG
CCGGGGGCAAGGCCAAAAAGAAG

1381727

AD-
137
159
GGGGCAAGGCCAAAAAGAAGA
UCUUCUUUUUGGCCUUGCCCCCG
CGGGGGCAAGGCCAAAAAGAAGA

1381728

AD-
141
163
CAAGGCCAAAAAGAAGAAGUG
CACUUCUUCUUUUUGGCCUUGCC
GGCAAGGCCAAAAAGAAGAAGTG

1381729

AD-
142
164
AAGGCCAAAAAGAAGAAGUGG
CCACUUCUUCUUUUUGGCCUUGC
GCAAGGCCAAAAAGAAGAAGTGG

1381730

AD-
143
165
AGGCCAAAAAGAAGAAGUGGU
ACCACUUCUUCUUUUUGGCCUUG
CAAGGCCAAAAAGAAGAAGTGGT

1381731

AD-
145
167
GCCAAAAAGAAGAAGUGGUCC
GGACCACUUCUUCUUUUUGGCCU
AGGCCAAAAAGAAGAAGTGGTCC

1381732

AD-
146
168
CCAAAAAGAAGAAGUGGUCCA
UGGACCACUUCUUCUUUUUGGCC
GGCCAAAAAGAAGAAGTGGTCCA

1381733

AD-
147
169
CAAAAAGAAGAAGUGGUCCAA
UUGGACCACUUCUUCUUUUUGGC
GCCAAAAAGAAGAAGTGGTCCAA

1381734

AD-
149
171
AAAAGAAGAAGUGGUCCAAAG
CUUUGGACCACUUCUUCUUUUUG
CAAAAAGAAGAAGTGGTCCAAAG

1381735

AD-
150
172
AAAGAAGAAGUGGUCCAAAGG
CCUUUGGACCACUUCUUCUUUUU
AAAAAGAAGAAGTGGTCCAAAGG

1381736

AD-
151
173
AAGAAGAAGUGGUCCAAAGGC
GCCUUUGGACCACUUCUUCUUUU
AAAAGAAGAAGTGGTCCAAAGGC

1381737

AD-
153
175
GAAGAAGUGGUCCAAAGGCAA
UUGCCUUUGGACCACUUCUUCUU
AAGAAGAAGTGGTCCAAAGGCAA

1381738

AD-
154
176
AAGAAGUGGUCCAAAGGCAAA
UUUGCCUUUGGACCACUUCUUCU
AGAAGAAGTGGTCCAAAGGCAAA

1381739

AD-
155
177
AGAAGUGGUCCAAAGGCAAAG
CUUUGCCUUUGGACCACUUCUUC
GAAGAAGTGGTCCAAAGGCAAAG

1381740

AD-
157
179
AAGUGGUCCAAAGGCAAAGUU
AACUUUGCCUUUGGACCACUUCU
AGAAGTGGTCCAAAGGCAAAGTT

1381741

AD-
158
180
AGUGGUCCAAAGGCAAAGUUC
GAACUUUGCCUUUGGACCACUUC
GAAGTGGTCCAAAGGCAAAGTTC

1381742

AD-
159
181
GUGGUCCAAAGGCAAAGUUCG
CGAACUUUGCCUUUGGACCACUU
AAGTGGTCCAAAGGCAAAGTTCG

1381743

AD-
161
183
GGUCCAAAGGCAAAGUUCGGG
CCCGAACUUUGCCUUUGGACCAC
GTGGTCCAAAGGCAAAGTTCGGG

1381744

AD-
162
184
GUCCAAAGGCAAAGUUCGGGA
UCCCGAACUUUGCCUUUGGACCA
TGGTCCAAAGGCAAAGTTCGGGA

1381745

AD-
163
185
UCCAAAGGCAAAGUUCGGGAC
GUCCCGAACUUUGCCUUUGGACC
GGTCCAAAGGCAAAGTTCGGGAC

1381746

AD-
165
187
CAAAGGCAAAGUUCGGGACAA
UUGUCCCGAACUUUGCCUUUGGA
TCCAAAGGCAAAGTTCGGGACAA

1381747

AD-
166
188
AAAGGCAAAGUUCGGGACAAG
CUUGUCCCGAACUUUGCCUUUGG
CCAAAGGCAAAGTTCGGGACAAG

1381748

AD-
167
189
AAGGCAAAGUUCGGGACAAGC
GCUUGUCCCGAACUUUGCCUUUG
CAAAGGCAAAGTTCGGGACAAGC

1381749

AD-
169
191
GGCAAAGUUCGGGACAAGCUC
GAGCUUGUCCCGAACUUUGCCUU
AAGGCAAAGTTCGGGACAAGCTC

1381750

AD-
170
192
GCAAAGUUCGGGACAAGCUCA
UGAGCUUGUCCCGAACUUUGCCU
AGGCAAAGTTCGGGACAAGCTCA

1381751

AD-
171
193
CAAAGUUCGGGACAAGCUCAA
UUGAGCUUGUCCCGAACUUUGCC
GGCAAAGTTCGGGACAAGCTCAA

1381752

AD-
172
194
AAAGUUCGGGACAAGCUCAAU
AUUGAGCUUGUCCCGAACUUUGC
GCAAAGTTCGGGACAAGCTCAAT

1381753

AD-
174
196
AGUUCGGGACAAGCUCAAUAA
UUAUUGAGCUUGUCCCGAACUUU
AAAGTTCGGGACAAGCTCAATAA

1381754

AD-
175
197
GUUCGGGACAAGCUCAAUAAC
GUUAUUGAGCUUGUCCCGAACUU
AAGTTCGGGACAAGCTCAATAAC

1381755

AD-
176
198
UUCGGGACAAGCUCAAUAACU
AGUUAUUGAGCUUGUCCCGAACU
AGTTCGGGACAAGCTCAATAACT

1381756

AD-
178
200
CGGGACAAGCUCAAUAACUUA
UAAGUUAUUGAGCUUGUCCCGAA
TTCGGGACAAGCTCAATAACTTA

1381757

AD-
179
201
GGGACAAGCUCAAUAACUUAG
CUAAGUUAUUGAGCUUGUCCCGA
TCGGGACAAGCTCAATAACTTAG

1381758

AD-
180
202
GGACAAGCUCAAUAACUUAGU
ACUAAGUUAUUGAGCUUGUCCCG
CGGGACAAGCTCAATAACTTAGT

1381759

AD-
182
204
ACAAGCUCAAUAACUUAGUCU
AGACUAAGUUAUUGAGCUUGUCC
GGACAAGCTCAATAACTTAGTCT

1381760

AD-
183
205
CAAGCUCAAUAACUUAGUCUU
AAGACUAAGUUAUUGAGCUUGUC
GACAAGCTCAATAACTTAGTCTT

1381761

AD-
184
206
AAGCUCAAUAACUUAGUCUUG
CAAGACUAAGUUAUUGAGCUUGU
ACAAGCTCAATAACTTAGTCTTG

1381762

AD-
186
208
GCUCAAUAACUUAGUCUUGUU
AACAAGACUAAGUUAUUGAGCUU
AAGCTCAATAACTTAGTCTTGTT

1381763

AD-
187
209
CUCAAUAACUUAGUCUUGUUU
AAACAAGACUAAGUUAUUGAGCU
AGCTCAATAACTTAGTCTTGTTT

1381764

AD-
188
210
UCAAUAACUUAGUCUUGUUUG
CAAACAAGACUAAGUUAUUGAGC
GCTCAATAACTTAGTCTTGTTTG

1381765

AD-
190
212
AAUAACUUAGUCUUGUUUGAC
GUCAAACAAGACUAAGUUAUUGA
TCAATAACTTAGTCTTGTTTGAC

1381766

AD-
191
213
AUAACUUAGUCUUGUUUGACA
UGUCAAACAAGACUAAGUUAUUG
CAATAACTTAGTCTTGTTTGACA

1381767

AD-
192
214
UAACUUAGUCUUGUUUGACAA
UUGUCAAACAAGACUAAGUUAUU
AATAACTTAGTCTTGTTTGACAA

1381768

AD-
194
216
ACUUAGUCUUGUUUGACAAAG
CUUUGUCAAACAAGACUAAGUUA
TAACTTAGTCTTGTTTGACAAAG

1381769

AD-
195
217
CUUAGUCUUGUUUGACAAAGC
GCUUUGUCAAACAAGACUAAGUU
AACTTAGTCTTGTTTGACAAAGC

1381770

AD-
196
218
UUAGUCUUGUUUGACAAAGCU
AGCUUUGUCAAACAAGACUAAGU
ACTTAGTCTTGTTTGACAAAGCT

1381771

AD-
198
220
AGUCUUGUUUGACAAAGCUAC
GUAGCUUUGUCAAACAAGACUAA
TTAGTCTTGTTTGACAAAGCTAC

1381772

AD-
199
221
GUCUUGUUUGACAAAGCUACC
GGUAGCUUUGUCAAACAAGACUA
TAGTCTTGTTTGACAAAGCTACC

1381773

AD-
200
222
UCUUGUUUGACAAAGCUACCU
AGGUAGCUUUGUCAAACAAGACU
AGTCTTGTTTGACAAAGCTACCT

1381774

AD-
202
224
UUGUUUGACAAAGCUACCUAU
AUAGGUAGCUUUGUCAAACAAGA
TCTTGTTTGACAAAGCTACCTAT

1381775

AD-
203
225
UGUUUGACAAAGCUACCUAUG
CAUAGGUAGCUUUGUCAAACAAG
CTTGTTTGACAAAGCTACCTATG

1381776

AD-
204
226
GUUUGACAAAGCUACCUAUGA
UCAUAGGUAGCUUUGUCAAACAA
TTGTTTGACAAAGCTACCTATGA

1381777

AD-
205
227
UUUGACAAAGCUACCUAUGAU
AUCAUAGGUAGCUUUGUCAAACA
TGTTTGACAAAGCTACCTATGAT

1381778

AD-
207
229
UGACAAAGCUACCUAUGAUAA
UUAUCAUAGGUAGCUUUGUCAAA
TTTGACAAAGCTACCTATGATAA

1381779

AD-
208
230
GACAAAGCUACCUAUGAUAAA
UUUAUCAUAGGUAGCUUUGUCAA
TTGACAAAGCTACCTATGATAAA

1381780

AD-
209
231
ACAAAGCUACCUAUGAUAAAC
GUUUAUCAUAGGUAGCUUUGUCA
TGACAAAGCTACCTATGATAAAC

1381781

AD-
211
233
AAAGCUACCUAUGAUAAACUC
GAGUUUAUCAUAGGUAGCUUUGU
ACAAAGCTACCTATGATAAACTC

1381782

AD-
212
234
AAGCUACCUAUGAUAAACUCU
AGAGUUUAUCAUAGGUAGCUUUG
CAAAGCTACCTATGATAAACTCT

1381783

AD-
213
235
AGCUACCUAUGAUAAACUCUG
CAGAGUUUAUCAUAGGUAGCUUU
AAAGCTACCTATGATAAACTCTG

1381784

AD-
215
237
CUACCUAUGAUAAACUCUGUA
UACAGAGUUUAUCAUAGGUAGCU
AGCTACCTATGATAAACTCTGTA

1381785

AD-
216
238
UACCUAUGAUAAACUCUGUAA
UUACAGAGUUUAUCAUAGGUAGC
GCTACCTATGATAAACTCTGTAA

1381786

AD-
217
239
ACCUAUGAUAAACUCUGUAAG
CUUACAGAGUUUAUCAUAGGUAG
CTACCTATGATAAACTCTGTAAG

1381787

AD-
219
241
CUAUGAUAAACUCUGUAAGGA
UCCUUACAGAGUUUAUCAUAGGU
ACCTATGATAAACTCTGTAAGGA

1381788

AD-
220
242
UAUGAUAAACUCUGUAAGGAA
UUCCUUACAGAGUUUAUCAUAGG
CCTATGATAAACTCTGTAAGGAA

1381789

AD-
221
243
AUGAUAAACUCUGUAAGGAAG
CUUCCUUACAGAGUUUAUCAUAG
CTATGATAAACTCTGTAAGGAAG

1381790

AD-
223
245
GAUAAACUCUGUAAGGAAGUU
AACUUCCUUACAGAGUUUAUCAU
ATGATAAACTCTGTAAGGAAGTT

1381791

AD-
224
246
AUAAACUCUGUAAGGAAGUUC
GAACUUCCUUACAGAGUUUAUCA
TGATAAACTCTGTAAGGAAGTTC

1381792

AD-
225
247
UAAACUCUGUAAGGAAGUUCC
GGAACUUCCUUACAGAGUUUAUC
GATAAACTCTGTAAGGAAGTTCC

1381793

AD-
227
249
AACUCUGUAAGGAAGUUCCCA
UGGGAACUUCCUUACAGAGUUUA
TAAACTCTGTAAGGAAGTTCCCA

1381794

AD-
228
250
ACUCUGUAAGGAAGUUCCCAA
UUGGGAACUUCCUUACAGAGUUU
AAACTCTGTAAGGAAGTTCCCAA

1381795

AD-
229
251
CUCUGUAAGGAAGUUCCCAAC
GUUGGGAACUUCCUUACAGAGUU
AACTCTGTAAGGAAGTTCCCAAC

1381796

AD-
231
253
CUGUAAGGAAGUUCCCAACUA
UAGUUGGGAACUUCCUUACAGAG
CTCTGTAAGGAAGTTCCCAACTA

1381797

AD-
232
254
UGUAAGGAAGUUCCCAACUAU
AUAGUUGGGAACUUCCUUACAGA
TCTGTAAGGAAGTTCCCAACTAT

1381798

AD-
233
255
GUAAGGAAGUUCCCAACUAUA
UAUAGUUGGGAACUUCCUUACAG
CTGTAAGGAAGTTCCCAACTATA

1381799

AD-
235
257
AAGGAAGUUCCCAACUAUAAA
UUUAUAGUUGGGAACUUCCUUAC
GTAAGGAAGTTCCCAACTATAAA

1381800

AD-
236
258
AGGAAGUUCCCAACUAUAAAC
GUUUAUAGUUGGGAACUUCCUUA
TAAGGAAGTTCCCAACTATAAAC

1381801

AD-
237
259
GGAAGUUCCCAACUAUAAACU
AGUUUAUAGUUGGGAACUUCCUU
AAGGAAGTTCCCAACTATAAACT

1381802

AD-
239
261
AAGUUCCCAACUAUAAACUUA
UAAGUUUAUAGUUGGGAACUUCC
GGAAGTTCCCAACTATAAACTTA

1381803

AD-
240
262
AGUUCCCAACUAUAAACUUAU
AUAAGUUUAUAGUUGGGAACUUC
GAAGTTCCCAACTATAAACTTAT

1381804

AD-
241
263
GUUCCCAACUAUAAACUUAUA
UAUAAGUUUAUAGUUGGGAACUU
AAGTTCCCAACTATAAACTTATA

1381805

AD-
243
265
UCCCAACUAUAAACUUAUAAC
GUUAUAAGUUUAUAGUUGGGAAC
GTTCCCAACTATAAACTTATAAC

1381806

AD-
244
266
CCCAACUAUAAACUUAUAACC
GGUUAUAAGUUUAUAGUUGGGAA
TTCCCAACTATAAACTTATAACC

1381807

AD-
245
267
CCAACUAUAAACUUAUAACCC
GGGUUAUAAGUUUAUAGUUGGGA
TCCCAACTATAAACTTATAACCC

1381808

AD-
262
284
ACCCCAGCUGUGGUCUCUGAG
CUCAGAGACCACAGCUGGGGUUA
TAACCCCAGCTGTGGTCTCTGAG

1381809

AD-
264
286
CCCAGCUGUGGUCUCUGAGAG
CUCUCAGAGACCACAGCUGGGGU
ACCCCAGCTGTGGTCTCTGAGAG

1381810

AD-
265
287
CCAGCUGUGGUCUCUGAGAGA
UCUCUCAGAGACCACAGCUGGGG
CCCCAGCTGTGGTCTCTGAGAGA

1381811

AD-
266
288
CAGCUGUGGUCUCUGAGAGAC
GUCUCUCAGAGACCACAGCUGGG
CCCAGCTGTGGTCTCTGAGAGAC

1381812

AD-
268
290
GCUGUGGUCUCUGAGAGACUG
CAGUCUCUCAGAGACCACAGCUG
CAGCTGTGGTCTCTGAGAGACTG

1381813

AD-
269
291
CUGUGGUCUCUGAGAGACUGA
UCAGUCUCUCAGAGACCACAGCU
AGCTGTGGTCTCTGAGAGACTGA

1381814

AD-
270
292
UGUGGUCUCUGAGAGACUGAA
UUCAGUCUCUCAGAGACCACAGC
GCTGTGGTCTCTGAGAGACTGAA

1381815

AD-
272
294
UGGUCUCUGAGAGACUGAAGA
UCUUCAGUCUCUCAGAGACCACA
TGTGGTCTCTGAGAGACTGAAGA

1381816

AD-
273
295
GGUCUCUGAGAGACUGAAGAU
AUCUUCAGUCUCUCAGAGACCAC
GTGGTCTCTGAGAGACTGAAGAT

1381817

AD-
274
296
GUCUCUGAGAGACUGAAGAUU
AAUCUUCAGUCUCUCAGAGACCA
TGGTCTCTGAGAGACTGAAGATT

1381818

AD-
276
298
CUCUGAGAGACUGAAGAUUCG
CGAAUCUUCAGUCUCUCAGAGAC
GTCTCTGAGAGACTGAAGATTCG

1381819

AD-
277
299
UCUGAGAGACUGAAGAUUCGA
UCGAAUCUUCAGUCUCUCAGAGA
TCTCTGAGAGACTGAAGATTCGA

1381820

AD-
278
300
CUGAGAGACUGAAGAUUCGAG
CUCGAAUCUUCAGUCUCUCAGAG
CTCTGAGAGACTGAAGATTCGAG

1381821

AD-
280
302
GAGAGACUGAAGAUUCGAGGC
GCCUCGAAUCUUCAGUCUCUCAG
CTGAGAGACTGAAGATTCGAGGC

1381822

AD-
281
303
AGAGACUGAAGAUUCGAGGCU
AGCCUCGAAUCUUCAGUCUCUCA
TGAGAGACTGAAGATTCGAGGCT

1381823

AD-
282
304
GAGACUGAAGAUUCGAGGCUC
GAGCCUCGAAUCUUCAGUCUCUC
GAGAGACTGAAGATTCGAGGCTC

1381824

AD-
284
306
GACUGAAGAUUCGAGGCUCCC
GGGAGCCUCGAAUCUUCAGUCUC
GAGACTGAAGATTCGAGGCTCCC

1381825

AD-
285
307
ACUGAAGAUUCGAGGCUCCCU
AGGGAGCCUCGAAUCUUCAGUCU
AGACTGAAGATTCGAGGCTCCCT

1381826

AD-
286
308
CUGAAGAUUCGAGGCUCCCUG
CAGGGAGCCUCGAAUCUUCAGUC
GACTGAAGATTCGAGGCTCCCTG

1381827

AD-
288
310
