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US20220213475A1 - DUAL TARGETING siRNA AGENTS - Google Patents

DUAL TARGETING siRNA AGENTS Download PDF

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US20220213475A1
US20220213475A1 US17/402,459 US202117402459A US2022213475A1 US 20220213475 A1 US20220213475 A1 US 20220213475A1 US 202117402459 A US202117402459 A US 202117402459A US 2022213475 A1 US2022213475 A1 US 2022213475A1
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dsrna
gene
dual targeting
pcsk9
targeting sirna
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Kevin Fitzgerald
Maria Frank-Kamenetsky
Klaus Charisse
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Alnylam Pharmaceuticals Inc
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21061—Kexin (3.4.21.61), i.e. proprotein convertase subtilisin/kexin type 9
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/3519—Fusion with another nucleic acid

Definitions

  • the invention relates to a composition of two covalently linked siRNAs, e.g., a dual targeting siRNA agent.
  • At least one siRNA is a dsRNA that targets a PCSK9 gene.
  • the covalently linked siRNA agent is used in methods of inhibition of PCSK9 gene expression and methods of treatment of pathological conditions associated with PCSK9 gene expression, e.g., hyperlipidemia.
  • PCSK9 Proprotein convertase subtilisin kexin 9
  • PCSK1-PCSK8 also called PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, and SIP/SKI-1
  • PCSK1-PCSK8 also called PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, and SIP/SKI-1
  • PCSK9 has been proposed to play a role in cholesterol metabolism.
  • PCSK9 mRNA expression is down-regulated by dietary cholesterol feeding in mice (Maxwell, K. N., (2003) J. Lipid Res. 44, 2109-2119), up-regulated by statins in HepG2 cells (Dubuc, G., (2004) Arterioscler. Thromb. Vasc. Biol. 24, 1454-1459), and up-regulated in sterol regulatory element binding protein (SREBP) transgenic mice (Horton, J. D., (2003) Proc. Natl. Acad. Sci.
  • SREBP sterol regulatory element binding protein
  • Hchola3 autosomal dominant hypercholesterolemia
  • PCSK9 may also play a role in determining LDL cholesterol levels in the general population, because single-nucleotide polymorphisms (SNPs) have been associated with cholesterol levels in a Japanese population (Shioji, K., (2004) J. Hum. Genet. 49, 109-114).
  • SNPs single-nucleotide polymorphisms
  • Autosomal dominant hypercholesterolemias are monogenic diseases in which patients exhibit elevated total and LDL cholesterol levels, tendon xanthomas, and premature atherosclerosis (Rader, D. J., (2003) J. Clin. Invest. 111, 1795-1803).
  • the pathogenesis of ADHs and a recessive form, autosomal recessive hypercholesterolemia (ARH) is due to defects in LDL uptake by the liver.
  • ADH may be caused by LDLR mutations, which prevent LDL uptake, or by mutations in the protein on LDL, apolipoprotein B, which binds to the LDLR.
  • ARH is caused by mutations in the ARH protein that are necessary for endocytosis of the LDLR-LDL complex via its interaction with clathrin. Therefore, if PCSK9 mutations are causative in Hchola3 families, it seems likely that PCSK9 plays a role in receptor-mediated LDL uptake.
  • PCSK9 overexpression results in a severe reduction in hepatic LDLR protein, without affecting LDLR mRNA levels, SREBP protein levels, or SREBP protein nuclear to cytoplasmic ratio.
  • PCSK9 Loss of function mutations in PCSK9 have been designed in mouse models (Rashid et al., (2005) PNAS, 102, 5374-5379), and identified in human individuals (Cohen et al. (2005) Nature Genetics 37:161-165). In both cases loss of PCSK9 function lead to lowering of total and LDLc cholesterol. In a retrospective outcome study over 15 years, loss of one copy of PCSK9 was shown to shift LDLc levels lower and to lead to an increased risk-benefit protection from developing cardiovascular heart disease (Cohen et al., (2006) N. Engl. J. Med., 354:1264-1272).
  • X-box binding protein 1 is a basic leucine zipper transcription factor that is involved in the cellular unfolded protein response (UPR).
  • XBP-1 is known to be active in the endoplasmic reticulum (ER).
  • the ER consists of a system of folded membranes and tubules in the cytoplasm of cells. Proteins and lipids are manufactured and processed in the ER. When unusual demands are placed on the ER, “ER stress” occurs. ER stress can be triggered by a viral infection, gene mutations, exposure to toxins, aggregation of improperly folded proteins or a shortage of intracellular nutrients. The result can be Type II diabetes, metabolic syndrome, a neurological disorder or cancer.
  • XBP-1 XBP-1 isoforms
  • spliced XBP-1S and unspliced XBP-1U.
  • Both isoforms of XBP-1 bind to the 21-bp Tax-responsive element of the human T-lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR) in vitro and transactivate HTLV-1 transcription.
  • HTLV-1 is associated with a rare form of blood dyscrasia known as Adult T-cell Leukemia/lymphoma (ATLL) and a myelopathy, tropical spastic paresis.
  • ATLL adult T-cell Leukemia/lymphoma
  • myelopathy tropical spastic paresis.
  • Double-stranded RNA molecules have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi).
  • RNAi RNA interference
  • WO 99/32619 (Fire et al.) disclosed the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans .
  • dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol .
  • siRNA targeting XPB-1 A description of siRNA targeting XPB-1 can be found in U.S. patent application Ser. No. 12/425,811 filed on Apr. 17, 2009 and published as US 2009-0275638.
  • Dual targeting siRNAs can be found in International patent application publication no. WO/2007/091269.
  • Described herein are dual targeting siRNA agent in which a first siRNA targeting PCSK9 is covalently joined to a second siRNA targeting a gene implicated in cholesterol metabolism, e.g., XBP-1.
  • the two siRNAs are covalently linked via, e.g., a disulfide linker.
  • one aspect of the invention is a dual targeting siRNA agent having a first dsRNA targeting a PCSK9 gene and a second dsRNA targeting a second gene, wherein the first dsRNA and the second dsRNA are linked with a covalent linker.
  • the second gene is can be, e.g., XBP-1, PCSK9, PCSK5, ApoC3, SCAP, or MIG12.
  • the second gene is XBP-1.
  • Each dsRNA is 30 nucleotides or less in length. In general, each strand of each dsRNA is 19-23 bases in length.
  • the dual targeting siRNA agent comprising a first dsRNA AD-10792 targeting a PCSK9 gene and a second dsRNA AD-18038 targeting an XBP-1 gene, wherein AD-10792 sense strand and AD-18038 sense strand are covalently linked with a disulfide linker.
  • the first dsRNA of the dual targeting siRNA agent targets a PCSK9 gene.
  • the first dsRNA includes at least 15 contiguous nucleotides of an antisense strand of one of Tables 1, 2, or 4-8, or includes an antisense strand of one of Tables 1, 2, or 4-8, or includes a sense strand and an antisense strand of one of Tables 1, 2, or 4-8.
  • the first dsRNA can be AD-9680 or AD-10792.
  • the second dsRNA target XBP-1.
  • the second dsRNA includes at least 15 contiguous nucleotides of an antisense strand of one of Tables 3 or 9-13, or includes an antisense strand of one of Tables 3 or 9-13, or includes a sense strand and an antisense strand of one of Tables 3 or 9-13.
  • the second dsRNA can be AD-18038.
  • Either the first and second dsRNA can include at least one modified nucleotide, e.g., a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
  • the first and second dsRNAs include “endo-light” modification with 2′-O-methyl modified nucleotide
  • the first and second dsRNAs are linked with a covalent linker.
  • the linker is a disulfide linker.
  • Various combinations of strands can be linked, e.g., the first and second dsRNA sense strands are covalently linked or, e.g., the first and second dsRNA antisense strands are covalently linked.
  • any of the dual targeting siRNA agents of the invention include a ligand.
  • isolated cells having and vectors encoding the dual targeting siRNA agent described herein.
  • administration of the dual targeting siRNA agent to a cell inhibits expression of the PCSK9 gene and the second gene at a level equivalent to inhibition of expression of both genes using administration of each siRNA individually.
  • administration of the dual targeting siRNA agent to a subject results in a greater reduction of total serum cholesterol that that obtained by administration of each siRNA alone.
  • the invention also includes a pharmaceutical composition comprising the dual targeting siRNA agents described herein and a pharmaceutical carrier.
  • the pharmaceutical carrier is a lipid formulation, e.g., a lipid formulation including cationic lipid DLinDMA or cationic lipid XTC. Examples of lipid formulations described in (but not limited to) Table A, below.
  • the lipid formulation can be XTC/DSPC/Cholesterol/PEG-DMG at % mol ratios of 50/10/38.5/1.5.
  • the invention is a method of inhibiting expression of the PCSK9 gene and a second gene in a cell, the method comprising (a) introducing into the cell the any of the dual targeting siRNA agents and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the PCSK9 gene and the second gene, thereby inhibiting expression of the PCSK9 gene and the second gene in the cell.
  • the invention includes methods of treating a disorder mediated by PCSK9 expression with the step of administering to a subject in need of such treatment a therapeutically effective amount of the pharmaceutical compositions described herein.
  • the disorder is hyperlipidemia.
  • the invention includes methods of reducing total serum cholesterol in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical compositions described herein.
  • FIG. 1A is a graph showing the effect on PCSK9 mRNA levels in primary mouse hepatocytes following treatment with a dual targeting siRNA, AD-23426.
  • AD-23426 was as effective at reducing mRNA expression as each single gene target siRNA.
  • AD-10792 PCSK9 siRNA.
  • AD-18038 XBP-1 siRNA.
  • FIG. 1B is a graph showing the effect on XBP-1 mRNA levels in primary mouse hepatocytes following treatment with a dual targeting siRNA, AD-23426.
  • AD-23426 was as effective at reducing mRNA expression as each single gene target siRNA.
  • AD-10792 PCSK9 siRNA.
  • AD-18038 XBP-1 siRNA.
  • Lipo2000 control transfection agent only.
  • FIG. 2 is a graph showing the effect on PCSK9 and XBP-1 mRNA levels in mice following treatment with a dual targeting siRNA, AD-23426.
  • LNP09 (lipid) formulated siRNA was administered to mice as described.
  • AD-23426 was as effective at reducing mRNA expression as each single gene target siRNA.
  • AD-10792 PCSK9 siRNA.
  • AD-18038 XBP-1 siRNA.
  • FIG. 3 is a graph showing the effect on serum cholesterol levels in mice following treatment with a dual targeting siRNA, AD-23426.
  • LNP09 (lipid) formulated siRNA was administered to mice as described.