GAAGAUUCGAGGCUCCCUGGC
GCCAGGGAGCCUCGAAUCUUCAG
CTGAAGATTCGAGGCTCCCTGGC

1381828

AD-
289
311
AAGAUUCGAGGCUCCCUGGCC
GGCCAGGGAGCCUCGAAUCUUCA
TGAAGATTCGAGGCTCCCTGGCC

1381829

AD-
290
312
AGAUUCGAGGCUCCCUGGCCA
UGGCCAGGGAGCCUCGAAUCUUC
GAAGATTCGAGGCTCCCTGGCCA

1381830

AD-
292
314
AUUCGAGGCUCCCUGGCCAGG
CCUGGCCAGGGAGCCUCGAAUCU
AGATTCGAGGCTCCCTGGCCAGG

1381831

AD-
302
324
CCCUGGCCAGGGCAGCCCUUC
GAAGGGCUGCCCUGGCCAGGGAG
CTCCCTGGCCAGGGCAGCCCTTC

1381832

AD-
303
325
CCUGGCCAGGGCAGCCCUUCA
UGAAGGGCUGCCCUGGCCAGGGA
TCCCTGGCCAGGGCAGCCCTTCA

1381833

AD-
304
326
CUGGCCAGGGCAGCCCUUCAG
CUGAAGGGCUGCCCUGGCCAGGG
CCCTGGCCAGGGCAGCCCTTCAG

1381834

AD-
306
328
GGCCAGGGCAGCCCUUCAGGA
UCCUGAAGGGCUGCCCUGGCCAG
CTGGCCAGGGCAGCCCTTCAGGA

1381835

AD-
307
329
GCCAGGGCAGCCCUUCAGGAG
CUCCUGAAGGGCUGCCCUGGCCA
TGGCCAGGGCAGCCCTTCAGGAG

1381836

AD-
308
330
CCAGGGCAGCCCUUCAGGAGC
GCUCCUGAAGGGCUGCCCUGGCC
GGCCAGGGCAGCCCTTCAGGAGC

1381837

AD-
310
332
AGGGCAGCCCUUCAGGAGCUC
GAGCUCCUGAAGGGCUGCCCUGG
CCAGGGCAGCCCTTCAGGAGCTC

1381838

AD-
311
333
GGGCAGCCCUUCAGGAGCUCC
GGAGCUCCUGAAGGGCUGCCCUG
CAGGGCAGCCCTTCAGGAGCTCC

1381839

AD-
312
334
GGCAGCCCUUCAGGAGCUCCU
AGGAGCUCCUGAAGGGCUGCCCU
AGGGCAGCCCTTCAGGAGCTCCT

1381840

AD-
314
336
CAGCCCUUCAGGAGCUCCUUA
UAAGGAGCUCCUGAAGGGCUGCC
GGCAGCCCTTCAGGAGCTCCTTA

1381841

AD-
315
337
AGCCCUUCAGGAGCUCCUUAG
CUAAGGAGCUCCUGAAGGGCUGC
GCAGCCCTTCAGGAGCTCCTTAG

1381842

AD-
316
338
GCCCUUCAGGAGCUCCUUAGU
ACUAAGGAGCUCCUGAAGGGCUG
CAGCCCTTCAGGAGCTCCTTAGT

1381843

AD-
318
340
CCUUCAGGAGCUCCUUAGUAA
UUACUAAGGAGCUCCUGAAGGGC
GCCCTTCAGGAGCTCCTTAGTAA

1381844

AD-
319
341
CUUCAGGAGCUCCUUAGUAAA
UUUACUAAGGAGCUCCUGAAGGG
CCCTTCAGGAGCTCCTTAGTAAA

1381845

AD-
320
342
UUCAGGAGCUCCUUAGUAAAG
CUUUACUAAGGAGCUCCUGAAGG
CCTTCAGGAGCTCCTTAGTAAAG

1381846

AD-
322
344
CAGGAGCUCCUUAGUAAAGGA
UCCUUUACUAAGGAGCUCCUGAA
TTCAGGAGCTCCTTAGTAAAGGA

1381847

AD-
323
345
AGGAGCUCCUUAGUAAAGGAC
GUCCUUUACUAAGGAGCUCCUGA
TCAGGAGCTCCTTAGTAAAGGAC

1381848

AD-
324
346
GGAGCUCCUUAGUAAAGGACU
AGUCCUUUACUAAGGAGCUCCUG
CAGGAGCTCCTTAGTAAAGGACT

1381849

AD-
326
348
AGCUCCUUAGUAAAGGACUUA
UAAGUCCUUUACUAAGGAGCUCC
GGAGCTCCTTAGTAAAGGACTTA

1381850

AD-
327
349
GCUCCUUAGUAAAGGACUUAU
AUAAGUCCUUUACUAAGGAGCUC
GAGCTCCTTAGTAAAGGACTTAT

1381851

AD-
328
350
CUCCUUAGUAAAGGACUUAUC
GAUAAGUCCUUUACUAAGGAGCU
AGCTCCTTAGTAAAGGACTTATC

1381852

AD-
330
352
CCUUAGUAAAGGACUUAUCAA
UUGAUAAGUCCUUUACUAAGGAG
CTCCTTAGTAAAGGACTTATCAA

1381853

AD-
331
353
CUUAGUAAAGGACUUAUCAAA
UUUGAUAAGUCCUUUACUAAGGA
TCCTTAGTAAAGGACTTATCAAA

1381854

AD-
332
354
UUAGUAAAGGACUUAUCAAAC
GUUUGAUAAGUCCUUUACUAAGG
CCTTAGTAAAGGACTTATCAAAC

1381855

AD-
334
356
AGUAAAGGACUUAUCAAACUG
CAGUUUGAUAAGUCCUUUACUAA
TTAGTAAAGGACTTATCAAACTG

1381856

AD-
335
357
GUAAAGGACUUAUCAAACUGG
CCAGUUUGAUAAGUCCUUUACUA
TAGTAAAGGACTTATCAAACTGG

1381857

AD-
336
358
UAAAGGACUUAUCAAACUGGU
ACCAGUUUGAUAAGUCCUUUACU
AGTAAAGGACTTATCAAACTGGT

1381858

AD-
338
360
AAGGACUUAUCAAACUGGUUU
AAACCAGUUUGAUAAGUCCUUUA
TAAAGGACTTATCAAACTGGTTT

1381859

AD-
339
361
AGGACUUAUCAAACUGGUUUC
GAAACCAGUUUGAUAAGUCCUUU
AAAGGACTTATCAAACTGGTTTC

1381860

AD-
340
362
GGACUUAUCAAACUGGUUUCA
UGAAACCAGUUUGAUAAGUCCUU
AAGGACTTATCAAACTGGTTTCA

1381861

AD-
342
364
ACUUAUCAAACUGGUUUCAAA
UUUGAAACCAGUUUGAUAAGUCC
GGACTTATCAAACTGGTTTCAAA

1381862

AD-
343
365
CUUAUCAAACUGGUUUCAAAG
CUUUGAAACCAGUUUGAUAAGUC
GACTTATCAAACTGGTTTCAAAG

1381863

AD-
344
366
UUAUCAAACUGGUUUCAAAGC
GCUUUGAAACCAGUUUGAUAAGU
ACTTATCAAACTGGTTTCAAAGC

1381864

AD-
345
367
UAUCAAACUGGUUUCAAAGCA
UGCUUUGAAACCAGUUUGAUAAG
CTTATCAAACTGGTTTCAAAGCA

1381865

AD-
347
369
UCAAACUGGUUUCAAAGCACA
UGUGCUUUGAAACCAGUUUGAUA
TATCAAACTGGTTTCAAAGCACA

1381866

AD-
348
370
CAAACUGGUUUCAAAGCACAG
CUGUGCUUUGAAACCAGUUUGAU
ATCAAACTGGTTTCAAAGCACAG

1381867

AD-
349
371
AAACUGGUUUCAAAGCACAGA
UCUGUGCUUUGAAACCAGUUUGA
TCAAACTGGTTTCAAAGCACAGA

1381868

AD-
351
373
ACUGGUUUCAAAGCACAGAGC
GCUCUGUGCUUUGAAACCAGUUU
AAACTGGTTTCAAAGCACAGAGC

1381869

AD-
352
374
CUGGUUUCAAAGCACAGAGCU
AGCUCUGUGCUUUGAAACCAGUU
AACTGGTTTCAAAGCACAGAGCT

1381870

AD-
353
375
UGGUUUCAAAGCACAGAGCUC
GAGCUCUGUGCUUUGAAACCAGU
ACTGGTTTCAAAGCACAGAGCTC

1381871

AD-
355
377
GUUUCAAAGCACAGAGCUCAA
UUGAGCUCUGUGCUUUGAAACCA
TGGTTTCAAAGCACAGAGCTCAA

1381872

AD-
356
378
UUUCAAAGCACAGAGCUCAAG
CUUGAGCUCUGUGCUUUGAAACC
GGTTTCAAAGCACAGAGCTCAAG

1381873

AD-
357
379
UUCAAAGCACAGAGCUCAAGU
ACUUGAGCUCUGUGCUUUGAAAC
GTTTCAAAGCACAGAGCTCAAGT

1381874

AD-
359
381
CAAAGCACAGAGCUCAAGUAA
UUACUUGAGCUCUGUGCUUUGAA
TTCAAAGCACAGAGCTCAAGTAA

1381875

AD-
360
382
AAAGCACAGAGCUCAAGUAAU
AUUACUUGAGCUCUGUGCUUUGA
TCAAAGCACAGAGCTCAAGTAAT

1381876

AD-
361
383
AAGCACAGAGCUCAAGUAAUU
AAUUACUUGAGCUCUGUGCUUUG
CAAAGCACAGAGCTCAAGTAATT

1381877

AD-
363
385
GCACAGAGCUCAAGUAAUUUA
UAAAUUACUUGAGCUCUGUGCUU
AAGCACAGAGCTCAAGTAATTTA

1381878

AD-
364
386
CACAGAGCUCAAGUAAUUUAC
GUAAAUUACUUGAGCUCUGUGCU
AGCACAGAGCTCAAGTAATTTAC

1381879

AD-
365
387
ACAGAGCUCAAGUAAUUUACA
UGUAAAUUACUUGAGCUCUGUGC
GCACAGAGCTCAAGTAATTTACA

1381880

AD-
367
389
AGAGCUCAAGUAAUUUACACC
GGUGUAAAUUACUUGAGCUCUGU
ACAGAGCTCAAGTAATTTACACC

1381881

AD-
368
390
GAGCUCAAGUAAUUUACACCA
UGGUGUAAAUUACUUGAGCUCUG
CAGAGCTCAAGTAATTTACACCA

1381882

AD-
369
391
AGCUCAAGUAAUUUACACCAG
CUGGUGUAAAUUACUUGAGCUCU
AGAGCTCAAGTAATTTACACCAG

1381883

AD-
371
393
CUCAAGUAAUUUACACCAGAA
UUCUGGUGUAAAUUACUUGAGCU
AGCTCAAGTAATTTACACCAGAA

1381884

AD-
372
394
UCAAGUAAUUUACACCAGAAA
UUUCUGGUGUAAAUUACUUGAGC
GCTCAAGTAATTTACACCAGAAA

1381885

AD-
373
395
CAAGUAAUUUACACCAGAAAU
AUUUCUGGUGUAAAUUACUUGAG
CTCAAGTAATTTACACCAGAAAT

1381886

AD-
375
397
AGUAAUUUACACCAGAAAUAC
GUAUUUCUGGUGUAAAUUACUUG
CAAGTAATTTACACCAGAAATAC

1381887

AD-
376
398
GUAAUUUACACCAGAAAUACC
GGUAUUUCUGGUGUAAAUUACUU
AAGTAATTTACACCAGAAATACC

1381888

AD-
377
399
UAAUUUACACCAGAAAUACCA
UGGUAUUUCUGGUGUAAAUUACU
AGTAATTTACACCAGAAATACCA

1381889

AD-
378
400
AAUUUACACCAGAAAUACCAA
UUGGUAUUUCUGGUGUAAAUUAC
GTAATTTACACCAGAAATACCAA

1381890

AD-
380
402
UUUACACCAGAAAUACCAAGG
CCUUGGUAUUUCUGGUGUAAAUU
AATTTACACCAGAAATACCAAGG

1381891

AD-
381
403
UUACACCAGAAAUACCAAGGG
CCCUUGGUAUUUCUGGUGUAAAU
ATTTACACCAGAAATACCAAGGG

1381892

AD-
382
404
UACACCAGAAAUACCAAGGGU
ACCCUUGGUAUUUCUGGUGUAAA
TTTACACCAGAAATACCAAGGGT

1381893

AD-
384
406
CACCAGAAAUACCAAGGGUGG
CCACCCUUGGUAUUUCUGGUGUA
TACACCAGAAATACCAAGGGTGG

1381894

AD-
385
407
ACCAGAAAUACCAAGGGUGGA
UCCACCCUUGGUAUUUCUGGUGU
ACACCAGAAATACCAAGGGTGGA

1381895

AD-
386
408
CCAGAAAUACCAAGGGUGGAG
CUCCACCCUUGGUAUUUCUGGUG
CACCAGAAATACCAAGGGTGGAG

1381896

AD-
388
410
AGAAAUACCAAGGGUGGAGAU
AUCUCCACCCUUGGUAUUUCUGG
CCAGAAATACCAAGGGTGGAGAT

1381897

AD-
389
411
GAAAUACCAAGGGUGGAGAUG
CAUCUCCACCCUUGGUAUUUCUG
CAGAAATACCAAGGGTGGAGATG

1381898

AD-
390
412
AAAUACCAAGGGUGGAGAUGC
GCAUCUCCACCCUUGGUAUUUCU
AGAAATACCAAGGGTGGAGATGC

1381899

AD-
392
414
AUACCAAGGGUGGAGAUGCUC
GAGCAUCUCCACCCUUGGUAUUU
AAATACCAAGGGTGGAGATGCTC

1381900

AD-
393
415
UACCAAGGGUGGAGAUGCUCC
GGAGCAUCUCCACCCUUGGUAUU
AATACCAAGGGTGGAGATGCTCC

1381901

AD-
394
416
ACCAAGGGUGGAGAUGCUCCA
UGGAGCAUCUCCACCCUUGGUAU
ATACCAAGGGTGGAGATGCTCCA

1381902

AD-
396
418
CAAGGGUGGAGAUGCUCCAGC
GCUGGAGCAUCUCCACCCUUGGU
ACCAAGGGTGGAGATGCTCCAGC

1381903

AD-
397
419
AAGGGUGGAGAUGCUCCAGCU
AGCUGGAGCAUCUCCACCCUUGG
CCAAGGGTGGAGATGCTCCAGCT

1381904

AD-
398
420
AGGGUGGAGAUGCUCCAGCUG
CAGCUGGAGCAUCUCCACCCUUG
CAAGGGTGGAGATGCTCCAGCTG

1381905

AD-
400
422
GGUGGAGAUGCUCCAGCUGCU
AGCAGCUGGAGCAUCUCCACCCU
AGGGTGGAGATGCTCCAGCTGCT

1381906

AD-
401
423
GUGGAGAUGCUCCAGCUGCUG
CAGCAGCUGGAGCAUCUCCACCC
GGGTGGAGATGCTCCAGCTGCTG

1381907

AD-
402
424
UGGAGAUGCUCCAGCUGCUGG
CCAGCAGCUGGAGCAUCUCCACC
GGTGGAGATGCTCCAGCTGCTGG

1381908

AD-
404
426
GAGAUGCUCCAGCUGCUGGUG
CACCAGCAGCUGGAGCAUCUCCA
TGGAGATGCTCCAGCTGCTGGTG

1381909

AD-
405
427
AGAUGCUCCAGCUGCUGGUGA
UCACCAGCAGCUGGAGCAUCUCC
GGAGATGCTCCAGCTGCTGGTGA

1381910

AD-
406
428
GAUGCUCCAGCUGCUGGUGAA
UUCACCAGCAGCUGGAGCAUCUC
GAGATGCTCCAGCTGCTGGTGAA

1381911

AD-
408
430
UGCUCCAGCUGCUGGUGAAGA
UCUUCACCAGCAGCUGGAGCAUC
GATGCTCCAGCTGCTGGTGAAGA

1381912

AD-
409
431
GCUCCAGCUGCUGGUGAAGAU
AUCUUCACCAGCAGCUGGAGCAU
ATGCTCCAGCTGCTGGTGAAGAT

1381913

AD-
410
432
CUCCAGCUGCUGGUGAAGAUG
CAUCUUCACCAGCAGCUGGAGCA
TGCTCCAGCTGCTGGTGAAGATG

1381914

AD-
412
434
CCAGCUGCUGGUGAAGAUGCA
UGCAUCUUCACCAGCAGCUGGAG
CTCCAGCTGCTGGTGAAGATGCA

1381915

AD-
413
435
CAGCUGCUGGUGAAGAUGCAU
AUGCAUCUUCACCAGCAGCUGGA
TCCAGCTGCTGGTGAAGATGCAT

1381916

AD-
414
436
AGCUGCUGGUGAAGAUGCAUG
CAUGCAUCUUCACCAGCAGCUGG
CCAGCTGCTGGTGAAGATGCATG

1381917

AD-
416
438
CUGCUGGUGAAGAUGCAUGAA
UUCAUGCAUCUUCACCAGCAGCU
AGCTGCTGGTGAAGATGCATGAA

1381918

AD-
417
439
UGCUGGUGAAGAUGCAUGAAU
AUUCAUGCAUCUUCACCAGCAGC
GCTGCTGGTGAAGATGCATGAAT

1381919

AD-
418
440
GCUGGUGAAGAUGCAUGAAUA
UAUUCAUGCAUCUUCACCAGCAG
CTGCTGGTGAAGATGCATGAATA

1381920

AD-
420
442
UGGUGAAGAUGCAUGAAUAGG
CCUAUUCAUGCAUCUUCACCAGC
GCTGGTGAAGATGCATGAATAGG

1381921

AD-
421
443
GGUGAAGAUGCAUGAAUAGGU
ACCUAUUCAUGCAUCUUCACCAG
CTGGTGAAGATGCATGAATAGGT

1381922

AD-
422
444
GUGAAGAUGCAUGAAUAGGUC
GACCUAUUCAUGCAUCUUCACCA
TGGTGAAGATGCATGAATAGGTC

1381923

AD-
423
445
UGAAGAUGCAUGAAUAGGUCC
GGACCUAUUCAUGCAUCUUCACC
GGTGAAGATGCATGAATAGGTCC

1381924

AD-
425
447
AAGAUGCAUGAAUAGGUCCAA
UUGGACCUAUUCAUGCAUCUUCA
TGAAGATGCATGAATAGGTCCAA

1381925

AD-
426
448
AGAUGCAUGAAUAGGUCCAAC
GUUGGACCUAUUCAUGCAUCUUC
GAAGATGCATGAATAGGTCCAAC

1381926

AD-
427
449
GAUGCAUGAAUAGGUCCAACC
GGUUGGACCUAUUCAUGCAUCUU
AAGATGCATGAATAGGTCCAACC

1381927

AD-
429
451
UGCAUGAAUAGGUCCAACCAG
CUGGUUGGACCUAUUCAUGCAUC
GATGCATGAATAGGTCCAACCAG

1381928

AD-
430
452
GCAUGAAUAGGUCCAACCAGC
GCUGGUUGGACCUAUUCAUGCAU
ATGCATGAATAGGTCCAACCAGC

1381929

AD-
431
453
CAUGAAUAGGUCCAACCAGCU
AGCUGGUUGGACCUAUUCAUGCA
TGCATGAATAGGTCCAACCAGCT

1381930

AD-
433
455
UGAAUAGGUCCAACCAGCUGU
ACAGCUGGUUGGACCUAUUCAUG
CATGAATAGGTCCAACCAGCTGT

1381931

AD-
434
456
GAAUAGGUCCAACCAGCUGUA
UACAGCUGGUUGGACCUAUUCAU
ATGAATAGGTCCAACCAGCTGTA

1381932

AD-
435
457
AAUAGGUCCAACCAGCUGUAC
GUACAGCUGGUUGGACCUAUUCA
TGAATAGGTCCAACCAGCTGTAC

1381933

AD-
436
458
AUAGGUCCAACCAGCUGUACA
UGUACAGCUGGUUGGACCUAUUC
GAATAGGTCCAACCAGCTGTACA

1381934

AD-
437
459
UAGGUCCAACCAGCUGUACAU
AUGUACAGCUGGUUGGACCUAUU
AATAGGTCCAACCAGCTGTACAT

1381935

AD-
438
460
AGGUCCAACCAGCUGUACAUU
AAUGUACAGCUGGUUGGACCUAU
ATAGGTCCAACCAGCTGTACATT

1381936

AD-
439
461
GGUCCAACCAGCUGUACAUUU
AAAUGUACAGCUGGUUGGACCUA
TAGGTCCAACCAGCTGTACATTT

1381937

AD-
440
462
GUCCAACCAGCUGUACAUUUG
CAAAUGUACAGCUGGUUGGACCU
AGGTCCAACCAGCTGTACATTTG

1381938

AD-
441
463
UCCAACCAGCUGUACAUUUGG
CCAAAUGUACAGCUGGUUGGACC
GGTCCAACCAGCTGTACATTTGG

1381939

AD-
442
464
CCAACCAGCUGUACAUUUGGA
UCCAAAUGUACAGCUGGUUGGAC
GTCCAACCAGCTGTACATTTGGA

1381940

AD-
443
465
CAACCAGCUGUACAUUUGGAA
UUCCAAAUGUACAGCUGGUUGGA
TCCAACCAGCTGTACATTTGGAA

1381941

AD-
444
466
AACCAGCUGUACAUUUGGAAA
UUUCCAAAUGUACAGCUGGUUGG
CCAACCAGCTGTACATTTGGAAA

1381942

AD-
445
467
ACCAGCUGUACAUUUGGAAAA
UUUUCCAAAUGUACAGCUGGUUG
CAACCAGCTGTACATTTGGAAAA

1381943

AD-
446
468
CCAGCUGUACAUUUGGAAAAA
UUUUUCCAAAUGUACAGCUGGUU
AACCAGCTGTACATTTGGAAAAA

1381944

AD-
447
469
CAGCUGUACAUUUGGAAAAAU
AUUUUUCCAAAUGUACAGCUGGU
ACCAGCTGTACATTTGGAAAAAT

1381945

AD-
448
470
AGCUGUACAUUUGGAAAAAUA
UAUUUUUCCAAAUGUACAGCUGG
CCAGCTGTACATTTGGAAAAATA

1381946

AD-
449
471
GCUGUACAUUUGGAAAAAUAA
UUAUUUUUCCAAAUGUACAGCUG
CAGCTGTACATTTGGAAAAATAA

1381947

AD-
450
472
CUGUACAUUUGGAAAAAUAAA
UUUAUUUUUCCAAAUGUACAGCU
AGCTGTACATTTGGAAAAATAAA

1381948

AD-
453
475
UACAUUUGGAAAAAUAAAACU
AGUUUUAUUUUUCCAAAUGUACA
TGTACATTTGGAAAAATAAAACT

1381949

TABLE 14

RPS25 Modified Duplex Sequences

Start Site
End Site

Duplex Name
Sense Sequence 5′ to 3′
Antisense Sequence 5′ to 3′
Target Sequence 5′ to 3′
in NM_001028.3
in NM_00128.3

AD-1381680
csasaug(Chd)CfgCfCfUfaaggacgascsa
VPusGfsucgUfcCfUfuaggCfgGfcauugscsg
CGCAATGCCGCCTAAGGACGACA
56
78

AD-1381681
asasugc(Chd)GfcCfUfAfaggacgacsasa
VPusUfsgucGfuCfCfuuagGfcGfgcauusgsc
GCAATGCCGCCTAAGGACGACAA
57
79

AD-1381682
usgsccg(Chd)CfuAfAfGfgacgacaasgsa
VPusCfsuugUfcGfUfccuuAfgGfcggcasusu
AATGCCGCCTAAGGACGACAAGA
59
81

AD-1381683
gscscgc(Chd)UfaAfGfGfacgacaagsasa
VPusUfscuuGfuCfGfuccuUfaGfgcggcsasu
ATGCCGCCTAAGGACGACAAGAA
60
82

AD-1381684
cscsgcc(Uhd)AfaGfGfAfcgacaagasasa
VPusUfsucuUfgUfCfguccUfuAfggcggscsa
TGCCGCCTAAGGACGACAAGAAG
61
83

AD-1381685
gscscua(Ahd)GfgAfCfGfacaagaagsasa
VPusUfscuuCfuUfGfucguCfcUfuaggcsgsg
CCGCCTAAGGACGACAAGAAGAA
63
85

AD-1381686
cscsuaa(Ghd)GfaCfGfAfcaagaagasasa
VPusUfsucuUfcUfUfgucgUfcCfuuaggscsg
CGCCTAAGGACGACAAGAAGAAG
64
86

AD-1381687
csusaag(Ghd)AfcGfAfCfaagaagaasgsa
VPusCfsuucUfuCfUfugucGfuCfcuuagsgsc
GCCTAAGGACGACAAGAAGAAGA
65
87

AD-1381688
asasgga(Chd)GfaCfAfAfgaagaagasasa
VPusUfsucuUfcUfUfcuugUfcGfuccuusasg
CTAAGGACGACAAGAAGAAGAAG
67
89

AD-1381689
asgsgac(Ghd)AfcAfAfGfaagaagaasgsa
VPusCfsuucUfuCfUfucuuGfuCfguccususa
TAAGGACGACAAGAAGAAGAAGG
68
90

AD-1381690
gsgsacg(Ahd)CfaAfGfAfagaagaagsgsa
VPusCfscuuCfuUfCfuucuUfgUfcguccsusu
AAGGACGACAAGAAGAAGAAGGA
69
91

AD-1381691
ascsgac(Ahd)AfgAfAfGfaagaaggascsa
VPusGfsuccUfuCfUfucuuCfuUfgucguscsc
GGACGACAAGAAGAAGAAGGACG
71
93

AD-1381692
csgsaca(Ahd)GfaAfGfAfagaaggacsgsa
VPusCfsgucCfuUfCfuucuUfcUfugucgsusc
GACGACAAGAAGAAGAAGGACGC
72
94

AD-1381693
gsascaa(Ghd)AfaGfAfAfgaaggacgscsa
VPusGfscguCfcUfUfcuucUfuCfuugucsgsu
ACGACAAGAAGAAGAAGGACGCT
73
95

AD-1381694
csasaga(Ahd)GfaAfGfAfaggacgcusgsa
VPusCfsagcGfuCfCfuucuUfcUfucuugsusc
GACAAGAAGAAGAAGGACGCTGG
75
97

AD-1381695
asasgaa(Ghd)AfaGfAfAfggacgcugsgsa
VPusCfscagCfgUfCfcuucUfuCfuucuusgsu
ACAAGAAGAAGAAGGACGCTGGA
76
98

AD-1381696
asgsaag(Ahd)AfgAfAfGfgacgcuggsasa
VPusUfsccaGfcGfUfccuuCfuUfcuucususg
CAAGAAGAAGAAGGACGCTGGAA
77
99

AD-1381697
gsasaga(Ahd)GfaAfGfGfacgcuggasasa
VPusUfsuccAfgCfGfuccuUfcUfucuucsusu
AAGAAGAAGAAGGACGCTGGAAA
78
100

AD-1381698
asgsaag(Ahd)AfgGfAfCfgcuggaaasgsa
VPusCfsuuuCfcAfGfcgucCfuUfcuucususc
GAAGAAGAAGGACGCTGGAAAGT
80
102

AD-1381699
gsasaga(Ahd)GfgAfCfGfcuggaaagsusa
VPusAfscuuUfcCfAfgcguCfcUfucuucsusu
AAGAAGAAGGACGCTGGAAAGTC
81
103

AD-1381700
asasgaa(Ghd)GfaCfGfCfuggaaaguscsa
VPusGfsacuUfuCfCfagcgUfcCfuucuuscsu
AGAAGAAGGACGCTGGAAAGTCG
82
104

AD-1381701
gsasagg(Ahd)CfgCfUfGfgaaagucgsgsa
VPusCfscgaCfuUfUfccagCfgUfccuucsusu
AAGAAGGACGCTGGAAAGTCGGC
84
106

AD-1381702
asasgga(Chd)GfcUfGfGfaaagucggscsa
VPusGfsccgAfcUfUfuccaGfcGfuccuuscsu
AGAAGGACGCTGGAAAGTCGGCC
85
107

AD-1381703
asgsgac(Ghd)CfuGfGfAfaagucggcscsa
VPusGfsgccGfaCfUfuuccAfgCfguccususc
GAAGGACGCTGGAAAGTCGGCCA
86
108

AD-1381704
gsascgc(Uhd)GfgAfAfAfgucggccasasa
VPusUfsuggCfcGfAfcuuuCfcAfgcgucscsu
AGGACGCTGGAAAGTCGGCCAAG
88
110

AD-1381705
ascsgcu(Ghd)GfaAfAfGfucggccaasgsa
VPusCfsuugGfcCfGfacuuUfcCfagcguscsc
GGACGCTGGAAAGTCGGCCAAGA
89
111

AD-1381706
csgscug(Ghd)AfaAfGfUfcggccaagsasa
VPusUfscuuGfgCfCfgacuUfuCfcagcgsusc
GACGCTGGAAAGTCGGCCAAGAA
90
112

AD-1381707
csusgga(Ahd)AfgUfCfGfgccaagaasasa
VPusUfsuucUfuGfGfccgaCfuUfuccagscsg
CGCTGGAAAGTCGGCCAAGAAAG
92
114

AD-1381708
usgsgaa(Ahd)GfuCfGfGfccaagaaasgsa
VPusCfsuuuCfuUfGfgccgAfcUfuuccasgsc
GCTGGAAAGTCGGCCAAGAAAGA
93
115

AD-1381709
gsgsaaa(Ghd)UfcGfGfCfcaagaaagsasa
VPusUfscuuUfcUfUfggccGfaCfuuuccsasg
CTGGAAAGTCGGCCAAGAAAGAC
94
116

AD-1381710
asasagu(Chd)GfgCfCfAfagaaagacsasa
VPusUfsgucUfuUfCfuuggCfcGfacuuuscsc
GGAAAGTCGGCCAAGAAAGACAA
96
118

AD-1381711
asasguc(Ghd)GfcCfAfAfgaaagacasasa
VPusUfsuguCfuUfUfcuugGfcCfgacuususc
GAAAGTCGGCCAAGAAAGACAAA
97
119

AD-1381712
asgsucg(Ghd)CfcAfAfGfaaagacaasasa
VPusUfsuugUfcUfUfucuuGfgCfcgacususu
AAAGTCGGCCAAGAAAGACAAAG
98
120

AD-1381713
uscsggc(Chd)AfaGfAfAfagacaaagsasa
VPusUfscuuUfgUfCfuuucUfuGfgccgascsu
AGTCGGCCAAGAAAGACAAAGAC
100
122

AD-1381714
csgsgcc(Ahd)AfgAfAfAfgacaaagascsa
VPusGfsucuUfuGfUfcuuuCfuUfggccgsasc
GTCGGCCAAGAAAGACAAAGACC
101
123

AD-1381715
gsgscca(Ahd)GfaAfAfGfacaaagacscsa
VPusGfsgucUfuUfGfucuuUfcUfuggccsgsa
TCGGCCAAGAAAGACAAAGACCC
102
124

AD-1381716
csasaga(Ahd)AfgAfCfAfaagacccasgsa
VPusCfsuggGfuCfUfuuguCfuUfucuugsgsc
GCCAAGAAAGACAAAGACCCAGT
105
127

AD-1381717
asasgaa(Ahd)GfaCfAfAfagacccagsusa
VPusAfscugGfgUfCfuuugUfcUfuucuusgsg
CCAAGAAAGACAAAGACCCAGTG
106
128

AD-1381718
asgsaaa(Ghd)AfcAfAfAfgacccagusgsa
VPusCfsacuGfgGfUfcuuuGfuCfuuucususg
CAAGAAAGACAAAGACCCAGTGA
107
129

AD-1381719
asasaga(Chd)AfaAfGfAfcccagugasasa
VPusUfsucaCfuGfGfgucuUfuGfucuuuscsu
AGAAAGACAAAGACCCAGTGAAC
109
131

AD-1381720
asasgac(Ahd)AfaGfAfCfccagugaascsa
VPusGfsuucAfcUfGfggucUfuUfgucuususc
GAAAGACAAAGACCCAGTGAACA
110
132

AD-1381721
asgsaca(Ahd)AfgAfCfCfcagugaacsasa
VPusUfsguuCfaCfUfggguCfuUfugucususu
AAAGACAAAGACCCAGTGAACAA
111
133

AD-1381722
gsascaa(Ahd)GfaCfCfCfagugaacasasa
VPusUfsuguUfcAfCfugggUfcUfuugucsusu
AAGACAAAGACCCAGTGAACAAA
112
134

AD-1381723
csasaag(Ahd)CfcCfAfGfugaacaaasusa
VPusAfsuuuGfuUfCfacugGfgUfcuuugsusc
GACAAAGACCCAGTGAACAAATC
114
136

AD-1381724
asasaga(Chd)CfcAfGfUfgaacaaauscsa
VPusGfsauuUfgUfUfcacuGfgGfucuuusgsu
ACAAAGACCCAGTGAACAAATCC
115
137

AD-1381725
asasgac(Chd)CfaGfUfGfaacaaaucscsa
VPusGfsgauUfuGfUfucacUfgGfgucuususg
CAAAGACCCAGTGAACAAATCCG
116
138

AD-1381726
gsasccc(Ahd)GfuGfAfAfcaaauccgsgsa
VPusCfscggAfuUfUfguucAfcUfgggucsusu
AAGACCCAGTGAACAAATCCGGG
118
140

AD-1381727
gsgsggg(Chd)AfaGfGfCfcaaaaagasasa
VPusUfsucuUfuUfUfggccUfuGfcccccsgsg
CCGGGGGCAAGGCCAAAAAGAAG
136
158

AD-1381728
gsgsggc(Ahd)AfgGfCfCfaaaaagaasgsa
VPusCfsuucUfuUfUfuggcCfuUfgccccscsg
CGGGGGCAAGGCCAAAAAGAAGA
137
159

AD-1381729
csasagg(Chd)CfaAfAfAfagaagaagsusa
VPusAfscuuCfuUfCfuuuuUfgGfccuugscsc
GGCAAGGCCAAAAAGAAGAAGTG
141
163

AD-1381730
asasggc(Chd)AfaAfAfAfgaagaagusgsa
VPusCfsacuUfcUfUfcuuuUfuGfgccuusgsc
GCAAGGCCAAAAAGAAGAAGTGG
142
164

AD-1381731
asgsgcc(Ahd)AfaAfAfGfaagaagugsgsa
VPusCfscacUfuCfUfucuuUfuUfggccususg
CAAGGCCAAAAAGAAGAAGTGGT
143
165

AD-1381732
gscscaa(Ahd)AfaGfAfAfgaagugguscsa
VPusGfsaccAfcUfUfcuucUfuUfuuggcscsu
AGGCCAAAAAGAAGAAGTGGTCC
145
167

AD-1381733
cscsaaa(Ahd)AfgAfAfGfaaguggucscsa
VPusGfsgacCfaCfUfucuuCfuUfuuuggscsc
GGCCAAAAAGAAGAAGTGGTCCA
146
168

AD-1381734
csasaaa(Ahd)GfaAfGfAfagugguccsasa
VPusUfsggaCfcAfCfuucuUfcUfuuuugsgsc
GCCAAAAAGAAGAAGTGGTCCAA
147
169

AD-1381735
asasaag(Ahd)AfgAfAfGfugguccaasasa
VPusUfsuugGfaCfCfacuuCfuUfcuuuususg
CAAAAAGAAGAAGTGGTCCAAAG
149
171

AD-1381736
asasaga(Ahd)GfaAfGfUfgguccaaasgsa
VPusCfsuuuGfgAfCfcacuUfcUfucuuususu
AAAAAGAAGAAGTGGTCCAAAGG
150
172

AD-1381737
asasgaa(Ghd)AfaGfUfGfguccaaagsgsa
VPusCfscuuUfgGfAfccacUfuCfuucuususu
AAAAGAAGAAGTGGTCCAAAGGC
151
173

AD-1381738
gsasaga(Ahd)GfuGfGfUfccaaaggcsasa
VPusUfsgccUfuUfGfgaccAfcUfucuucsusu
AAGAAGAAGTGGTCCAAAGGCAA
153
175

AD-1381739
asasgaa(Ghd)UfgGfUfCfcaaaggcasasa
VPusUfsugcCfuUfUfggacCfaCfuucuuscsu
AGAAGAAGTGGTCCAAAGGCAAA
154
176

AD-1381740
asgsaag(Uhd)GfgUfCfCfaaaggcaasasa
VPusUfsuugCfcUfUfuggaCfcAfcuucususc
GAAGAAGTGGTCCAAAGGCAAAG
155
177

AD-1381741
asasgug(Ghd)UfcCfAfAfaggcaaagsusa
VPusAfscuuUfgCfCfuuugGfaCfcacuuscsu
AGAAGTGGTCCAAAGGCAAAGTT
157
179

AD-1381742
asgsugg(Uhd)CfcAfAfAfggcaaagususa
VPusAfsacuUfuGfCfcuuuGfgAfccacususc
GAAGTGGTCCAAAGGCAAAGTTC
158
180