  • AD-23426 was more effective at reducing serum cholesterol compared to each single gene target siRNA.
  • AD-10792 PCSK9 siRNA.
  • AD-18038 XBP-1 siRNA.
  • FIG. 4A is a graph showing the effect on IFN- ⁇ , in human PBMC following treatment with a dual targeting siRNA, AD-23426.
  • FIG. 4B is a graph showing the effect on TNF- ⁇ in human PBMC following treatment with a dual targeting siRNA, AD-23426.
  • DOTAP and LNP09 (lipid) formulated siRNAs was administered huPBMC as described below.
  • AD-23426 did not induce IFN- ⁇ or TNF- ⁇ .
  • the invention provides a solution to the problem of treating diseases that can be modulated by the down regulation of the PCSK9 gene, such as hyperlipidemia, by using dual targeting siRNA to silence the PCSK9 gene.
  • the invention provides compositions and methods for inhibiting the expression of the PCSK9 gene in a subject using two siRNA, e.g., a dual targeting siRNA.
  • the invention also provides compositions and methods for treating pathological conditions and diseases, such as hyperlipidemia, that can be modulated by down regulating the expression of the PCSK9 gene.
  • the dual targeting siRNA agents target a PCSK9 gene and at least one other gene.
  • the other gene can be another region of the PCSK9 gene, or can be another gene, e.g., XBP-1.
  • the dual targeting siRNA agents have the advantage of lower toxicity, lower off-target effects, and lower effective concentration compared to individual siRNAs.
  • the use of the dual targeting siRNA dsRNAs enables the targeted degradation of an mRNA that is involved in the regulation of the LDL receptor and circulating cholesterol levels.
  • Using cell-based and animal assays it was demonstrated that inhibiting both a PCSK9 gene and an XBP-1 gene using a dual targeting siRNA is at least as effective at inhibiting their corresponding targets as the use of single siRNAs. It was also demonstrated that administration of a dual targeting siRNA results in a synergistic lowering of total serum cholesterol. Thus, reduction of total serum cholesterol is enhanced with a dual targeting siRNA compared to a single target siRNA.
  • G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively.
  • T and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine.
  • ribonucleotide or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety.
  • guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety.
  • a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil.
  • nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine.
  • 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 invention.
  • PCSK9 refers to the proprotein convertase subtilisin kexin 9 gene or protein (also known as FH3, HCHOLA3, NARC-1, NARC1).
  • Examples of mRNA sequences to PCSK9 include but are not limited to the following: human: NM_174936; mouse: NM_153565, and rat: NM_199253. Additional examples of PCSK9 mRNA sequences are readily available using, e.g., GenBank.
  • XBP-1 refers to -Box Protein 1, which is also known as Tax-responsive element-binding protein 5, TREB5, and XBP2.
  • XBP-1 sequence can be found as NCBI GeneID:7494 and RefSeq ID number: NM_005080 (human) and NM_013842 (mouse).
  • a dsRNA featured in the invention can target a specific XBP-1 isoform, e.g., the spliced form (XBP-1S) or the unspliced form (XBP-1U), or a dsRNA featured in the invention can target both isoforms by binding to a common region of the mRNA transcript.
  • PCSK5 refers to the Proprotein convertase subtilisin/kexin type 5 gene, mRNA or protein belonging to the subtilisin-like proprotein convertase family.
  • ApoC3 refers to the Apolipoprotein C-III protein gene, mRNA or protein, and is a very low density lipoprotein (VLDL).
  • SCAP refers to the SREBP cleavage-activating protein gene, mRNA or protein.
  • SCAP is a regulatory protein that is required for the proteolytic cleavage of the sterol regulatory element binding protein (SREBP).
  • SREBP sterol regulatory element binding protein
  • MIG12 is a gene also known as TMSB10 and TB10 refers to the thymosin beta 10 gene.
  • TMSB10 a gene also known as TMSB10
  • TB10 refers to the thymosin beta 10 gene.
  • siRNA targeting MIG12 are described International patent application no. PCT/US10/25444, filed on Feb. 25, 2010, published as WO/20XX/XXXXXX This application and the siRNA sequences described therein are incorporated by reference for all purposes.
  • RNA refers to an agent that contains RNA and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • siRNA includes siRNA.
  • RNA and siRNA agent refers to a dsRNA that mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • double-stranded RNA or “dsRNA,” as used herein, refers to an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having “sense” and “antisense” orientations with respect to a target RNA.
  • the term “dual targeting siRNA agent” refers to a composition of two siRNAs, e.g., two dsRNAs.
  • One dsRNA includes an antisense strand with a first region of complementarity to a first target gene, e.g., PCSK9.
  • the second dsRNA include an antisense strand with a second region of complementarity to a second target gene.
  • the first and second target genes are identical, e.g., both are PCSK9 and each dsRNA targets a different region of PCSK9.
  • the first and second target genes are different, e.g., the first dsRNA targets PCSK9 and the second dsRNA targets a different gene, e.g., XBP-1.
  • Covalent linker refers to a molecule for covalently joining two molecules, e.g., two dsRNAs. As described in more detail below, the term includes, e.g., a nucleic acid linker, a peptide linker, and the like and includes disulfide linkers.
  • target gene refers to a gene of interest, e.g., PCSK9 or a second gene, e.g., XBP-1, targeted by an siRNA of the invention for inhibition of expression.