AD-1381743
gsusggu(Chd)CfaAfAfGfgcaaaguuscsa
VPusGfsaacUfuUfGfccuuUfgGfaccacsusu
AAGTGGTCCAAAGGCAAAGTTCG
159
181

AD-1381744
gsgsucc(Ahd)AfaGfGfCfaaaguucgsgsa
VPusCfscgaAfcUfUfugccUfuUfggaccsasc
GTGGTCCAAAGGCAAAGTTCGGG
161
183

AD-1381745
gsuscca(Ahd)AfgGfCfAfaaguucggsgsa
VPusCfsccgAfaCfUfuugcCfuUfuggacscsa
TGGTCCAAAGGCAAAGTTCGGGA
162
184

AD-1381746
uscscaa(Ahd)GfgCfAfAfaguucgggsasa
VPusUfscccGfaAfCfuuugCfcUfuuggascsc
GGTCCAAAGGCAAAGTTCGGGAC
163
185

AD-1381747
csasaag(Ghd)CfaAfAfGfuucgggacsasa
VPusUfsgucCfcGfAfacuuUfgCfcuuugsgsa
TCCAAAGGCAAAGTTCGGGACAA
165
187

AD-1381748
asasagg(Chd)AfaAfGfUfucgggacasasa
VPusUfsuguCfcCfGfaacuUfuGfccuuusgsg
CCAAAGGCAAAGTTCGGGACAAG
166
188

AD-1381749
asasggc(Ahd)AfaGfUfUfcgggacaasgsa
VPusCfsuugUfcCfCfgaacUfuUfgccuususg
CAAAGGCAAAGTTCGGGACAAGC
167
189

AD-1381750
gsgscaa(Ahd)GfuUfCfGfggacaagcsusa
VPusAfsgcuUfgUfCfccgaAfcUfuugccsusu
AAGGCAAAGTTCGGGACAAGCTC
169
191

AD-1381751
gscsaaa(Ghd)UfuCfGfGfgacaagcuscsa
VPusGfsagcUfuGfUfcccgAfaCfuuugcscsu
AGGCAAAGTTCGGGACAAGCTCA
170
192

AD-1381752
csasaag(Uhd)UfcGfGfGfacaagcucsasa
VPusUfsgagCfuUfGfucccGfaAfcuuugscsc
GGCAAAGTTCGGGACAAGCTCAA
171
193

AD-1381753
asasagu(Uhd)CfgGfGfAfcaagcucasasa
VPusUfsugaGfcUfUfguccCfgAfacuuusgsc
GCAAAGTTCGGGACAAGCTCAAT
172
194

AD-1381754
asgsuuc(Ghd)GfgAfCfAfagcucaausasa
VPusUfsauuGfaGfCfuuguCfcCfgaacususu
AAAGTTCGGGACAAGCTCAATAA
174
196

AD-1381755
gsusucg(Ghd)GfaCfAfAfgcucaauasasa
VPusUfsuauUfgAfGfcuugUfcCfcgaacsusu
AAGTTCGGGACAAGCTCAATAAC
175
197

AD-1381756
ususcgg(Ghd)AfcAfAfGfcucaauaascsa
VPusGfsuuaUfuGfAfgcuuGfuCfccgaascsu
AGTTCGGGACAAGCTCAATAACT
176
198

AD-1381757
csgsgga(Chd)AfaGfCfUfcaauaacususa
VPusAfsaguUfaUfUfgagcUfuGfucccgsasa
TTCGGGACAAGCTCAATAACTTA
178
200

AD-1381758
gsgsgac(Ahd)AfgCfUfCfaauaacuusasa
VPusUfsaagUfuAfUfugagCfuUfgucccsgsa
TCGGGACAAGCTCAATAACTTAG
179
201

AD-1381759
gsgsaca(Ahd)GfcUfCfAfauaacuuasgsa
VPusCfsuaaGfuUfAfuugaGfcUfuguccscsg
CGGGACAAGCTCAATAACTTAGT
180
202

AD-1381760
ascsaag(Chd)UfcAfAfUfaacuuaguscsa
VPusGfsacuAfaGfUfuauuGfaGfcuuguscsc
GGACAAGCTCAATAACTTAGTCT
182
204

AD-1381761
csasagc(Uhd)CfaAfUfAfacuuagucsusa
VPusAfsgacUfaAfGfuuauUfgAfgcuugsusc
GACAAGCTCAATAACTTAGTCTT
183
205

AD-1381762
asasgcu(Chd)AfaUfAfAfcuuagucususa
VPusAfsagaCfuAfAfguuaUfuGfagcuusgsu
ACAAGCTCAATAACTTAGTCTTG
184
206

AD-1381763
gscsuca(Ahd)UfaAfCfUfuagucuugsusa
VPusAfscaaGfaCfUfaaguUfaUfugagcsusu
AAGCTCAATAACTTAGTCTTGTT
186
208

AD-1381764
csuscaa(Uhd)AfaCfUfUfagucuugususa
VPusAfsacaAfgAfCfuaagUfuAfuugagscsu
AGCTCAATAACTTAGTCTTGTTT
187
209

AD-1381765
uscsaau(Ahd)AfcUfUfAfgucuuguususa
VPusAfsaacAfaGfAfcuaaGfuUfauugasgsc
GCTCAATAACTTAGTCTTGTTTG
188
210

AD-1381766
asasuaa(Chd)UfuAfGfUfcuuguuugsasa
VPusUfscaaAfcAfAfgacuAfaGfuuauusgsa
TCAATAACTTAGTCTTGTTTGAC
190
212

AD-1381767
asusaac(Uhd)UfaGfUfCfuuguuugascsa
VPusGfsucaAfaCfAfagacUfaAfguuaususg
CAATAACTTAGTCTTGTTTGACA
191
213

AD-1381768
usasacu(Uhd)AfgUfCfUfuguuugacsasa
VPusUfsgucAfaAfCfaagaCfuAfaguuasusu
AATAACTTAGTCTTGTTTGACAA
192
214

AD-1381769
ascsuua(Ghd)UfcUfUfGfuuugacaasasa
VPusUfsuugUfcAfAfacaaGfaCfuaagususa
TAACTTAGTCTTGTTTGACAAAG
194
216

AD-1381770
csusuag(Uhd)CfuUfGfUfuugacaaasgsa
VPusCfsuuuGfuCfAfaacaAfgAfcuaagsusu
AACTTAGTCTTGTTTGACAAAGC
195
217

AD-1381771
ususagu(Chd)UfuGfUfUfugacaaagscsa
VPusGfscuuUfgUfCfaaacAfaGfacuaasgsu
ACTTAGTCTTGTTTGACAAAGCT
196
218

AD-1381772
asgsucu(Uhd)GfuUfUfGfacaaagcusasa
VPusUfsagcUfuUfGfucaaAfcAfagacusasa
TTAGTCTTGTTTGACAAAGCTAC
198
220

AD-1381773
gsuscuu(Ghd)UfuUfGfAfcaaagcuascsa
VPusGfsuagCfuUfUfgucaAfaCfaagacsusa
TAGTCTTGTTTGACAAAGCTACC
199
221

AD-1381774
uscsuug(Uhd)UfuGfAfCfaaagcuacscsa
VPusGfsguaGfcUfUfugucAfaAfcaagascsu
AGTCTTGTTTGACAAAGCTACCT
200
222

AD-1381775
ususguu(Uhd)GfaCfAfAfagcuaccusasa
VPusUfsaggUfaGfCfuuugUfcAfaacaasgsa
TCTTGTTTGACAAAGCTACCTAT
202
224

AD-1381776
usgsuuu(Ghd)AfcAfAfAfgcuaccuasusa
VPusAfsuagGfuAfGfcuuuGfuCfaaacasasg
CTTGTTTGACAAAGCTACCTATG
203
225

AD-1381777
gsusuug(Ahd)CfaAfAfGfcuaccuausgsa
VPusCfsauaGfgUfAfgcuuUfgUfcaaacsasa
TTGTTTGACAAAGCTACCTATGA
204
226

AD-1381778
ususuga(Chd)AfaAfGfCfuaccuaugsasa
VPusUfscauAfgGfUfagcuUfuGfucaaascsa
TGTTTGACAAAGCTACCTATGAT
205
227

AD-1381779
usgsaca(Ahd)AfgCfUfAfccuaugausasa
VPusUfsaucAfuAfGfguagCfuUfugucasasa
TTTGACAAAGCTACCTATGATAA
207
229

AD-1381780
gsascaa(Ahd)GfcUfAfCfcuaugauasasa
VPusUfsuauCfaUfAfgguaGfcUfuugucsasa
TTGACAAAGCTACCTATGATAAA
208
230

AD-1381781
ascsaaa(Ghd)CfuAfCfCfuaugauaasasa
VPusUfsuuaUfcAfUfagguAfgCfuuuguscsa
TGACAAAGCTACCTATGATAAAC
209
231

AD-1381782
asasagc(Uhd)AfcCfUfAfugauaaacsusa
VPusAfsguuUfaUfCfauagGfuAfgcuuusgsu
ACAAAGCTACCTATGATAAACTC
211
233

AD-1381783
asasgcu(Ahd)CfcUfAfUfgauaaacuscsa
VPusGfsaguUfuAfUfcauaGfgUfagcuususg
CAAAGCTACCTATGATAAACTCT
212
234

AD-1381784
asgscua(Chd)CfuAfUfGfauaaacucsusa
VPusAfsgagUfuUfAfucauAfgGfuagcususu
AAAGCTACCTATGATAAACTCTG
213
235

AD-1381785
csusacc(Uhd)AfuGfAfUfaaacucugsusa
VPusAfscagAfgUfUfuaucAfuAfgguagscsu
AGCTACCTATGATAAACTCTGTA
215
237

AD-1381786
usasccu(Ahd)UfgAfUfAfaacucugusasa
VPusUfsacaGfaGfUfuuauCfaUfagguasgsc
GCTACCTATGATAAACTCTGTAA
216
238

AD-1381787
ascscua(Uhd)GfaUfAfAfacucuguasasa
VPusUfsuacAfgAfGfuuuaUfcAfuaggusasg
CTACCTATGATAAACTCTGTAAG
217
239

AD-1381788
csusaug(Ahd)UfaAfAfCfucuguaagsgsa
VPusCfscuuAfcAfGfaguuUfaUfcauagsgsu
ACCTATGATAAACTCTGTAAGGA
219
241

AD-1381789
usasuga(Uhd)AfaAfCfUfcuguaaggsasa
VPusUfsccuUfaCfAfgaguUfuAfucauasgsg
CCTATGATAAACTCTGTAAGGAA
220
242

AD-1381790
asusgau(Ahd)AfaCfUfCfuguaaggasasa
VPusUfsuccUfuAfCfagagUfuUfaucausasg
CTATGATAAACTCTGTAAGGAAG
221
243

AD-1381791
gsasuaa(Ahd)CfuCfUfGfuaaggaagsusa
VPusAfscuuCfcUfUfacagAfgUfuuaucsasu
ATGATAAACTCTGTAAGGAAGTT
223
245

AD-1381792
asusaaa(Chd)UfcUfGfUfaaggaagususa
VPusAfsacuUfcCfUfuacaGfaGfuuuauscsa
TGATAAACTCTGTAAGGAAGTTC
224
246

AD-1381793
usasaac(Uhd)CfuGfUfAfaggaaguuscsa
VPusGfsaacUfuCfCfuuacAfgAfguuuasusc
GATAAACTCTGTAAGGAAGTTCC
225
247

AD-1381794
asascuc(Uhd)GfuAfAfGfgaaguuccscsa
VPusGfsggaAfcUfUfccuuAfcAfgaguususa
TAAACTCTGTAAGGAAGTTCCCA
227
249

AD-1381795
ascsucu(Ghd)UfaAfGfGfaaguucccsasa
VPusUfsgggAfaCfUfuccuUfaCfagagususu
AAACTCTGTAAGGAAGTTCCCAA
228
250

AD-1381796
csuscug(Uhd)AfaGfGfAfaguucccasasa
VPusUfsuggGfaAfCfuuccUfuAfcagagsusu
AACTCTGTAAGGAAGTTCCCAAC
229
251

AD-1381797
csusgua(Ahd)GfgAfAfGfuucccaacsusa
VPusAfsguuGfgGfAfacuuCfcUfuacagsasg
CTCTGTAAGGAAGTTCCCAACTA
231
253

AD-1381798
usgsuaa(Ghd)GfaAfGfUfucccaacusasa
VPusUfsaguUfgGfGfaacuUfcCfuuacasgsa
TCTGTAAGGAAGTTCCCAACTAT
232
254

AD-1381799
gsusaag(Ghd)AfaGfUfUfcccaacuasusa
VPusAfsuagUfuGfGfgaacUfuCfcuuacsasg
CTGTAAGGAAGTTCCCAACTATA
233
255

AD-1381800
asasgga(Ahd)GfuUfCfCfcaacuauasasa
VPusUfsuauAfgUfUfgggaAfcUfuccuusasc
GTAAGGAAGTTCCCAACTATAAA
235
257

AD-1381801
asgsgaa(Ghd)UfuCfCfCfaacuauaasasa
VPusUfsuuaUfaGfUfugggAfaCfuuccususa
TAAGGAAGTTCCCAACTATAAAC
236
258

AD-1381802
gsgsaag(Uhd)UfcCfCfAfacuauaaascsa
VPusGfsuuuAfuAfGfuuggGfaAfcuuccsusu
AAGGAAGTTCCCAACTATAAACT
237
259

AD-1381803
asasguu(Chd)CfcAfAfCfuauaaacususa
VPusAfsaguUfuAfUfaguuGfgGfaacuuscsc
GGAAGTTCCCAACTATAAACTTA
239
261

AD-1381804
asgsuuc(Chd)CfaAfCfUfauaaacuusasa
VPusUfsaagUfuUfAfuaguUfgGfgaacususc
GAAGTTCCCAACTATAAACTTAT
240
262

AD-1381805
gsusucc(Chd)AfaCfUfAfuaaacuuasusa
VPusAfsuaaGfuUfUfauagUfuGfggaacsusu
AAGTTCCCAACTATAAACTTATA
241
263

AD-1381806
uscscca(Ahd)CfuAfUfAfaacuuauasasa
VPusUfsuauAfaGfUfuuauAfgUfugggasasc
GTTCCCAACTATAAACTTATAAC
243
265

AD-1381807
cscscaa(Chd)UfaUfAfAfacuuauaascsa
VPusGfsuuaUfaAfGfuuuaUfaGfuugggsasa
TTCCCAACTATAAACTTATAACC
244
266

AD-1381808
cscsaac(Uhd)AfuAfAfAfcuuauaacscsa
VPusGfsguuAfuAfAfguuuAfuAfguuggsgsa
TCCCAACTATAAACTTATAACCC
245
267

AD-1381809
ascsccc(Ahd)GfcUfGfUfggucucugsasa
VPusUfscagAfgAfCfcacaGfcUfggggususa
TAACCCCAGCTGTGGTCTCTGAG
262
284

AD-1381810
cscscag(Chd)UfgUfGfGfucucugagsasa
VPusUfscucAfgAfGfaccaCfaGfcugggsgsu
ACCCCAGCTGTGGTCTCTGAGAG
264
286

AD-1381811
cscsagc(Uhd)GfuGfGfUfcucugagasgsa
VPusCfsucuCfaGfAfgaccAfcAfgcuggsgsg
CCCCAGCTGTGGTCTCTGAGAGA
265
287

AD-1381812
csasgcu(Ghd)UfgGfUfCfucugagagsasa
VPusUfscucUfcAfGfagacCfaCfagcugsgsg
CCCAGCTGTGGTCTCTGAGAGAC
266
288

AD-1381813
gscsugu(Ghd)GfuCfUfCfugagagacsusa
VPusAfsgucUfcUfCfagagAfcCfacagcsusg
CAGCTGTGGTCTCTGAGAGACTG
268
290

AD-1381814
csusgug(Ghd)UfcUfCfUfgagagacusgsa
VPusCfsaguCfuCfUfcagaGfaCfcacagscsu
AGCTGTGGTCTCTGAGAGACTGA
269
291

AD-1381815
usgsugg(Uhd)CfuCfUfGfagagacugsasa
VPusUfscagUfcUfCfucagAfgAfccacasgsc
GCTGTGGTCTCTGAGAGACTGAA
270
292

AD-1381816
usgsguc(Uhd)CfuGfAfGfagacugaasgsa
VPusCfsuucAfgUfCfucucAfgAfgaccascsa
TGTGGTCTCTGAGAGACTGAAGA
272
294

AD-1381817
gsgsucu(Chd)UfgAfGfAfgacugaagsasa
VPusUfscuuCfaGfUfcucuCfaGfagaccsasc
GTGGTCTCTGAGAGACTGAAGAT
273
295

AD-1381818
gsuscuc(Uhd)GfaGfAfGfacugaagasusa
VPusAfsucuUfcAfGfucucUfcAfgagacscsa
TGGTCTCTGAGAGACTGAAGATT
274
296

AD-1381819
csuscug(Ahd)GfaGfAfCfugaagauuscsa
VPusGfsaauCfuUfCfagucUfcUfcagagsasc
GTCTCTGAGAGACTGAAGATTCG
276
298

AD-1381820
uscsuga(Ghd)AfgAfCfUfgaagauucsgsa
VPusCfsgaaUfcUfUfcaguCfuCfucagasgsa
TCTCTGAGAGACTGAAGATTCGA
277
299

AD-1381821
csusgag(Ahd)GfaCfUfGfaagauucgsasa
VPusUfscgaAfuCfUfucagUfcUfcucagsasg
CTCTGAGAGACTGAAGATTCGAG
278
300

AD-1381822
gsasgag(Ahd)CfuGfAfAfgauucgagsgsa
VPusCfscucGfaAfUfcuucAfgUfcucucsasg
CTGAGAGACTGAAGATTCGAGGC
280
302

AD-1381823
asgsaga(Chd)UfgAfAfGfauucgaggscsa
VPusGfsccuCfgAfAfucuuCfaGfucucuscsa
TGAGAGACTGAAGATTCGAGGCT
281
303

AD-1381824
gsasgac(Uhd)GfaAfGfAfuucgaggcsusa
VPusAfsgccUfcGfAfaucuUfcAfgucucsusc
GAGAGACTGAAGATTCGAGGCTC
282
304

AD-1381825
gsascug(Ahd)AfgAfUfUfcgaggcucscsa
VPusGfsgagCfcUfCfgaauCfuUfcagucsusc
GAGACTGAAGATTCGAGGCTCCC
284
306

AD-1381826
ascsuga(Ahd)GfaUfUfCfgaggcuccscsa
VPusGfsggaGfcCfUfcgaaUfcUfucaguscsu
AGACTGAAGATTCGAGGCTCCCT
285
307

AD-1381827
csusgaa(Ghd)AfuUfCfGfaggcucccsusa
VPusAfsgggAfgCfCfucgaAfuCfuucagsusc
GACTGAAGATTCGAGGCTCCCTG
286
308

AD-1381828
gsasaga(Uhd)UfcGfAfGfgcucccugsgsa
VPusCfscagGfgAfGfccucGfaAfucuucsasg
CTGAAGATTCGAGGCTCCCTGGC
288
310

AD-1381829
asasgau(Uhd)CfgAfGfGfcucccuggscsa
VPusGfsccaGfgGfAfgccuCfgAfaucuuscsa
TGAAGATTCGAGGCTCCCTGGCC
289
311

AD-1381830
asgsauu(Chd)GfaGfGfCfucccuggcscsa
VPusGfsgccAfgGfGfagccUfcGfaaucususc
GAAGATTCGAGGCTCCCTGGCCA
290
312

AD-1381831
asusucg(Ahd)GfgCfUfCfccuggccasgsa
VPusCfsuggCfcAfGfggagCfcUfcgaauscsu
AGATTCGAGGCTCCCTGGCCAGG
292
314

AD-1381832
cscscug(Ghd)CfcAfGfGfgcagcccususa
VPusAfsaggGfcUfGfcccuGfgCfcagggsasg
CTCCCTGGCCAGGGCAGCCCTTC
302
324

AD-1381833
cscsugg(Chd)CfaGfGfGfcagcccuuscsa
VPusGfsaagGfgCfUfgcccUfgGfccaggsgsa
TCCCTGGCCAGGGCAGCCCTTCA
303
325

AD-1381834
csusggc(Chd)AfgGfGfCfagcccuucsasa
VPusUfsgaaGfgGfCfugccCfuGfgccagsgsg
CCCTGGCCAGGGCAGCCCTTCAG
304
326

AD-1381835
gsgscca(Ghd)GfgCfAfGfcccuucagsgsa
VPusCfscugAfaGfGfgcugCfcCfuggccsasg
CTGGCCAGGGCAGCCCTTCAGGA
306
328

AD-1381836
gscscag(Ghd)GfcAfGfCfccuucaggsasa
VPusUfsccuGfaAfGfggcuGfcCfcuggcscsa
TGGCCAGGGCAGCCCTTCAGGAG
307
329

AD-1381837
cscsagg(Ghd)CfaGfCfCfcuucaggasgsa
VPusCfsuccUfgAfAfgggcUfgCfccuggscsc
GGCCAGGGCAGCCCTTCAGGAGC
308
330

AD-1381838
asgsggc(Ahd)GfcCfCfUfucaggagcsusa
VPusAfsgcuCfcUfGfaaggGfcUfgcccusgsg
CCAGGGCAGCCCTTCAGGAGCTC
310
332

AD-1381839
gsgsgca(Ghd)CfcCfUfUfcaggagcuscsa
VPusGfsagcUfcCfUfgaagGfgCfugcccsusg
CAGGGCAGCCCTTCAGGAGCTCC
311
333

AD-1381840
gsgscag(Chd)CfcUfUfCfaggagcucscsa
VPusGfsgagCfuCfCfugaaGfgGfcugccscsu
AGGGCAGCCCTTCAGGAGCTCCT
312
334

AD-1381841
csasgcc(Chd)UfuCfAfGfgagcuccususa
VPusAfsaggAfgCfUfccugAfaGfggcugscsc
GGCAGCCCTTCAGGAGCTCCTTA
314
336

AD-1381842
asgsccc(Uhd)UfcAfGfGfagcuccuusasa
VPusUfsaagGfaGfCfuccuGfaAfgggcusgsc
GCAGCCCTTCAGGAGCTCCTTAG
315
337

AD-1381843
gscsccu(Uhd)CfaGfGfAfgcuccuuasgsa
VPusCfsuaaGfgAfGfcuccUfgAfagggcsusg
CAGCCCTTCAGGAGCTCCTTAGT
316
338

AD-1381844
cscsuuc(Ahd)GfgAfGfCfuccuuagusasa
VPusUfsacuAfaGfGfagcuCfcUfgaaggsgsc
GCCCTTCAGGAGCTCCTTAGTAA
318
340

AD-1381845
csusuca(Ghd)GfaGfCfUfccuuaguasasa
VPusUfsuacUfaAfGfgagcUfcCfugaagsgsg
CCCTTCAGGAGCTCCTTAGTAAA
319
341

AD-1381846
ususcag(Ghd)AfgCfUfCfcuuaguaasasa
VPusUfsuuaCfuAfAfggagCfuCfcugaasgsg
CCTTCAGGAGCTCCTTAGTAAAG
320
342

AD-1381847
csasgga(Ghd)CfuCfCfUfuaguaaagsgsa
VPusCfscuuUfaCfUfaaggAfgCfuccugsasa
TTCAGGAGCTCCTTAGTAAAGGA
322
344

AD-1381848
asgsgag(Chd)UfcCfUfUfaguaaaggsasa
VPusUfsccuUfuAfCfuaagGfaGfcuccusgsa
TCAGGAGCTCCTTAGTAAAGGAC
323
345

AD-1381849
gsgsagc(Uhd)CfcUfUfAfguaaaggascsa
VPusGfsuccUfuUfAfcuaaGfgAfgcuccsusg
CAGGAGCTCCTTAGTAAAGGACT
324
346

AD-1381850
asgscuc(Chd)UfuAfGfUfaaaggacususa
VPusAfsaguCfcUfUfuacuAfaGfgagcuscsc
GGAGCTCCTTAGTAAAGGACTTA
326
348

AD-1381851
gscsucc(Uhd)UfaGfUfAfaaggacuusasa
VPusUfsaagUfcCfUfuuacUfaAfggagcsusc
GAGCTCCTTAGTAAAGGACTTAT
327
349

AD-1381852
csusccu(Uhd)AfgUfAfAfaggacuuasusa
VPusAfsuaaGfuCfCfuuuaCfuAfaggagscsu
AGCTCCTTAGTAAAGGACTTATC
328
350

AD-1381853
cscsuua(Ghd)UfaAfAfGfgacuuaucsasa
VPusUfsgauAfaGfUfccuuUfaCfuaaggsasg
CTCCTTAGTAAAGGACTTATCAA
330
352

AD-1381854
csusuag(Uhd)AfaAfGfGfacuuaucasasa
VPusUfsugaUfaAfGfuccuUfuAfcuaagsgsa
TCCTTAGTAAAGGACTTATCAAA
331
353

AD-1381855
ususagu(Ahd)AfaGfGfAfcuuaucaasasa
VPusUfsuugAfuAfAfguccUfuUfacuaasgsg
CCTTAGTAAAGGACTTATCAAAC
332
354

AD-1381856
asgsuaa(Ahd)GfgAfCfUfuaucaaacsusa
VPusAfsguuUfgAfUfaaguCfcUfuuacusasa
TTAGTAAAGGACTTATCAAACTG
334
356

AD-1381857
gsusaaa(Ghd)GfaCfUfUfaucaaacusgsa
VPusCfsaguUfuGfAfuaagUfcCfuuuacsusa
TAGTAAAGGACTTATCAAACTGG
335
357

AD-1381858
usasaag(Ghd)AfcUfUfAfucaaacugsgsa
VPusCfscagUfuUfGfauaaGfuCfcuuuascsu
AGTAAAGGACTTATCAAACTGGT
336
358

AD-1381859
asasgga(Chd)UfuAfUfCfaaacuggususa
VPusAfsaccAfgUfUfugauAfaGfuccuususa
TAAAGGACTTATCAAACTGGTTT
338
360

AD-1381860
asgsgac(Uhd)UfaUfCfAfaacugguususa
VPusAfsaacCfaGfUfuugaUfaAfguccususu
AAAGGACTTATCAAACTGGTTTC
339
361

AD-1381861
gsgsacu(Uhd)AfuCfAfAfacugguuuscsa
VPusGfsaaaCfcAfGfuuugAfuAfaguccsusu
AAGGACTTATCAAACTGGTTTCA
340
362

AD-1381862
ascsuua(Uhd)CfaAfAfCfugguuucasasa
VPusUfsugaAfaCfCfaguuUfgAfuaaguscsc
GGACTTATCAAACTGGTTTCAAA
342
364

AD-1381863
csusuau(Chd)AfaAfCfUfgguuucaasasa
VPusUfsuugAfaAfCfcaguUfuGfauaagsusc
GACTTATCAAACTGGTTTCAAAG
343
365

AD-1381864
ususauc(Ahd)AfaCfUfGfguuucaaasgsa
VPusCfsuuuGfaAfAfccagUfuUfgauaasgsu
ACTTATCAAACTGGTTTCAAAGC
344
366

AD-1381865
usasuca(Ahd)AfcUfGfGfuuucaaagscsa
VPusGfscuuUfgAfAfaccaGfuUfugauasasg
CTTATCAAACTGGTTTCAAAGCA
345
367

AD-1381866
uscsaaa(Chd)UfgGfUfUfucaaagcascsa
VPusGfsugcUfuUfGfaaacCfaGfuuugasusa
TATCAAACTGGTTTCAAAGCACA
347
369

AD-1381867
csasaac(Uhd)GfgUfUfUfcaaagcacsasa
VPusUfsgugCfuUfUfgaaaCfcAfguuugsasu
ATCAAACTGGTTTCAAAGCACAG
348
370

AD-1381868
asasacu(Ghd)GfuUfUfCfaaagcacasgsa
VPusCfsuguGfcUfUfugaaAfcCfaguuusgsa
TCAAACTGGTTTCAAAGCACAGA
349
371

AD-1381869
ascsugg(Uhd)UfuCfAfAfagcacagasgsa
VPusCfsucuGfuGfCfuuugAfaAfccagususu
AAACTGGTTTCAAAGCACAGAGC
351
373

AD-1381870
csusggu(Uhd)UfcAfAfAfgcacagagscsa
VPusGfscucUfgUfGfcuuuGfaAfaccagsusu
AACTGGTTTCAAAGCACAGAGCT
352
374

AD-1381871
usgsguu(Uhd)CfaAfAfGfcacagagcsusa
VPusAfsgcuCfuGfUfgcuuUfgAfaaccasgsu
ACTGGTTTCAAAGCACAGAGCTC
353
375

AD-1381872
gsusuuc(Ahd)AfaGfCfAfcagagcucsasa
VPusUfsgagCfuCfUfgugcUfuUfgaaacscsa
TGGTTTCAAAGCACAGAGCTCAA
355
377

AD-1381873
ususuca(Ahd)AfgCfAfCfagagcucasasa
VPusUfsugaGfcUfCfugugCfuUfugaaascsc
GGTTTCAAAGCACAGAGCTCAAG
356
378

AD-1381874
ususcaa(Ahd)GfcAfCfAfgagcucaasgsa
VPusCfsuugAfgCfUfcuguGfcUfuugaasasc
GTTTCAAAGCACAGAGCTCAAGT
357
379

AD-1381875
csasaag(Chd)AfcAfGfAfgcucaagusasa
VPusUfsacuUfgAfGfcucuGfuGfcuuugsasa
TTCAAAGCACAGAGCTCAAGTAA
359
381

AD-1381876
asasagc(Ahd)CfaGfAfGfcucaaguasasa
VPusUfsuacUfuGfAfgcucUfgUfgcuuusgsa
TCAAAGCACAGAGCTCAAGTAAT
360
382

AD-1381877
asasgca(Chd)AfgAfGfCfucaaguaasusa
VPusAfsuuaCfuUfGfagcuCfuGfugcuususg
CAAAGCACAGAGCTCAAGTAATT
361
383

AD-1381878
gscsaca(Ghd)AfgCfUfCfaaguaauususa
VPusAfsaauUfaCfUfugagCfuCfugugcsusu
AAGCACAGAGCTCAAGTAATTTA
363
385

AD-1381879
csascag(Ahd)GfcUfCfAfaguaauuusasa
VPusUfsaaaUfuAfCfuugaGfcUfcugugscsu
AGCACAGAGCTCAAGTAATTTAC
364
386

AD-1381880
ascsaga(Ghd)CfuCfAfAfguaauuuascsa
VPusGfsuaaAfuUfAfcuugAfgCfucugusgsc
GCACAGAGCTCAAGTAATTTACA
365
387

AD-1381881
asgsagc(Uhd)CfaAfGfUfaauuuacascsa
VPusGfsuguAfaAfUfuacuUfgAfgcucusgsu
ACAGAGCTCAAGTAATTTACACC
367
389

AD-1381882
gsasgcu(Chd)AfaGfUfAfauuuacacscsa
VPusGfsgugUfaAfAfuuacUfuGfagcucsusg
CAGAGCTCAAGTAATTTACACCA
368
390

AD-1381883
asgscuc(Ahd)AfgUfAfAfuuuacaccsasa
VPusUfsgguGfuAfAfauuaCfuUfgagcuscsu
AGAGCTCAAGTAATTTACACCAG
369
391

AD-1381884
csuscaa(Ghd)UfaAfUfUfuacaccagsasa
VPusUfscugGfuGfUfaaauUfaCfuugagscsu
AGCTCAAGTAATTTACACCAGAA
371
393

AD-1381885
uscsaag(Uhd)AfaUfUfUfacaccagasasa
VPusUfsucuGfgUfGfuaaaUfuAfcuugasgsc
GCTCAAGTAATTTACACCAGAAA
372
394

AD-1381886
csasagu(Ahd)AfuUfUfAfcaccagaasasa
VPusUfsuucUfgGfUfguaaAfuUfacuugsasg
CTCAAGTAATTTACACCAGAAAT
373
395

AD-1381887
asgsuaa(Uhd)UfuAfCfAfccagaaausasa
VPusUfsauuUfcUfGfguguAfaAfuuacususg
CAAGTAATTTACACCAGAAATAC
375
397

AD-1381888
gsusaau(Uhd)UfaCfAfCfcagaaauascsa
VPusGfsuauUfuCfUfggugUfaAfauuacsusu
AAGTAATTTACACCAGAAATACC
376
398

AD-1381889
usasauu(Uhd)AfcAfCfCfagaaauacscsa
VPusGfsguaUfuUfCfugguGfuAfaauuascsu
AGTAATTTACACCAGAAATACCA
377
399

AD-1381890
asasuuu(Ahd)CfaCfCfAfgaaauaccsasa
VPusUfsgguAfuUfUfcuggUfgUfaaauusasc
GTAATTTACACCAGAAATACCAA
378
400

AD-1381891
ususuac(Ahd)CfcAfGfAfaauaccaasgsa
VPusCfsuugGfuAfUfuucuGfgUfguaaasusu
AATTTACACCAGAAATACCAAGG
380
402

AD-1381892
ususaca(Chd)CfaGfAfAfauaccaagsgsa
VPusCfscuuGfgUfAfuuucUfgGfuguaasasu
ATTTACACCAGAAATACCAAGGG
381
403

AD-1381893
usascac(Chd)AfgAfAfAfuaccaaggsgsa
VPusCfsccuUfgGfUfauuuCfuGfguguasasa
TTTACACCAGAAATACCAAGGGT
382
404

AD-1381894
csascca(Ghd)AfaAfUfAfccaagggusgsa
VPusCfsaccCfuUfGfguauUfuCfuggugsusa
TACACCAGAAATACCAAGGGTGG
384
406

AD-1381895
ascscag(Ahd)AfaUfAfCfcaagggugsgsa
VPusCfscacCfcUfUfgguaUfuUfcuggusgsu
ACACCAGAAATACCAAGGGTGGA
385
407

AD-1381896
cscsaga(Ahd)AfuAfCfCfaaggguggsasa
VPusUfsccaCfcCfUfugguAfuUfucuggsusg
CACCAGAAATACCAAGGGTGGAG
386
408

AD-1381897
asgsaaa(Uhd)AfcCfAfAfggguggagsasa
VPusUfscucCfaCfCfcuugGfuAfuuucusgsg
CCAGAAATACCAAGGGTGGAGAT
388
410

AD-1381898
gsasaau(Ahd)CfcAfAfGfgguggagasusa
VPusAfsucuCfcAfCfccuuGfgUfauuucsusg
CAGAAATACCAAGGGTGGAGATG
389
411

AD-1381899
asasaua(Chd)CfaAfGfGfguggagausgsa
VPusCfsaucUfcCfAfcccuUfgGfuauuuscsu
AGAAATACCAAGGGTGGAGATGC
390
412

AD-1381900
asusacc(Ahd)AfgGfGfUfggagaugcsusa
VPusAfsgcaUfcUfCfcaccCfuUfgguaususu
AAATACCAAGGGTGGAGATGCTC
392
414

AD-1381901
usascca(Ahd)GfgGfUfGfgagaugcuscsa
VPusGfsagcAfuCfUfccacCfcUfugguasusu
AATACCAAGGGTGGAGATGCTCC
393
415

AD-1381902
ascscaa(Ghd)GfgUfGfGfagaugcucscsa
VPusGfsgagCfaUfCfuccaCfcCfuuggusasu
ATACCAAGGGTGGAGATGCTCCA
394
416

AD-1381903
csasagg(Ghd)UfgGfAfGfaugcuccasgsa
VPusCfsuggAfgCfAfucucCfaCfccuugsgsu
ACCAAGGGTGGAGATGCTCCAGC
396
418

AD-1381904
asasggg(Uhd)GfgAfGfAfugcuccagscsa
VPusGfscugGfaGfCfaucuCfcAfcccuusgsg
CCAAGGGTGGAGATGCTCCAGCT
397
419

AD-1381905
asgsggu(Ghd)GfaGfAfUfgcuccagcsusa
VPusAfsgcuGfgAfGfcaucUfcCfacccususg
CAAGGGTGGAGATGCTCCAGCTG
398
420

AD-1381906
gsgsugg(Ahd)GfaUfGfCfuccagcugscsa
VPusGfscagCfuGfGfagcaUfcUfccaccscsu
AGGGTGGAGATGCTCCAGCTGCT
400
422

AD-1381907
gsusgga(Ghd)AfuGfCfUfccagcugcsusa
VPusAfsgcaGfcUfGfgagcAfuCfuccacscsc
GGGTGGAGATGCTCCAGCTGCTG
401
423

AD-1381908
usgsgag(Ahd)UfgCfUfCfcagcugcusgsa
VPusCfsagcAfgCfUfggagCfaUfcuccascsc
GGTGGAGATGCTCCAGCTGCTGG
402
424

AD-1381909
gsasgau(Ghd)CfuCfCfAfgcugcuggsusa
VPusAfsccaGfcAfGfcuggAfgCfaucucscsa
TGGAGATGCTCCAGCTGCTGGTG
404
426

AD-1381910
asgsaug(Chd)UfcCfAfGfcugcuggusgsa
VPusCfsaccAfgCfAfgcugGfaGfcaucuscsc
GGAGATGCTCCAGCTGCTGGTGA
405
427