  • target sequence refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene, including mRNA that is a product of RNA processing of a primary transcription product.
  • the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion.
  • the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all sub-ranges therebetween.
  • 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.
  • the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person.
  • Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing.
  • Complementary sequences within an iRNA 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.
  • first sequence is referred to as “substantially complementary” with respect to a second sequence herein
  • the two sequences can be fully complementary, or they may form one or more, but generally not more than 5, 4, 1 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.
  • 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.
  • a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.
  • “Complementary” sequences may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.
  • Such non-Watson-Crick base pairs includes, but are not limited to, G:U Wobble or Hoogstein base pairing.
  • 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 the target gene (e.g., an mRNA encoding PCSK9 or a second gene, e.g., XBP-1).
  • a polynucleotide is complementary to at least a part of a PCSK9 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding PCSK9.
  • RNA molecule or “ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art.
  • a “ribonucleoside” includes a nucleoside base and a ribose sugar
  • ribonucleotide is a ribonucleoside with one, two or three phosphate moieties.
  • the terms “ribonucleoside” and “ribonucleotide” can be considered to be equivalent as used herein.
  • RNA can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described herein below.
  • the molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex.
  • an RNA molecule can also include at least one modified ribonucleoside including but not limited to a 2′-O-methyl modified nucleotide, a nucleoside comprising a 5′ phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2′-deoxy-2′-fluoro modified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof.
  • a 2′-O-methyl modified nucleotide a nucleoside comprising a 5′ phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bis
  • an RNA molecule can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the dsRNA molecule.
  • the modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule.
  • modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA via a RISC pathway.
  • PNAs peptide nucleic acids
  • a modified ribonucleoside includes a deoxyribonucleoside.
  • an iRNA agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double stranded portion of a dsRNA.
  • iRNA double stranded DNA molecule encompassed by the term “iRNA.”
  • nucleotide overhang refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA.
  • 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) may be on the sense strand, the antisense strand or any combination thereof.
  • 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.
  • One or more of the nucleotides in the overhang can be replaced with a nucleoside thiophosphate.
  • dsRNA dsRNA 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.
  • 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.
  • antisense strand or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence.
  • region of complementarity refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.
  • sense strand or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.
  • SNALP refers to a stable nucleic acid-lipid particle.
  • a SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed.
  • SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in International Application No. WO 2009082817. These applications are incorporated herein by reference in their entirety.
  • iRNA “Introducing into a cell,” when referring to an iRNA, means facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; an iRNA may also be “introduced into a cell,” wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism.
  • iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be by a beta-glucan delivery system, such as those described in U.S. Pat.
  • the term “modulate the expression of,” refers to at an least partial “inhibition” or partial “activation” of target gene expression in a cell treated with an iRNA composition as described herein compared to the expression of the target gene in an untreated cell.
  • activate activate
  • increase increase the expression of
  • control cells refer to the at least partial activation of the expression of a target gene, as manifested by an increase in the amount of target mRNA, which may be isolated from or detected in a first cell or group of cells in which a target gene is transcribed and which has or have been treated such that the expression of a target gene is increased, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).
  • expression of a target gene is activated by at least about 10%, 15%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA as described herein.
  • a target gene is activated by at least about 60%, 70%, or 80% by administration of an iRNA featured in the invention.
  • expression of a target gene is activated by at least about 85%, 90%, or 95% or more by administration of an iRNA as described herein.
  • the target gene expression is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000 fold or more in cells treated with an iRNA as described herein compared to the expression in an untreated cell.
  • Activation of expression by small dsRNAs is described, for example, in Li et al., 2006 Proc. Natl. Acad. Sci. U.S.A. 103:17337-42, and in US20070111963 and US2005226848, each of which is incorporated herein by reference.
  • the degree of inhibition is usually expressed in terms of
  • the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to target gene expression, e.g., the amount of protein encoded by a target gene, or the number of cells displaying a certain phenotype, e.g., lack of or decreased cytokine production.
  • target gene silencing may be determined in any cell expressing target, either constitutively or by genomic engineering, and by any appropriate assay.
  • the assays provided in the Examples below shall serve as such reference.
  • expression of a target gene is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% by administration of an iRNA featured in the invention.
  • a target gene is suppressed by at least about 60%, 65%, 70%, 75%, or 80% by administration of an iRNA featured in the invention.
  • a target gene is suppressed by at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more by administration of an iRNA as described herein.
  • the terms “treat,” “treatment,” and the like refer to relief from or alleviation of pathological processes mediated by target expression.
  • the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression or anticipated progression of such condition.
  • lower in the context of a disease marker or symptom is meant a statistically significant decrease in such level.
  • the decrease can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without such disorder.
  • the phrase “therapeutically effective amount” refers to an amount that provides a therapeutic benefit in the treatment or management of pathological processes mediated by target gene expression, e.g., PCSK9 and/or a second gene, e.g., XBP-1, or an overt symptom of pathological processes mediated target gene expression.
  • target gene expression e.g., PCSK9 and/or a second gene, e.g., XBP-1, or an overt symptom of pathological processes mediated target gene expression.
  • prophylactically effective amount refer to an amount that provides a therapeutic benefit in the prevention of pathological processes mediated by target gene expression or an overt symptom of pathological processes mediated by target gene expression.