AD-1381911
gsasugc(Uhd)CfcAfGfCfugcuggugsasa
VPusUfscacCfaGfCfagcuGfgAfgcaucsusc
GAGATGCTCCAGCTGCTGGTGAA
406
428

AD-1381912
usgscuc(Chd)AfgCfUfGfcuggugaasgsa
VPusCfsuucAfcCfAfgcagCfuGfgagcasusc
GATGCTCCAGCTGCTGGTGAAGA
408
430

AD-1381913
gscsucc(Ahd)GfcUfGfCfuggugaagsasa
VPusUfscuuCfaCfCfagcaGfcUfggagcsasu
ATGCTCCAGCTGCTGGTGAAGAT
409
431

AD-1381914
csuscca(Ghd)CfuGfCfUfggugaagasusa
VPusAfsucuUfcAfCfcagcAfgCfuggagscsa
TGCTCCAGCTGCTGGTGAAGATG
410
432

AD-1381915
cscsagc(Uhd)GfcUfGfGfugaagaugscsa
VPusGfscauCfuUfCfaccaGfcAfgcuggsasg
CTCCAGCTGCTGGTGAAGATGCA
412
434

AD-1381916
csasgcu(Ghd)CfuGfGfUfgaagaugcsasa
VPusUfsgcaUfcUfUfcaccAfgCfagcugsgsa
TCCAGCTGCTGGTGAAGATGCAT
413
435

AD-1381917
asgscug(Chd)UfgGfUfGfaagaugcasusa
VPusAfsugcAfuCfUfucacCfaGfcagcusgsg
CCAGCTGCTGGTGAAGATGCATG
414
436

AD-1381918
csusgcu(Ghd)GfuGfAfAfgaugcaugsasa
VPusUfscauGfcAfUfcuucAfcCfagcagscsu
AGCTGCTGGTGAAGATGCATGAA
416
438

AD-1381919
usgscug(Ghd)UfgAfAfGfaugcaugasasa
VPusUfsucaUfgCfAfucuuCfaCfcagcasgsc
GCTGCTGGTGAAGATGCATGAAT
417
439

AD-1381920
gscsugg(Uhd)GfaAfGfAfugcaugaasusa
VPusAfsuucAfuGfCfaucuUfcAfccagcsasg
CTGCTGGTGAAGATGCATGAATA
418
440

AD-1381921
usgsgug(Ahd)AfgAfUfGfcaugaauasgsa
VPusCfsuauUfcAfUfgcauCfuUfcaccasgsc
GCTGGTGAAGATGCATGAATAGG
420
442

AD-1381922
gsgsuga(Ahd)GfaUfGfCfaugaauagsgsa
VPusCfscuaUfuCfAfugcaUfcUfucaccsasg
CTGGTGAAGATGCATGAATAGGT
421
443

AD-1381923
gsusgaa(Ghd)AfuGfCfAfugaauaggsusa
VPusAfsccuAfuUfCfaugcAfuCfuucacscsa
TGGTGAAGATGCATGAATAGGTC
422
444

AD-1381924
usgsaag(Ahd)UfgCfAfUfgaauagguscsa
VPusGfsaccUfaUfUfcaugCfaUfcuucascsc
GGTGAAGATGCATGAATAGGTCC
423
445

AD-1381925
asasgau(Ghd)CfaUfGfAfauagguccsasa
VPusUfsggaCfcUfAfuucaUfgCfaucuuscsa
TGAAGATGCATGAATAGGTCCAA
425
447

AD-1381926
asgsaug(Chd)AfuGfAfAfuagguccasasa
VPusUfsuggAfcCfUfauucAfuGfcaucususc
GAAGATGCATGAATAGGTCCAAC
426
448

AD-1381927
gsasugc(Ahd)UfgAfAfUfagguccaascsa
VPusGfsuugGfaCfCfuauuCfaUfgcaucsusu
AAGATGCATGAATAGGTCCAACC
427
449

AD-1381928
usgscau(Ghd)AfaUfAfGfguccaaccsasa
VPusUfsgguUfgGfAfccuaUfuCfaugcasusc
GATGCATGAATAGGTCCAACCAG
429
451

AD-1381929
gscsaug(Ahd)AfuAfGfGfuccaaccasgsa
VPusCfsuggUfuGfGfaccuAfuUfcaugcsasu
ATGCATGAATAGGTCCAACCAGC
430
452

AD-1381930
csasuga(Ahd)UfaGfGfUfccaaccagscsa
VPusGfscugGfuUfGfgaccUfaUfucaugscsa
TGCATGAATAGGTCCAACCAGCT
431
453

AD-1381931
usgsaau(Ahd)GfgUfCfCfaaccagcusgsa
VPusCfsagcUfgGfUfuggaCfcUfauucasusg
CATGAATAGGTCCAACCAGCTGT
433
455

AD-1381932
gsasaua(Ghd)GfuCfCfAfaccagcugsusa
VPusAfscagCfuGfGfuuggAfcCfuauucsasu
ATGAATAGGTCCAACCAGCTGTA
434
456

AD-1381933
asasuag(Ghd)UfcCfAfAfccagcugusasa
VPusUfsacaGfcUfGfguugGfaCfcuauuscsa
TGAATAGGTCCAACCAGCTGTAC
435
457

AD-1381934
asusagg(Uhd)CfcAfAfCfcagcuguascsa
VPusGfsuacAfgCfUfgguuGfgAfccuaususc
GAATAGGTCCAACCAGCTGTACA
436
458

AD-1381935
usasggu(Chd)CfaAfCfCfagcuguacsasa
VPusUfsguaCfaGfCfugguUfgGfaccuasusu
AATAGGTCCAACCAGCTGTACAT
437
459

AD-1381936
asgsguc(Chd)AfaCfCfAfgcuguacasusa
VPusAfsuguAfcAfGfcuggUfuGfgaccusasu
ATAGGTCCAACCAGCTGTACATT
438
460

AD-1381937
gsgsucc(Ahd)AfcCfAfGfcuguacaususa
VPusAfsaugUfaCfAfgcugGfuUfggaccsusa
TAGGTCCAACCAGCTGTACATTT
439
461

AD-1381938
gsuscca(Ahd)CfcAfGfCfuguacauususa
VPusAfsaauGfuAfCfagcuGfgUfuggacscsu
AGGTCCAACCAGCTGTACATTTG
440
462

AD-1381939
uscscaa(Chd)CfaGfCfUfguacauuusgsa
VPusCfsaaaUfgUfAfcagcUfgGfuuggascsc
GGTCCAACCAGCTGTACATTTGG
441
463

AD-1381940
cscsaac(Chd)AfgCfUfGfuacauuugsgsa
VPusCfscaaAfuGfUfacagCfuGfguuggsasc
GTCCAACCAGCTGTACATTTGGA
442
464

AD-1381941
csasacc(Ahd)GfcUfGfUfacauuuggsasa
VPusUfsccaAfaUfGfuacaGfcUfgguugsgsa
TCCAACCAGCTGTACATTTGGAA
443
465

AD-1381942
asascca(Ghd)CfuGfUfAfcauuuggasasa
VPusUfsuccAfaAfUfguacAfgCfugguusgsg
CCAACCAGCTGTACATTTGGAAA
444
466

AD-1381943
ascscag(Chd)UfgUfAfCfauuuggaasasa
VPusUfsuucCfaAfAfuguaCfaGfcuggususg
CAACCAGCTGTACATTTGGAAAA
445
467

AD-1381944
cscsagc(Uhd)GfuAfCfAfuuuggaaasasa
VPusUfsuuuCfcAfAfauguAfcAfgcuggsusu
AACCAGCTGTACATTTGGAAAAA
446
468

AD-1381945
csasgcu(Ghd)UfaCfAfUfuuggaaaasasa
VPusUfsuuuUfcCfAfaaugUfaCfagcugsgsu
ACCAGCTGTACATTTGGAAAAAT
447
469

AD-1381946
asgscug(Uhd)AfcAfUfUfuggaaaaasusa
VPusAfsuuuUfuCfCfaaauGfuAfcagcusgsg
CCAGCTGTACATTTGGAAAAATA
448
470

AD-1381947
gscsugu(Ahd)CfaUfUfUfggaaaaausasa
VPusUfsauuUfuUfCfcaaaUfgUfacagcsusg
CAGCTGTACATTTGGAAAAATAA
449
471

AD-1381948
csusgua(Chd)AfuUfUfGfgaaaaauasasa
VPusUfsuauUfuUfUfccaaAfuGfuacagscsu
AGCTGTACATTTGGAAAAATAAA
450
472

AD-1381949
usascau(Uhd)UfgGfAfAfaaauaaaascsa
VPusGfsuuuUfaUfUfuuucCfaAfauguascsa
TGTACATTTGGAAAAATAAAACT
453
475

TABLE 15

RPS25 Single Dose Screen in HeLa Cells

Average RPS25 mRNA

Remaining

Duplex
10 nM

ID
mean
SD

XD-18245
0.238
0.013

XD-18246
0.131
0.042

XD-18247
0.040
0.003

XD-18248
0.053
0.007

XD-18249
0.035
0.008

XD-18250
0.049
0.011

XD-18251
0.186
0.008

XD-18252
0.098
0.022

XD-18253
0.032
0.007

XD-18254
0.048
0.002

XD-18255
0.031
0.007

XD-18256
0.060
0.011

XD-18257
0.038
0.004

XD-18258
0.058
0.005

XD-18259
0.040
0.007

XD-18260
0.051
0.009

XD-18261
0.088
0.028

XD-18262
0.039
0.004

XD-18263
0.065
0.016

XD-18264
0.042
0.003

XD-18265
0.049
0.003

XD-18266
0.058
0.001

XD-18267
0.064
0.006

XD-18268
0.078
0.007

XD-18269
0.091
0.022

XD-18270
0.078
0.013

XD-18271
0.073
0.010

XD-18272
0.049
0.011

XD-18273
0.039
0.003

XD-18274
0.121
0.007

XD-18275
0.052
0.003

XD-18276
0.098
0.011

XD-18277
0.100
0.016

XD-18278
0.036
0.009

XD-18279
0.049
0.006

XD-18280
0.035
0.003

XD-18281
0.054
0.010

XD-18282
0.395
0.025

XD-18283
0.211
0.033

XD-18284
0.030
0.010

XD-18285
0.046
0.004

XD-18286
0.064
0.003

XD-18287
0.061
0.003

XD-18288
0.072
0.031

XD-18289
0.076
0.008

XD-18290
0.055
0.004

XD-18291
0.101
0.016

XD-18292
0.052
0.003

XD-18293
0.074
0.004

XD-18294
0.043
0.004

XD-18295
0.059
0.009

XD-18296
0.049
0.003

XD-18297
0.072
0.006

XD-18298
0.271
0.018

XD-18299
0.093
0.004

XD-18300
0.043
0.003

XD-18301
0.048
0.002

XD-18302
0.081
0.002

XD-18303
0.039
0.008

XD-18304
0.033
0.004

XD-18305
0.045
0.006

XD-18306
0.046
0.004

XD-18307
0.067
0.005

XD-18308
0.042
0.004

XD-18309
0.565
0.028

XD-18310
0.118
0.009

XD-18311
0.064
0.005

XD-18312
0.051
0.005

XD-18313
0.041
0.005

XD-18314
0.039
0.002

XD-18315
0.053
0.004

XD-18316
0.044
0.002

XD-18317
0.051
0.001

XD-18318
0.052
0.005

XD-18319
0.057
0.005

XD-18320
0.065
0.009

XD-18321
0.036
0.006

XD-18322
0.028
0.003

XD-18323
0.043
0.006

XD-18324
0.073
0.008

XD-18325
0.086
0.006

XD-18326
0.038
0.001

XD-18327
0.044
0.013

XD-18328
0.067
0.002

XD-18329
0.065
0.004

XD-18330
0.467
0.072

XD-18331
0.075
0.022

XD-18332
0.042
0.003

XD-18333
0.044
0.005

XD-18334
0.075
0.004

XD-18335
0.053
0.007

XD-18336
0.036
0.004

XD-18337
0.035
0.004

XD-18338
0.043
0.003

XD-18339
0.138
0.004

XD-18340
0.044
0.002

XD-18341
0.066
0.001

XD-18342
0.052
0.003

XD-18343
0.017
0.013

XD-18344
0.052
0.024

XD-18345
0.030
0.004

XD-18346
0.033
0.007

XD-18347
0.029
0.001

XD-18348
0.024
0.010

XD-18349
0.033
0.001

XD-18350
0.040
0.008

XD-18351
0.067
0.028

XD-18352
0.043
0.026

XD-18353
0.052
0.002

XD-18354
0.028
0.015

XD-18355
0.038
0.006

XD-18356
0.071
0.009

XD-18357
0.062
0.001

XD-18358
0.044
0.001

XD-18359
0.059
0.021

XD-18360
0.046
0.003

XD-18361
0.033
0.003

XD-18362
0.070
0.007

XD-18363
0.033
0.005

XD-18364
0.034
0.001

XD-18365
0.035
0.010

XD-18366
0.030
0.003

XD-18367
0.104
0.005

XD-18368
0.030
0.005

XD-18369
0.041
0.008

XD-18370
0.045
0.008

XD-18371
0.044
0.005

XD-18372
0.036
0.002

XD-18373
0.481
0.016

XD-18374
0.081
0.004

XD-18375
0.052
0.002

XD-18376
0.047
0.002

XD-18377
0.053
0.011

XD-18378
0.213
0.037

XD-18379
0.449
0.007

XD-18380
0.063
0.015

XD-18381
0.044
0.002

XD-18382
0.046
0.014

XD-18383
0.042
0.007

XD-18384
0.040
0.002

XD-18385
0.031
0.013

XD-18386
0.036
0.006

XD-18387
0.038
0.006

XD-18388
0.092
0.008

XD-18389
0.051
0.006

XD-18390
0.084
0.011

XD-18391
0.385
0.042

XD-18392
0.063
0.010

XD-18393
0.319
0.017

XD-18394
0.196
0.011

XD-18395
0.731
0.029

XD-18396
0.919
0.019

XD-18397
0.704
0.022

XD-18398
0.170
0.016

XD-18399
0.148
0.026

XD-18400
0.129
0.019

XD-18401
0.104
0.025

XD-18402
0.044
0.003

XD-18403
0.063
0.005

XD-18404
0.063
0.004

XD-18405
0.050
0.003

XD-18406
0.046
0.004

XD-18407
0.041
0.009

XD-18408
0.063
0.008

XD-18409
0.064
0.016

XD-18410
0.048
0.001

XD-18411
0.069
0.005

XD-18412
0.045
0.003

XD-18413
0.082
0.007

XD-18414
0.340
0.036

XD-18415
0.035
0.002

XD-18416
0.045
0.004

XD-18417
0.029
0.006

XD-18418
0.055
0.007

XD-18419
0.044
0.005

XD-18420
0.044
0.002

XD-18421
0.061
0.006

XD-18422
0.065
0.006

XD-18423
0.065
0.009

XD-18424
0.034
0.010

XD-18425
0.037
0.003

XD-18426
0.040
0.006

XD-18427
0.037
0.005

XD-18428
0.052
0.007

XD-18429
0.133
0.038

XD-18430
0.153
0.012

XD-18431
0.044
0.006

XD-18432
0.058
0.015

XD-18433
0.076
0.005

XD-18434
0.043
0.006

XD-18435
0.034
0.002

XD-18436
0.030
0.002

XD-18437
0.069
0.003

XD-18438
0.048
0.011

XD-18439
0.041
0.006

XD-18440
0.039
0.010

XD-18441
0.028
0.004

XD-18442
0.026
0.003

XD-18443
0.048
0.008

XD-18444
0.047
0.006

XD-18445
0.045
0.005

XD-18446
0.042
0.002

XD-18447
0.062
0.003

XD-18448
0.056
0.001

XD-18449
0.060
0.005

XD-18450
0.068
0.006

XD-18451
0.050
0.006

XD-18452
0.066
0.005

XD-18453
0.048
0.006

XD-18454
0.132
0.012

XD-18455
0.135
0.010

XD-18456
0.214
0.014

XD-18457
0.066
0.006

XD-18458
0.051
0.012

XD-18459
0.050
0.005

XD-18460
0.048
0.005

XD-18461
0.063
0.004

XD-18462
0.044
0.003

XD-18463
0.066
0.010

XD-18464
0.054
0.007

XD-18465
0.087
0.009

XD-18466
0.057
0.004

XD-18467
0.038
0.004

XD-18468
0.110
0.008

XD-18469
0.060
0.016

XD-18470
0.070
0.004

XD-18471
0.112
0.029

XD-18472
0.074
0.024

XD-18473
0.107
0.005

XD-18474
0.145
0.024

XD-18475
0.120
0.016

XD-18476
0.064
0.008

XD-18477
0.102
0.004

XD-18478
0.077
0.009

XD-18479
0.041
0.005

XD-18480
0.045
0.002

XD-18481
0.039
0.003

XD-18482
0.049
0.009

XD-18483
0.042
0.007

XD-18484
0.036
0.004

XD-18485
0.039
0.001

XD-18486
0.067
0.002

XD-18487
0.174
0.044

XD-18488
0.047
0.004

XD-18489
0.140
0.044

XD-18490
0.048
0.006

XD-18491
0.057
0.004

XD-18492
0.125
0.017

XD-18493
0.101
0.011

XD-18494
0.049
0.011

XD-18495
0.086
0.006

XD-18496
0.079
0.006

XD-18497
0.084
0.006

XD-18498
0.313
0.008

XD-18499
0.079
0.004

XD-18500
0.093
0.005

XD-18501
0.133
0.005

XD-18502
0.114
0.005

XD-18503
0.075
0.003

XD-18504
0.073
0.002

XD-18505
0.078
0.014

XD-18506
0.075
0.008

XD-18507
0.064
0.003

XD-18508
0.064
0.008

XD-18509
0.052
0.005

XD-18510
0.062
0.006

XD-18511
0.072
0.002

XD-18512
0.067
0.006

XD-18513
0.057
0.017

XD-18514
0.048
0.005

XD-00376
0.910
0.049

XD-00033
0.976
0.060

XD-00033
0.067
0.002

Example 2. In Vivo Evaluation in Transgenic Mice

This Example describes methods for the in vivo evaluation of RPS25 RNAi agents in a transgenic mouse model of a nucleotide repeat expansion disease, C9ORF72 ALS/FTD (a transgenic mouse model expressing human C9orf72 RNAs with up to, e.g., 450 GGGGCC repeats (SEQ ID NO: 17); see, e.g., Jiang, et al. (2016) Neuron 90:535-550).

The ability of selected dsRNA agents designed and assayed in Example 1 are assessed for their ability to reduce the level of both sense- or antisense-containing foci in mice expressing human C9orf72 RNAs with up to 450 GGGGCC repeats (SEQ ID NO: 17).

Briefly, control littermates, mice heterozygous for the human C9orf72 RNA with up to 450 GGGGCC repeats (SEQ ID NO: 17), and mice homozygous for the human C9orf72 RNA with up to 450 GGGGCC repeats (SEQ ID NO: 17) are administered intrathecally or subcutaneously a single dose of the dsRNA agents of interest, or a placebo. Two weeks post-administration, animals are sacrificed, blood and tissue samples, including cerebral cortex, spinal cord, liver, spleen, and cervical lymph nodes, are collected.

There are three C9orf72 transcripts generated by differential use of transcription alternative start and termination sites generates. Therefore, to determine the effect of administration of the dsRNA agents targeting RPS25 on the level of detrimental repeat-containing mRNA, the levels of repeat-containing C9orf 72 mRNA, total C9orf72 mRNA, and exon 1b-containing, mRNA levels are determined in cortex and spinal cord samples by qRT-PCR (see, e.g., above and Jiang, supra).

The results demonstrate that administration of a single dose of the dsRNA agents targeting RPS25 inhibits the production of repeat-containing C9orf 72 mRNA, the level of total C9orf72 mRNA and the level of exon 1b-containing mRNA levels.

In order to determine the effect of the dsRNA agents targeting RPS25 to reduce the number and/or formation of both C9ORF72 sense strand- and antisense strand-containing foci, the FISH methods described in Jiang, supra are employed in samples obtained from the animals administered the duplexes of interest from above. The probes that are used include those that are against the sense and antisense RNA hexanucleotide repeat (Exiqon, Inc.). All hybridization steps are performed under RNase-free conditions. Fifteen micrometer brain and spinal cord OCT frozen sections are permeabilized and the sections are blocked. The sections are then hybridized with denatured probes. After hybridization, slides are washed. Autofluorescence of lipofuscin is quenched and cell nuclei are stained with DAPI. Quantitation of sense and antisense RNA foci in mouse frontal cortex, hippocampal dentate gyrus, retrosplenial cortex and cerebellar molecular layer is performed by a blinded investigator. Three to six random pictures are taken by confocal microscopy under 100× magnification and 200-400 cells are counted.

The results demonstrate that administration of a single dose of the dsRNA agents targeting RPS25 reduce the level of C9orf72 sense strand- and C9orf72 antisense strand-containing foci in the frontal cortex, hippocampal dentate gyrus, retrosplenial cortex and cerebellar molecular layer.

The effect of administration of the agents targeting RPS25 on the level of aberrant dipeptide repeat protein level and poly(GP) and poly(GA) burden and size is also assessed as described in, for example, Jiang, supra) in the animals administered the duplexes of interest above.

Immunohistochemistry is used to identify and assess aberrant dipeptide repeat protein level in mouse hemibrain and spinal cord. Briefly, eight to ten micron thick sagittal slices of mouse hemibrain or coronal slices of spinal cord are cut from formalin-fixed, paraffin-embedded blocks and mounted on glass slides. After drying, slides are deparaffinized and rehydrated in xylene and alcohol washes before washing. Then slides are steamed and blocked. After staining with commercially available antibodies against poly(GP), poly(GA), poly(GR), poly(PA), poly(PR), GFAP, IBA-1, CD3, F4/80, and CD45R/B220 overnight, HRP-conjugated secondary antibody is applied and peroxidase activity is developed with substrate. Sections are counterstained with Harris' modified hematoxylin and coverslipped.

To quantify poly(GP) and poly(GA) inclusion burden and size, mice hemibrain sections immunostained for poly(GP) or poly(GA) are scanned at 40× magnification to obtain high-resolution digitized images. Using suitable software, the number of inclusions in the hippocampus or a delineated area in the retrosplenial cortex are counted. To measure the size of inclusions in these regions, images are taken with a microscope under 63× magnification. Although each inclusion in a given field is only analyzed once, multiple images of the field may be taken to ensure the analysis is done only on inclusions that are in focus. Images are opened and enlarged, and an outline tool is used to trace each inclusion to determine its area (μm²). For each mouse, the average size of inclusions in μm2 within each tested region is calculated.

The data is used to determine whether administration of a single dose of the dsRNA agents targeting RPS25 reduces the level of aberrant dipeptide repeat protein levels, in particular the level of poly(GP) and poly(GA) inclusion burden and size.