  • the specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by target gene expression, the patient's history and age, the stage of pathological processes mediated by target gene expression, and the administration of other agents that inhibit pathological processes mediated by target gene expression.
  • a “pharmaceutical composition” comprises a pharmacologically effective amount of an iRNA and a pharmaceutically acceptable carrier.
  • pharmaceutically effective amount refers to that amount of an iRNA effective to produce the intended pharmacological or therapeutic result. For example, if a given clinical treatment is considered effective when there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 10% reduction in that parameter.
  • pharmaceutically carrier refers to a carrier for administration of a therapeutic agent, e.g., a dual targeting siRNA agent.
  • Carriers are described in more detail below, and include lipid formulations, e.g., LNP09 and SNALP formulations.
  • Double-Stranded Ribonucleic Acid dsRNA
  • dual targeting siRNA agents e.g., siRNAs that inhibit the expression of a PCSK9 gene and a second gene.
  • the dual targeting siRNA agent includes two siRNA covalently linked via, e.g., a disulfide linker.
  • the first siRNA targets a first region of a PCSK9 gene.
  • the second siRNA targets a second gene, e.g., XBP-1, or, e.g., targets a second region of the PCSK9 gene.
  • dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Applied Biosystems, Inc. Further descriptions of synthesis are found below and in the examples.
  • Each siRNA is a dsRNA.
  • a dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used.
  • One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of a target gene.
  • the other 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.
  • the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a “hairpin loop”) between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure.
  • the hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.
  • dsRNA substantially complementary strands of a dsRNA
  • those molecules need not, but can be covalently connected.
  • the connecting structure is referred to as a “linker.”
  • the duplex structure of the siRNA is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive.
  • the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base
  • the two siRNAs in the dual targeting siRNA agent can have duplex lengths that are identical or that differ.
  • the region of complementarity to the target sequence in an siRNA is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive.
  • the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive.
  • the region of complementarity can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides,
  • the two siRNAs in the dual targeting siRNA agent can have regions of complementarity that are identical in length or that differ in length.
  • any of the dsRNA may include one or more single-stranded nucleotide overhangs.
  • at least one end of a dsRNA has a single-stranded nucleotide overhang of 1 to 4, or 1 or 2 or 3 or 4 nucleotides.
  • dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts.
  • the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand.
  • the dsRNA can also have a blunt end, generally located at the 5′-end of the antisense strand.
  • one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
  • the two siRNAs in the dual targeting siRNA agent can have different or identical overhangs as described by location, length, and nucleotide.
  • the dual targeting siRNA agent includes at least a first siRNA targeting a first region of a PCSK9 gene.
  • a PCSK9 gene is a human PCSK9 gene.
  • the PCSK9 gene is a mouse or a rat PCSK9 gene.
  • Exemplary siRNA targeting PCSK9 are described in U.S. patent application Ser. No. 11/746,864 filed on May 10, 2007 (now U.S. Pat. No. 7,605,251) and International Patent Application No. PCT/US2007/068655 filed May 10, 2007 (published as WO 2007/134161). Additional disclosure can be found in U.S. patent application Ser. No. 12/478,452 filed Jun. 4, 2009 (published as US 2010/0010066) and International Patent Application No. PCT/US2009/032743 filed Jan. 30, 2009 (published as WO 2009/134487).
  • the sequences of the target, sense, and antisense strands are incorporated by reference for all purposes.
  • Tables 1, 2, and 4-8 disclose sequences of the target, sense strands, and antisense strands of PCSK9 targeting siRNA.
  • the first siRNA is AD-9680.
  • the dsRNA AD-9680 targets the human PCSK 9 gene at nucleotides 3530-3548 of a human PCSK9 gene (accession number NM_174936).
  • AD-9680 siRNA sequences SEQ ID Table 1: AD-9680 Sequence 5′ to 3′ NO: Target sequence UUCUAGACCUGUUUUGCUU 4142 Sense strand UUCUAGACCUGUUUUGCUU 4143 Sense strand, uucuAGAccuGuuuuGcuuTsT 4144 modified Antisense strand AAGCAAAACAGGUCUAGAA 4145 Antisense strand, AAGcAAAAcAGGUCuAGAATsT 4146 modified
  • the first siRNA is AD-10792.
  • the dsRNA AD-10792 targets the PCSK9 gene at nucleotides 1091-1109 of a human PCSK9 gene (accession number NM 174936).
  • AD-10792 is also complementary to rodent PCSK9.
  • AD-10792 siRNA sequences SEQ ID Table 2: AD-10792 Sequence 5′ to 3′ NO: Target sequence GCCUGGAGUUUAUUCGGAA 4147 Sense strand GCCUGGAGUUUAUUCGGAA 4148 Sense strand, GccuGGAGuuuAuucGGAATsT 4149 modified Antisense strand UUCCGAAUAAACUCCAGGC 4150 Antisense strand, UUCCGAAuAAACUCcAGGCTsT 4151 modified
  • the second siRNA of the dual targeting siRNA agent targets a second gene.
  • the second gene is PCSK9
  • the second siRNA target a region of PCSK9 that is different from the region targeted by the first siRNA.
  • the second siRNA targets a different second gene.
  • the second target gene can be XBP-1, PCSK5, ApoC3, SCAP, MIG12, HMG CoA Reductase, or IDOL (Inducible Degrader of the LDLR) and the like.
  • the second gene is a human gene.
  • the second gene is a mouse or a rat gene.
  • the second siRNA targets the XBP-1 gene.
  • XBP-1 XBP-1 gene.
  • Exemplary siRNA targeting XBP-1 can be found in U.S. patent application Ser. No. 12/425,811 filed Apr. 17, 2009 (published as US 2009-0275638). The sequences of the target, sense, and antisense strands are incorporated by reference for all purposes.
  • Tables 3 and 9-13 disclose sequences of the target, sense strands, and antisense strands of XBP-1 targeting siRNA.
  • the first siRNA is AD-18038.
  • the dsRNA AD-18038 targets the human XBP-1 gene at nucleotides 896-914 of a human XBP-1 gene (accession number NM_001004210).
  • AD-18038 siRNA sequences SEQ ID Table 3: AD-18038 Sequence 5′ to 3′ NO: Target sequence CACCCUGAAUUCAUUGUCU 4153 Sense strand CACCCUGAAUUCAUUGUCU 4154 Sense strand, cAcccuGAAuucAuuGucudTsdT 4155 modified Antisense strand AGACAAUGAAUUCAGGGUG 4156 Antisense strand, AGAcAAUGAAUUcAGGGUGdTsdT 4157 modified
  • a dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences in Tables 1-13, and differing in their ability to inhibit the expression of a target gene by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated according to the invention.
  • RNAs provided in Tables 1-13 identify a site in the target gene transcript that is susceptible to RISC-mediated cleavage.
  • the present invention further features iRNAs that target within one of such sequences.
  • an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site.
  • Such an iRNA will generally include at least 15 contiguous nucleotides from one of the sequences provided herein coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a target gene.
  • target sequence is generally 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA.
  • Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that may serve as target sequences.
  • the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected.
  • This process coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression.
  • sequences identified, for example, above represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.
  • optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.
  • modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.
  • An iRNA as described in Tables 1-13 can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described in Tables 1-13 contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′ or 3′ end of the region of complementarity.
  • RNA strand which is complementary to a region of a PCSK9 gene
  • the RNA strand generally does not contain any mismatch within the central 13 nucleotides.
  • the methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a PCSK9 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of a PCSK9 gene is important, especially if the particular region of complementarity in a PCSK9 gene is known to have polymorphic sequence variation within the population.
  • the dual targeting siRNA agents of the invention include two siRNAs joined via a covalent linker.
  • Covalent linkers are well-known to one of skill in the art and include, e.g., a nucleic acid linker, a peptide linker, and the like.
  • the covalent linker joins the two siRNAs.
  • the covalent linker can join two sense strands, two antisense strands, one sense and one antisense strand, two sense strands and one antisense strand, two antisense strands and one sense strand, or two sense and two antisense strands.
  • the covalent linker can include RNA and/or DNA and/or a peptide.
  • the linker can be single stranded, double stranded, partially single strands, or partially double stranded.
  • the linker includes a disulfide bond.
  • the linker can be cleavable or non-cleavable.
  • the covalent linker can be a polyRNA, such as poly(5′-adenyl-3′-phosphate—AAAAAAAA) or poly(5′-cytidyl-3′-phosphate-5′-uridyl-3′-phosphate—CUCUCUCU)), e.g., X n single stranded poly RNA linker wherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive. Modified nucleotides or a mixture of nucleotides can also be present in said polyRNA linker.
  • the covalent linker can be a polyDNA, such as poly(5′-2′deoxythymidy1-3′-phosphate—TTTTTTTT), e.g., wherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive. Modified nucleotides or a mixture of nucleotides can also be present in said polyDNA linker. a single stranded polyDNA linker wherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive. Modified nucleotides or a mixture of nucleotides can also be present in said polyDNA linker.
  • the covalent linker can include a disulfide bond, optionally a bis-hexyl-disulfide linker.
  • the disulfide linker is
  • the covalent linker can include a peptide bond, e.g., include amino acids.
  • the covalent linker is a 1-10 amino acid long linker, preferably comprising 4-5 amino acids, optionally X-Gly-Phe-Gly-Y wherein X and Y represent any amino acid.
  • the covalent linker can include HEG, a hexaethylenglycol linker.
  • At least one of the siRNA of the dual targeting siRNA agent is chemically modified to enhance stability or other beneficial characteristics.
  • the nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference.
  • Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages.
  • end modifications e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 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
  • RNA compounds useful in this invention 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.
  • modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • the modified RNA will have a phosphorus atom in its internucleoside backbone.
  • Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′.
  • Various salts, mixed salts and free acid forms are also included.
  • 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.
  • 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
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • RNA mimetics suitable or contemplated for use in iRNAs both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S.
  • PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.
  • RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH 2 —NH—CH 2 —, —CH 2 —N(CH 3 )—O—CH 2 —[known as a methylene (methylimino) or MMI backbone], —CH 2 —O—N(CH 3 )—CH 2 —, —CH 2 —N(CH 3 )—N(CH 3 )—CH 2 — and —N(CH 3 )—CH 2 —CH 2 —[wherein the native phosphodiester backbone is represented as —O—P—O—CH 2 —] of the above-referenced U.S.
  • RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
  • Modified RNAs may also contain one or more substituted sugar moieties.
  • the iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • Exemplary suitable modifications include O[(CH 2 ) n O] m CH 3 , O(CH 2 ).
  • n OCH 3 O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to about 10.