INFORMAL SEQUENCE LISTING

SEQ ID NO: 1

>NM_001028.3 Homo sapiens ribosomal protein S25 (RPS25), mRNA

CTTTTTGTCCGACATCTTGACGAGGCTGCGGTGTCTGCTGCTATTCTCCGAGCTTCGCAATGCCGCCTAA

GGACGACAAGAAGAAGAAGGACGCTGGAAAGTCGGCCAAGAAAGACAAAGACCCAGTGAACAAATCCGGG

GGCAAGGCCAAAAAGAAGAAGTGGTCCAAAGGCAAAGTTCGGGACAAGCTCAATAACTTAGTCTTGTTTG

ACAAAGCTACCTATGATAAACTCTGTAAGGAAGTTCCCAACTATAAACTTATAACCCCAGCTGTGGTCTC

TGAGAGACTGAAGATTCGAGGCTCCCTGGCCAGGGCAGCCCTTCAGGAGCTCCTTAGTAAAGGACTTATC

AAACTGGTTTCAAAGCACAGAGCTCAAGTAATTTACACCAGAAATACCAAGGGTGGAGATGCTCCAGCTG

CTGGTGAAGATGCATGAATAGGTCCAACCAGCTGTACATTTGGAAAAATAAAACTTTATTAAA

SEQ ID NO: 2

Reverse Complement of SEQ ID NO: 1

TTTAATAAAGTTTTATTTTTCCAAATGTACAGCTGGTTGGACCTATTCATGCATCTTCACCAGCAGCTGGAGCAT

CTCCACCCTTGGTATTTCTGGTGTAAATTACTTGAGCTCTGTGCTTTGAAACCAGTTTGATAAGTCCTTTACTAA

GGAGCTCCTGAAGGGCTGCCCTGGCCAGGGAGCCTCGAATCTTCAGTCTCTCAGAGACCACAGCTGGGGTTATAA

GTTTATAGTTGGGAACTTCCTTACAGAGTTTATCATAGGTAGCTTTGTCAAACAAGACTAAGTTATTGAGCTTGT

CCCGAACTTTGCCTTTGGACCACTTCTTCTTTTTGGCCTTGCCCCCGGATTTGTTCACTGGGTCTTTGTCTTTCT

TGGCCGACTTTCCAGCGTCCTTCTTCTTCTTGTCGTCCTTAGGCGGCATTGCGAAGCTCGGAGAATAGCAGCAGA

CACCGCAGCCTCGTCAAGATGTCGGACAAAAAG

SEQ ID NO: 3

>NM_024266.3 Mus musculus ribosomal protein S25 (Rps25), mRNA

AGCGAGGCTGCTGTGGTCTACACGACTCTCTGAGCTTCGCCATGCCTCCCAAAGACGACAAGAAGAAGAA

AGATGCCGGAAAGTCGGCCAAAAAGGATAAAGACCCAGTAAATAAATCTGGTGGCAAGGCCAAGAAGAAG

AAGTGGTCCAAAGGCAAAGTTCGGGACAAGTTGAACAATCTTGTCCTGTTCGACAAAGCGACATACGACA

AGCTCTGTAAGGAGGTTCCGAACTATAAGCTTATTACTCCAGCCGTGGTCTCTGAGAGACTGAAGATTCG

CGGTTCCTTGGCCAGGGCAGCCCTTCAGGAGCTCCTTAGTAAAGGACTTATCAAGCTGGTTTCAAAGCAC

AGAGCCCAAGTAATTTACACCAGAAACACAAAGGGTGGGGACGCTCCAGCTGCTGGCGAAGATGCATGAA

CAGGTTCAATCAGCTGTACATTTGGAAAAATAAAACTTTATTGAATCAAATGAATGGGTGCATCTGTTTC

CTAAGGCAGCCGGGGAGGATTTGGTCTTAGGAATAATAGCTGGAATTGGTTTGTTGGCCATGAAGTCAGA

TGCAATTGCGCCTGGGAACCTTCAGCTTTTCCCTTTACGTGGTTGCTTGCTTCTTGTTGCAGCTTCGGTT

TTGAATTGATGCCTGAAAGAAAATAAAAACTTAGCAAGACTAATGGTAAATCTAAAAAAAAAAAAAAAAA

A

SEQ ID NO: 4

Reverse Complement of SEQ ID NO: 3

TTTTTTTTTTTTTTTTTTAGATTTACCATTAGTCTTGCTAAGTTTTTATTTTCTTTCAGGCATCAATTCAAAACC

GAAGCTGCAACAAGAAGCAAGCAACCACGTAAAGGGAAAAGCTGAAGGTTCCCAGGCGCAATTGCATCTGACTTC

ATGGCCAACAAACCAATTCCAGCTATTATTCCTAAGACCAAATCCTCCCCGGCTGCCTTAGGAAACAGATGCACC

CATTCATTTGATTCAATAAAGTTTTATTTTTCCAAATGTACAGCTGATTGAACCTGTTCATGCATCTTCGCCAGC

AGCTGGAGCGTCCCCACCCTTTGTGTTTCTGGTGTAAATTACTTGGGCTCTGTGCTTTGAAACCAGCTTGATAAG

TCCTTTACTAAGGAGCTCCTGAAGGGCTGCCCTGGCCAAGGAACCGCGAATCTTCAGTCTCTCAGAGACCACGGC

TGGAGTAATAAGCTTATAGTTCGGAACCTCCTTACAGAGCTTGTCGTATGTCGCTTTGTCGAACAGGACAAGATT

GTTCAACTTGTCCCGAACTTTGCCTTTGGACCACTTCTTCTTCTTGGCCTTGCCACCAGATTTATTTACTGGGTC

TTTATCCTTTTTGGCCGACTTTCCGGCATCTTTCTTCTTCTTGTCGTCTTTGGGAGGCATGGCGAAGCTCAGAGA

GTCGTGTAGACCACAGCAGCCTCGCT

SEQ ID NO: 5

>NM_001005528.1 Rattus norvegicus ribosomal protein s25 (Rps25), mRNA

TGTGGCTGCAGTGGTCCACACTACTCTCTGAGTTTCGCCATGCCGCCCAAAGACGACAAGAAGAAGAAGG

ATGCCGGAAAGTCGGCCAAAAAAGACAAGGACCCAGTAAATAAATCTGGTGGCAAGGCCAAAAAGAAGAA

GTGGTCCAAAGGCAAAGTTCGGGACAAGCTGAACAATCTCGTCCTGTTTGACAAAGCTACTTACGACAAA

CTTTGTAAGGAAGTTCCCAACTATAAGCTTATTACTCCAGCTGTGGTCTCCGAGAGACTGAAGATTCGAG

GTTCCTTGGCCAGGGCAGCCCTTCAGGAGCTACTTAGTAAAGGACTTATCAAGCTGGTTTCAAAGCACAG

AGCCCAAGTAATTTACACCAGAAACACAAAGGGTGGAGATGCCCCAGCTGCTGGTGAAGATGCATAAACA

GATTGAATCAGCTGTACATTTGGGAAAATAAAACTTTATTGAATCA

SEQ ID NO: 6

Reverse Complement of SEQ ID NO: 5

TGATTCAATAAAGTTTTATTTTCCCAAATGTACAGCTGATTCAATCTGTTTATGCATCTTCACCAGCAGCTGGGG

CATCTCCACCCTTTGTGTTTCTGGTGTAAATTACTTGGGCTCTGTGCTTTGAAACCAGCTTGATAAGTCCTTTAC

TAAGTAGCTCCTGAAGGGCTGCCCTGGCCAAGGAACCTCGAATCTTCAGTCTCTCGGAGACCACAGCTGGAGTAA

TAAGCTTATAGTTGGGAACTTCCTTACAAAGTTTGTCGTAAGTAGCTTTGTCAAACAGGACGAGATTGTTCAGCT

TGTCCCGAACTTTGCCTTTGGACCACTTCTTCTTTTTGGCCTTGCCACCAGATTTATTTACTGGGTCCTTGTCTT

TTTTGGCCGACTTTCCGGCATCCTTCTTCTTCTTGTCGTCTTTGGGCGGCATGGCGAAACTCAGAGAGTAGTGTG

GACCACTGCAGCCACA

SEQ ID NO: 7

>XM_015115940.1 PREDICTED: Macaca mulatta ribosomal protein S25 (RPS25),

mRNA

GCACCTGCGGCGCCTGCGCATTGGGAGCGACACGCTCGGGCATAAGTAGTGCCGGAAAGTTAGTTGCCGA

GACCTGGTGGATTGTTTTCCGTTTATCAGTGCCGGAAAACAGTACTACAGTACTGCGTCACAACTAGCCC

GGACTCCGACAACCTGGCGCGGTATTTAGGCGGTGCGGCTTGGGAACTAGAATTCACTTCCTGTCTTCCT

CTTGAGGCTAGAGGGCGAGCACTTCGCCGTGGGACTTCCTCCGCCTGGCTCCGCCTCTTGCCCCGGAAGT

ACTTACAGCGGACGGAGGTTTCTGGGCCCGTTTCTGAGCAGCGCTTCCTTTTTGTCCGACATCTTAGCAA

GCCTGCGGTGTCTGCTGCTGCTCCCCGAGCTTCGCAATGCCGCCCAAGGACGACAAGAAGAAGAAGGACG

CCGGAAAGTCGGCCAAGAAAGACAAAGACCCAGTGAACAAATCTGGGGGCAAGGCCAAAAAGAAGAAGTG

GTCCAAAGGCAAAGTTCGGGACAAGCTCAATAACTTAGTCTTGTTTGACAAAGCTACCTACGACAAACTC

TGTAAGGAAGTTCCCAACTATAAACTTATAACCCCAGCTGTAGTCTCTGAGAGACTGAAGATTCGAGGCT

CCCTGGCCAGGGCAGCCCTTCAGGAGCTCCTTAGTAAAGGACTTATCAAACTGGTTTCAAAGCACAGAGC

TCAAGTAATTTACACCAGAAATACCAAGGGCGGAGATGCTCCAGCTGCTGGTGAAGATGCATGAATAGGT

CCAACCAATTGTACATTTGGAAAAATAAAACTATTAAATCAAA

SEQ ID NO: 8

Reverse Complement of SEQ ID NO: 7

TTTGATTTAATAGTTTTATTTTTCCAAATGTACAATTGGTTGGACCTATTCATGCATCTTCACCAGCAGCTGGAG

CATCTCCGCCCTTGGTATTTCTGGTGTAAATTACTTGAGCTCTGTGCTTTGAAACCAGTTTGATAAGTCCTTTAC

TAAGGAGCTCCTGAAGGGCTGCCCTGGCCAGGGAGCCTCGAATCTTCAGTCTCTCAGAGACTACAGCTGGGGTTA

TAAGTTTATAGTTGGGAACTTCCTTACAGAGTTTGTCGTAGGTAGCTTTGTCAAACAAGACTAAGTTATTGAGCT

TGTCCCGAACTTTGCCTTTGGACCACTTCTTCTTTTTGGCCTTGCCCCCAGATTTGTTCACTGGGTCTTTGTCTT

TCTTGGCCGACTTTCCGGCGTCCTTCTTCTTCTTGTCGTCCTTGGGCGGCATTGCGAAGCTCGGGGAGCAGCAGC

AGACACCGCAGGCTTGCTAAGATGTCGGACAAAAAGGAAGCGCTGCTCAGAAACGGGCCCAGAAACCTCCGTCCG

CTGTAAGTACTTCCGGGGCAAGAGGCGGAGCCAGGCGGAGGAAGTCCCACGGCGAAGTGCTCGCCCTCTAGCCTC

AAGAGGAAGACAGGAAGTGAATTCTAGTTCCCAAGCCGCACCGCCTAAATACCGCGCCAGGTTGTCGGAGTCCGG

GCTAGTTGTGACGCAGTACTGTAGTACTGTTTTCCGGCACTGATAAACGGAAAACAATCCACCAGGTCTCGGCAA

CTAACTTTCCGGCACTACTTATGCCCGAGCGTGTCGCTCCCAATGCGCAGGCGCCGCAGGTGC

SEQ ID NO: 9

>NM_001285107.1 Macaca fascicularis ribosomal protein S25 (RPS25), mRNA

CTTTTTGTCCGACATCTTAGCAAGCCAGCGGTGTCTGCTGCTGCTCCCCGAGCTTCGCAATGCCGCCCAA

GGACGACAAGAAGAAGGAGGACGCCGGAAAGTCGGCCAAGAAAGACAAAGACCCAGTGAACAAATCTGGG

GGCAAGGCCAAAAAGAAGAAGTGGTCCAAAGGCAAAGTTCGGGACAAGCTCAATAACTTAGTCTTGTTTG

ACAAAGCTACCTACGACAAACTCTGTAAGGAAGTTCCCAACTATAAACTTATAACCCCAGCTGTAGTCTC

TGAGAGACTGAAGATTCGAGGCTCCCTGGCCAGGGCAGCCCTTCAGGAGCTCCTTAGTAAAGGACTTATC

AAACTGGTTTCAAAGCACAGAGCTCAAGTAATTTACACCAGAAATACCAAGGGCGGAGATGCTCCAGCTG

CTGGTGAAGATGCATGAATAGGTCCAACCAATTGTACATTTGGAAAAATAAAACTTTATTAAATCAAAAA

AAAAAAAAAAA

SEQ ID NO: 10

Reverse Complement of SEQ ID NO:9

TTTTTTTTTTTTTTTTGATTTAATAAAGTTTTATTTTTCCAAATGTACAATTGGTTGGACCTATTCATGCATCTT

CACCAGCAGCTGGAGCATCTCCGCCCTTGGTATTTCTGGTGTAAATTACTTGAGCTCTGTGCTTTGAAACCAGTT

TGATAAGTCCTTTACTAAGGAGCTCCTGAAGGGCTGCCCTGGCCAGGGAGCCTCGAATCTTCAGTCTCTCAGAGA

CTACAGCTGGGGTTATAAGTTTATAGTTGGGAACTTCCTTACAGAGTTTGTCGTAGGTAGCTTTGTCAAACAAGA

CTAAGTTATTGAGCTTGTCCCGAACTTTGCCTTTGGACCACTTCTTCTTTTTGGCCTTGCCCCCAGATTTGTTCA

CTGGGTCTTTGTCTTTCTTGGCCGACTTTCCGGCGTCCTCCTTCTTCTTGTCGTCCTTGGGCGGCATTGCGAAGC

TCGGGGAGCAGCAGCAGACACCGCTGGCTTGCTAAGATGTCGGACAAAAAG

SEQ ID NO: 11

>NM_145005.6 Homo sapiens C9orf72-SMCR8 complex subunit (C9orf72),

transcript variant 1, mRNA

ACGTAACCTACGGTGTCCCGCTAGGAAAGAGAGGTGCGTCAAACAGCGACAAGTTCCGCCCACGTAAAAG

ATGACGCTTGATATCTCCGGAGCATTTGGATAATGTGACAGTTGGAATGCAGTGATGTCGACTCTTTGCC

CACCGCCATCTCCAGCTGTTGCCAAGACAGAGATTGCTTTAAGTGGCAAATCACCTTTATTAGCAGCTAC

TTTTGCTTACTGGGACAATATTCTTGGTCCTAGAGTAAGGCACATTTGGGCTCCAAAGACAGAACAGGTA

CTTCTCAGTGATGGAGAAATAACTTTTCTTGCCAACCACACTCTAAATGGAGAAATCCTTCGAAATGCAG

AGAGTGGTGCTATAGATGTAAAGTTTTTTGTCTTGTCTGAAAAGGGAGTGATTATTGTTTCATTAATCTT

TGATGGAAACTGGAATGGGGATCGCAGCACATATGGACTATCAATTATACTTCCACAGACAGAACTTAGT

TTCTACCTCCCACTTCATAGAGTGTGTGTTGATAGATTAACACATATAATCCGGAAAGGAAGAATATGGA

TGCATAAGGAAAGACAAGAAAATGTCCAGAAGATTATCTTAGAAGGCACAGAGAGAATGGAAGATCAGGG

TCAGAGTATTATTCCAATGCTTACTGGAGAAGTGATTCCTGTAATGGAACTGCTTTCATCTATGAAATCA

CACAGTGTTCCTGAAGAAATAGATATAGCTGATACAGTACTCAATGATGATGATATTGGTGACAGCTGTC

ATGAAGGCTTTCTTCTCAAGTAAGAATTTTTCTTTTCATAAAAGCTGGATGAAGCAGATACCATCTTATG

CTCACCTATGACAAGATTTGGAAGAAAGAAAATAACAGACTGTCTACTTAGATTGTTCTAGGGACATTAC

GTATTTGAACTGTTGCTTAAATTTGTGTTATTTTTCACTCATTATATTTCTATATATATTTGGTGTTATT

CCATTTGCTATTTAAAGAAACCGAGTTTCCATCCCAGACAAGAAATCATGGCCCCTTGCTTGATTCTGGT

TTCTTGTTTTACTTCTCATTAAAGCTAACAGAATCCTTTCATATTAAGTTGTACTGTAGATGAACTTAAG

TTATTTAGGCGTAGAACAAAATTATTCATATTTATACTGATCTTTTTCCATCCAGCAGTGGAGTTTAGTA

CTTAAGAGTTTGTGCCCTTAAACCAGACTCCCTGGATTAATGCTGTGTACCCGTGGGCAAGGTGCCTGAA

TTCTCTATACACCTATTTCCTCATCTGTAAAATGGCAATAATAGTAATAGTACCTAATGTGTAGGGTTGT

TATAAGCATTGAGTAAGATAAATAATATAAAGCACTTAGAACAGTGCCTGGAACATAAAAACACTTAATA

ATAGCTCATAGCTAACATTTCCTATTTACATTTCTTCTAGAAATAGCCAGTATTTGTTGAGTGCCTACAT

GTTAGTTCCTTTACTAGTTGCTTTACATGTATTATCTTATATTCTGTTTTAAAGTTTCTTCACAGTTACA

GATTTTCATGAAATTTTACTTTTAATAAAAGAGAAGTAAAAGTATAAAGTATTCACTTTTATGTTCACAG

TCTTTTCCTTTAGGCTCATGATGGAGTATCAGAGGCATGAGTGTGTTTAACCTAAGAGCCTTAATGGCTT

GAATCAGAAGCACTTTAGTCCTGTATCTGTTCAGTGTCAGCCTTTCATACATCATTTTAAATCCCATTTG

ACTTTAAGTAAGTCACTTAATCTCTCTACATGTCAATTTCTTCAGCTATAAAATGATGGTATTTCAATAA

ATAAATACATTAATTAAATGATATTATACTGACTAATTGGGCTGTTTTAAGGCTCAATAAGAAAATTTCT

GTGAAAGGTCTCTAGAAAATGTAGGTTCCTATACAAATAAAAGATAACATTGTGCTTATAAAAAAAA

SEQ ID NO: 12

>NM_018325.5 Homo sapiens C9orf72-SMCR8 complex subunit (C9orf72),

transcript variant 2, mRNA

GGTTGCGGTGCCTGCGCCCGCGGCGGCGGAGGCGCAGGCGGTGGCGAGTGGATATCTCCGGAGCATTTGG

ATAATGTGACAGTTGGAATGCAGTGATGTCGACTCTTTGCCCACCGCCATCTCCAGCTGTTGCCAAGACA

GAGATTGCTTTAAGTGGCAAATCACCTTTATTAGCAGCTACTTTTGCTTACTGGGACAATATTCTTGGTC

CTAGAGTAAGGCACATTTGGGCTCCAAAGACAGAACAGGTACTTCTCAGTGATGGAGAAATAACTTTTCT

TGCCAACCACACTCTAAATGGAGAAATCCTTCGAAATGCAGAGAGTGGTGCTATAGATGTAAAGTTTTTT

GTCTTGTCTGAAAAGGGAGTGATTATTGTTTCATTAATCTTTGATGGAAACTGGAATGGGGATCGCAGCA

CATATGGACTATCAATTATACTTCCACAGACAGAACTTAGTTTCTACCTCCCACTTCATAGAGTGTGTGT

TGATAGATTAACACATATAATCCGGAAAGGAAGAATATGGATGCATAAGGAAAGACAAGAAAATGTCCAG

AAGATTATCTTAGAAGGCACAGAGAGAATGGAAGATCAGGGTCAGAGTATTATTCCAATGCTTACTGGAG

AAGTGATTCCTGTAATGGAACTGCTTTCATCTATGAAATCACACAGTGTTCCTGAAGAAATAGATATAGC

TGATACAGTACTCAATGATGATGATATTGGTGACAGCTGTCATGAAGGCTTTCTTCTCAATGCCATCAGC

TCACACTTGCAAACCTGTGGCTGTTCCGTTGTAGTAGGTAGCAGTGCAGAGAAAGTAAATAAGATAGTCA

GAACATTATGCCTTTTTCTGACTCCAGCAGAGAGAAAATGCTCCAGGTTATGTGAAGCAGAATCATCATT

TAAATATGAGTCAGGGCTCTTTGTACAAGGCCTGCTAAAGGATTCAACTGGAAGCTTTGTGCTGCCTTTC

CGGCAAGTCATGTATGCTCCATATCCCACCACACACATAGATGTGGATGTCAATACTGTGAAGCAGATGC

CACCCTGTCATGAACATATTTATAATCAGCGTAGATACATGAGATCCGAGCTGACAGCCTTCTGGAGAGC

CACTTCAGAAGAAGACATGGCTCAGGATACGATCATCTACACTGACGAAAGCTTTACTCCTGATTTGAAT

ATTTTTCAAGATGTCTTACACAGAGACACTCTAGTGAAAGCCTTCCTGGATCAGGTCTTTCAGCTGAAAC

CTGGCTTATCTCTCAGAAGTACTTTCCTTGCACAGTTTCTACTTGTCCTTCACAGAAAAGCCTTGACACT

AATAAAATATATAGAAGACGATACGCAGAAGGGAAAAAAGCCCTTTAAATCTCTTCGGAACCTGAAGATA

GACCTTGATTTAACAGCAGAGGGCGATCTTAACATAATAATGGCTCTGGCTGAGAAAATTAAACCAGGCC

TACACTCTTTTATCTTTGGAAGACCTTTCTACACTAGTGTGCAAGAACGAGATGTTCTAATGACTTTTTA

AATGTGTAACTTAATAAGCCTATTCCATCACAATCATGATCGCTGGTAAAGTAGCTCAGTGGTGTGGGGA

AACGTTCCCCTGGATCATACTCCAGAATTCTGCTCTCAGCAATTGCAGTTAAGTAAGTTACACTACAGTT

CTCACAAGAGCCTGTGAGGGGATGTCAGGTGCATCATTACATTGGGTGTCTCTTTTCCTAGATTTATGCT

TTTGGGATACAGACCTATGTTTACAATATAATAAATATTATTGCTATCTTTTAAAGATATAATAATAGGA

TGTAAACTTGACCACAACTACTGTTTTTTTGAAATACATGATTCATGGTTTACATGTGTCAAGGTGAAAT

CTGAGTTGGCTTTTACAGATAGTTGACTTTCTATCTTTTGGCATTCTTTGGTGTGTAGAATTACTGTAAT

ACTTCTGCAATCAACTGAAAACTAGAGCCTTTAAATGATTTCAATTCCACAGAAAGAAAGTGAGCTTGAA

CATAGGATGAGCTTTAGAAAGAAAATTGATCAAGCAGATGTTTAATTGGAATTGATTATTAGATCCTACT

TTGTGGATTTAGTCCCTGGGATTCAGTCTGTAGAAATGTCTAATAGTTCTCTATAGTCCTTGTTCCTGGT

GAACCACAGTTAGGGTGTTTTGTTTATTTTATTGTTCTTGCTATTGTTGATATTCTATGTAGTTGAGCTC

TGTAAAAGGAAATTGTATTTTATGTTTTAGTAATTGTTGCCAACTTTTTAAATTAATTTTCATTATTTTT

GAGCCAAATTGAAATGTGCACCTCCTGTGCCTTTTTTCTCCTTAGAAAATCTAATTACTTGGAACAAGTT

CAGATTTCACTGGTCAGTCATTTTCATCTTGTTTTCTTCTTGCTAAGTCTTACCATGTACCTGCTTTGGC

AATCATTGCAACTCTGAGATTATAAAATGCCTTAGAGAATATACTAACTAATAAGATCTTTTTTTCAGAA

ACAGAAAATAGTTCCTTGAGTACTTCCTTCTTGCATTTCTGCCTATGTTTTTGAAGTTGTTGCTGTTTGC

CTGCAATAGGCTATAAGGAATAGCAGGAGAAATTTTACTGAAGTGCTGTTTTCCTAGGTGCTACTTTGGC

AGAGCTAAGTTATCTTTTGTTTTCTTAATGCGTTTGGACCATTTTGCTGGCTATAAAATAACTGATTAAT

ATAATTCTAACACAATGTTGACATTGTAGTTACACAAACACAAATAAATATTTTATTTAAAATTCTGGAA

GTAATATAAAAGGGAAAATATATTTATAAGAAAGGGATAAAGGTAATAGAGCCCTTCTGCCCCCCACCCA

CCAAATTTACACAACAAAATGACATGTTCGAATGTGAAAGGTCATAATAGCTTTCCCATCATGAATCAGA

AAGATGTGGACAGCTTGATGTTTTAGACAACCACTGAACTAGATGACTGTTGTACTGTAGCTCAGTCATT

TAAAAAATATATAAATACTACCTTGTAGTGTCCCATACTGTGTTTTTTACATGGTAGATTCTTATTTAAG

TGCTAACTGGTTATTTTCTTTGGCTGGTTTATTGTACTGTTATACAGAATGTAAGTTGTACAGTGAAATA

AGTTATTAAAGCATGTGTAAACATTGTTATATATCTTTTCTCCTAAATGGAGAATTTTGAATAAAATATA

TTTGAAATTTT

SEQ ID NO: 13

>NM_001256054.2 Homo sapiens C9orf72-SMCR8 complex subunit (C9orf72),

transcript variant 3, mRNA

ACGTAACCTACGGTGTCCCGCTAGGAAAGAGAGGTGCGTCAAACAGCGACAAGTTCCGCCCACGTAAAAG

ATGACGCTTGGTGTGTCAGCCGTCCCTGCTGCCCGGTTGCTTCTCTTTTGGGGGCGGGGTCTAGCAAGAG

CAGGTGTGGGTTTAGGAGATATCTCCGGAGCATTTGGATAATGTGACAGTTGGAATGCAGTGATGTCGAC

TCTTTGCCCACCGCCATCTCCAGCTGTTGCCAAGACAGAGATTGCTTTAAGTGGCAAATCACCTTTATTA

GCAGCTACTTTTGCTTACTGGGACAATATTCTTGGTCCTAGAGTAAGGCACATTTGGGCTCCAAAGACAG

AACAGGTACTTCTCAGTGATGGAGAAATAACTTTTCTTGCCAACCACACTCTAAATGGAGAAATCCTTCG

AAATGCAGAGAGTGGTGCTATAGATGTAAAGTTTTTTGTCTTGTCTGAAAAGGGAGTGATTATTGTTTCA

TTAATCTTTGATGGAAACTGGAATGGGGATCGCAGCACATATGGACTATCAATTATACTTCCACAGACAG

AACTTAGTTTCTACCTCCCACTTCATAGAGTGTGTGTTGATAGATTAACACATATAATCCGGAAAGGAAG

AATATGGATGCATAAGGAAAGACAAGAAAATGTCCAGAAGATTATCTTAGAAGGCACAGAGAGAATGGAA

GATCAGGGTCAGAGTATTATTCCAATGCTTACTGGAGAAGTGATTCCTGTAATGGAACTGCTTTCATCTA

TGAAATCACACAGTGTTCCTGAAGAAATAGATATAGCTGATACAGTACTCAATGATGATGATATTGGTGA

CAGCTGTCATGAAGGCTTTCTTCTCAATGCCATCAGCTCACACTTGCAAACCTGTGGCTGTTCCGTTGTA

GTAGGTAGCAGTGCAGAGAAAGTAAATAAGATAGTCAGAACATTATGCCTTTTTCTGACTCCAGCAGAGA

GAAAATGCTCCAGGTTATGTGAAGCAGAATCATCATTTAAATATGAGTCAGGGCTCTTTGTACAAGGCCT

GCTAAAGGATTCAACTGGAAGCTTTGTGCTGCCTTTCCGGCAAGTCATGTATGCTCCATATCCCACCACA

CACATAGATGTGGATGTCAATACTGTGAAGCAGATGCCACCCTGTCATGAACATATTTATAATCAGCGTA

GATACATGAGATCCGAGCTGACAGCCTTCTGGAGAGCCACTTCAGAAGAAGACATGGCTCAGGATACGAT

CATCTACACTGACGAAAGCTTTACTCCTGATTTGAATATTTTTCAAGATGTCTTACACAGAGACACTCTA

GTGAAAGCCTTCCTGGATCAGGTCTTTCAGCTGAAACCTGGCTTATCTCTCAGAAGTACTTTCCTTGCAC

AGTTTCTACTTGTCCTTCACAGAAAAGCCTTGACACTAATAAAATATATAGAAGACGATACGCAGAAGGG

AAAAAAGCCCTTTAAATCTCTTCGGAACCTGAAGATAGACCTTGATTTAACAGCAGAGGGCGATCTTAAC

ATAATAATGGCTCTGGCTGAGAAAATTAAACCAGGCCTACACTCTTTTATCTTTGGAAGACCTTTCTACA

CTAGTGTGCAAGAACGAGATGTTCTAATGACTTTTTAAATGTGTAACTTAATAAGCCTATTCCATCACAA

TCATGATCGCTGGTAAAGTAGCTCAGTGGTGTGGGGAAACGTTCCCCTGGATCATACTCCAGAATTCTGC

TCTCAGCAATTGCAGTTAAGTAAGTTACACTACAGTTCTCACAAGAGCCTGTGAGGGGATGTCAGGTGCA

TCATTACATTGGGTGTCTCTTTTCCTAGATTTATGCTTTTGGGATACAGACCTATGTTTACAATATAATA

AATATTATTGCTATCTTTTAAAGATATAATAATAGGATGTAAACTTGACCACAACTACTGTTTTTTTGAA

ATACATGATTCATGGTTTACATGTGTCAAGGTGAAATCTGAGTTGGCTTTTACAGATAGTTGACTTTCTA

TCTTTTGGCATTCTTTGGTGTGTAGAATTACTGTAATACTTCTGCAATCAACTGAAAACTAGAGCCTTTA

AATGATTTCAATTCCACAGAAAGAAAGTGAGCTTGAACATAGGATGAGCTTTAGAAAGAAAATTGATCAA

GCAGATGTTTAATTGGAATTGATTATTAGATCCTACTTTGTGGATTTAGTCCCTGGGATTCAGTCTGTAG

AAATGTCTAATAGTTCTCTATAGTCCTTGTTCCTGGTGAACCACAGTTAGGGTGTTTTGTTTATTTTATT

GTTCTTGCTATTGTTGATATTCTATGTAGTTGAGCTCTGTAAAAGGAAATTGTATTTTATGTTTTAGTAA

TTGTTGCCAACTTTTTAAATTAATTTTCATTATTTTTGAGCCAAATTGAAATGTGCACCTCCTGTGCCTT

TTTTCTCCTTAGAAAATCTAATTACTTGGAACAAGTTCAGATTTCACTGGTCAGTCATTTTCATCTTGTT

TTCTTCTTGCTAAGTCTTACCATGTACCTGCTTTGGCAATCATTGCAACTCTGAGATTATAAAATGCCTT

AGAGAATATACTAACTAATAAGATCTTTTTTTCAGAAACAGAAAATAGTTCCTTGAGTACTTCCTTCTTG

CATTTCTGCCTATGTTTTTGAAGTTGTTGCTGTTTGCCTGCAATAGGCTATAAGGAATAGCAGGAGAAAT

TTTACTGAAGTGCTGTTTTCCTAGGTGCTACTTTGGCAGAGCTAAGTTATCTTTTGTTTTCTTAATGCGT

TTGGACCATTTTGCTGGCTATAAAATAACTGATTAATATAATTCTAACACAATGTTGACATTGTAGTTAC

ACAAACACAAATAAATATTTTATTTAAAATTCTGGAAGTAATATAAAAGGGAAAATATATTTATAAGAAA

GGGATAAAGGTAATAGAGCCCTTCTGCCCCCCACCCACCAAATTTACACAACAAAATGACATGTTCGAAT

GTGAAAGGTCATAATAGCTTTCCCATCATGAATCAGAAAGATGTGGACAGCTTGATGTTTTAGACAACCA

CTGAACTAGATGACTGTTGTACTGTAGCTCAGTCATTTAAAAAATATATAAATACTACCTTGTAGTGTCC

CATACTGTGTTTTTTACATGGTAGATTCTTATTTAAGTGCTAACTGGTTATTTTCTTTGGCTGGTTTATT

GTACTGTTATACAGAATGTAAGTTGTACAGTGAAATAAGTTATTAAAGCATGTGTAAACATTGTTATATA

TCTTTTCTCCTAAATGGAGAATTTTGAATAAAATATATTTGAAATTTTAAAAAAAAAAAAAAAAAA

	Number	Date	Country
	62958336	Jan 2020	US
	62886072	Aug 2019	US

	Number	Date	Country
Parent	PCT/US2020/046055	Aug 2020	US
Child	17668413		US

SMALL RIBOSOMAL PROTEIN SUBUNIT 25 (RPS25) iRNA AGENT COMPOSITIONS AND METHODS OF USE THEREOF

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