  • dsRNAs include one of the following at the 2′ position: C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties.
  • the modification includes a 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group.
  • 2′-dimethylaminooxyethoxy i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2′-DMAOE, as described in examples herein below
  • 2′-dimethylaminoethoxyethoxy also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE
  • 2′-O—CH 2 —O—CH 2 —N(CH 2 ) 2 also described in examples herein below.
  • modifications include 2′-methoxy (2′-OCH 3 ), 2′-aminopropoxy (2′-OCH 2 CH 2 CH 2 NH 2 ) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.
  • An iRNA may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • 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-substi
  • 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., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993.
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention.
  • 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.
  • RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA).
  • LNA locked nucleic acids
  • 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).
  • 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 U.S. Provisional Patent Application No. 61/223,665 (“the '665 application”), filed Jul. 7, 2009, entitled “Oligonucleotide End Caps” and International patent application no. PCT/US10/41214, filed Jul. 7, 2010.
  • RNA of an iRNA featured in the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA.
  • 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.
  • 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).
  • a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated.
  • 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.
  • Preferred 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.
  • polyamino acids examples 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.
  • PLL polylysine
  • poly L-aspartic acid poly L-glutamic acid
  • styrene-maleic acid anhydride copolymer poly(L-lactide-co-glycolied) copolymer
  • divinyl ether-maleic anhydride copolymer divinyl ether-
  • 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 alpha 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 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-gulucosamine 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.
  • 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.
  • 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, bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted
  • biotin e.g., aspirin, vitamin E, folic acid
  • 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-gulucosamine multivalent mannose, or multivalent fucose.
  • the ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF- ⁇ B.
  • the ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments.
  • the drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
  • the ligand is a lipid or lipid-based molecule.
  • a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA).
  • HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body.
  • 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, neproxin or aspirin can be used.
  • a lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
  • a serum protein e.g., HSA.
  • a lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue.
  • 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.
  • the lipid based ligand binds HSA.
  • it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue.
  • the affinity it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
  • the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney.
  • Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
  • 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.
  • B vitamin e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells.
  • HSA high density lipoprotein
  • LDL low density lipoprotein
  • the ligand is a cell-permeation agent, preferably a helical cell-permeation agent.
  • the agent is amphipathic.
  • An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids.
  • the helical agent is preferably an alpha-helical agent, which preferably has 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.
  • 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:4158).
  • An RFGF analogue e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:4159)
  • the peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes.
  • sequences from the HIV Tat protein GRKKRRQRRRPPQ (SEQ ID NO:4160)
  • the Drosophila Antennapedia protein RQIKIWFQNRRMKWKK (SEQ ID NO:4161) 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).
  • OBOC one-bead-one-compound
  • 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.
  • RGD arginine-glycine-aspartic acid
  • 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 moiety can be used to target 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).
  • 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.
  • a glycosylated RGD peptide can deliver a iRNA agent to a tumor cell expressing ⁇ vß 3 (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 ⁇ -helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., ⁇ -defensin, ⁇ -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).
  • NLS nuclear localization signal
  • 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).
  • 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; 5,26
  • 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 dsRNAs, which 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.
  • iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
  • An additional region of the iRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • 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.
  • RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
  • the RNA of an iRNA can be modified by a non-ligand group.
  • 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.
  • 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., 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).
  • RNA conjugates Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.
  • an iRNA to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be performed directly by administering a composition comprising an iRNA, e.g. a dsRNA, to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.
  • any method of delivering a nucleic acid molecule can be adapted for use with an iRNA (see e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties).
  • iRNA e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties.
  • factors that are important to consider in order to successfully deliver an iRNA molecule in vivo (a) biological stability of the delivered molecule, (2) preventing non-specific effects, and (3) accumulation of the delivered molecule in the target tissue.
  • the non-specific effects of an iRNA can be minimized by local administration, for example by direct injection or implantation into a tissue (as a non-limiting example, a tumor) or topically administering the preparation.
  • Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that may otherwise be harmed by the agent or that may degrade the agent, and permits a lower total dose of the iRNA molecule to be administered.
  • Several studies have shown successful knockdown of gene products when an iRNA is administered locally.
  • VEGF dsRNA 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.
  • 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.
  • RNA interference has also shown success with local delivery to the CNS by direct injection (Dom, 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.
  • 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.
  • RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects.
  • iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation.
  • lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation.
  • an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O., et al (2006) Nat.
  • the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system.
  • drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system.
  • Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell.
  • Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim SH., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA.
  • vesicles or micelles further prevents degradation of the iRNA when administered systemically.
  • Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety).
  • DOTAP Disposon-based lipid particles
  • Oligofectamine “solid nucleic acid lipid particles”
  • cardiolipin Choen, P Y., et al (2006) 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.
  • an iRNA forms a complex with cyclodextrin for systemic administration.
  • Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.
  • the dsRNAs of the invention can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG . (1996), 12:5-10; Skillem, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type.
  • transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector.
  • the transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).
  • the individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector.
  • 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.
  • each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid.
  • a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
  • iRNA 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 an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
  • iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKOTM). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention.
  • Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
  • a reporter such as a fluorescent marker, such as Green Fluorescent Protein (GFP).
  • 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) picomavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g.
  • pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g.
  • the constructs can include viral sequences for transfection, if desired.
  • the construct may be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors.
  • Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.
  • Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue.
  • the regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.
  • Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24).
  • inducible expression systems suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG).
  • IPTG isopropyl-beta-D1-thiogalactopyranoside
  • viral vectors that contain nucleic acid sequences encoding an iRNA can be used.
  • a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA.
  • the nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitates delivery of the nucleic acid into a patient.
  • retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
  • Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
  • Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.
  • Adenoviruses are also contemplated for use in delivery of iRNAs.
  • Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy.
  • a suitable AV vector for expressing an iRNA featured in the invention a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
  • Adeno-associated virus AAV
  • the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.
  • a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.
  • a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.
  • viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
  • lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like.
  • AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
  • the pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • the invention provides pharmaceutical compositions containing a dual targeting siRNA agent, as described herein, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition containing the siRNA is useful for treating a disease or disorder associated with the expression or activity of a target gene, such as pathological processes mediated by PCSK9 expression.
  • Such pharmaceutical compositions are formulated based on the mode of delivery.
  • IV intravenous
  • compositions that are formulated for direct delivery into the brain parenchyma e.g., by infusion into the brain, such as by continuous pump infusion.
  • compositions featured herein are administered in dosages sufficient to inhibit expression of the target genes.
  • a suitable dose of siRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day.
  • the dsRNA can be administered at 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg
  • the pharmaceutical composition may be administered once daily, or the iRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage.
  • the dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
  • the effect of a single dose of siRNA on PCSK9 levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.
  • treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments.
  • Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
  • mouse models for the study of various human diseases, such as pathological processes mediated by PCSK9 expression. Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose.
  • a suitable mouse model is, for example, a mouse containing a transgene expressing human PCSK9.
  • the present invention also includes pharmaceutical compositions and formulations that include the iRNA compounds featured in the invention.
  • the pharmaceutical compositions of the present invention 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 (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 iRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).
  • a particular tissue such as the liver (e.g., the hepatocytes of the liver).
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms, gloves and the like may also be useful.
  • Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).
  • iRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes.
  • iRNAs may be complexed to lipids, in particular to cationic lipids.
  • Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or a C1-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.
  • Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.
  • liposome means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
  • Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
  • lipid vesicles In order to traverse intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
  • liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
  • Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
  • Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
  • Liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
  • liposomes to deliver agents including high-molecular weight DNA into the skin.
  • Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis
  • Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
  • Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).
  • liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine.
  • Neutral liposome compositions 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).
  • DOPE dioleoyl phosphatidylethanolamine
  • Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC.
  • PC phosphatidylcholine
  • Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • 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 NovasomeTM I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and NovasomeTM II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).
  • Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.
  • 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.
  • PEG polyethylene glycol
  • Liposomes comprising (1) sphingomyelin and (2) the ganglioside G M1 or a galactocerebroside sulfate ester.
  • U.S. Pat. No. 5,543,152 discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).
  • liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art.
  • Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C 1215G , that contains a PEG moiety.
  • Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives.
  • Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S.
  • Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher.
  • Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1).
  • Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No.
  • a number of liposomes comprising nucleic acids are known in the art.
  • WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes.
  • U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA.
  • U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes.
  • WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.
  • Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
  • HLB hydrophile/lipophile balance
  • 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.
  • 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.
  • Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
  • amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
  • a dual targeting siRNA agent featured in the invention is fully encapsulated in the lipid formulation, e.g., to form a nucleic acid-lipid particle, e.g., a SPLP, pSPLP, or SNALP.
  • a nucleic acid-lipid particle e.g., a SPLP, pSPLP, or SNALP.
  • SNALP refers to a stable nucleic acid-lipid particle, including SPLP.
  • SPLP refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle.
  • Nucleic acid-lipid particles typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate).
  • SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site).
  • SPLPs include “pSPLP”, which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683.
  • the particles of the present invention 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.
  • the mean diameter of the particles can be about 50 nm, 55 nm, 60 nn, 65 nn, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 140 nm, 145 nm, or 150 nm.
  • nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease.
  • Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.
  • the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.
  • the lipid to dsRNA ratio can be about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 113:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, or 50:1.
  • the nucleic acid lipid particles include a cationic lipid.
  • the cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dil
  • the cationic lipid may comprise from about 10 mol % to about 70 mol % or about 40 mol % of the total lipid present in the particle.
  • the cationic lipid may comprise 10 mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol %, 70 mol %, 75 mol %, 80 mol %, 85 mol %, 90 mol %, or 95 mol % of the total lipid present in the particle.
  • the cationic lipid may comprise 57.1 mol % or 57.5 mol % of the total lipid present in the particle.
  • the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.
  • the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0 ⁇ 20 nm and a 0.027 siRNA/Lipid Ratio.
  • the nucleic acid lipid particle generally includes a non-cationic lipid.
  • the non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine
  • the non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.
  • the non-cationic lipid may be about 5 mol %, 6 mol %, 7 mol %, 7.5 mol %, 7.7 mol %, 8 mol %.
  • the nucleic acid lipid particle generally includes a conjugated lipid.
  • the conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof.
  • the PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci 2 ), a PEG-dimyristyloxypropyl (Ci 4 ), a PEG-dipalmityloxypropyl (Ci 6 ), or a PEG-distearyloxypropyl (C] 8 ).
  • the conjugated lipid can be 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); or PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000).
  • PEG-DMG PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14)
  • PEG-DSG PEG-distyryl glycerol
  • C18-PEG, or PEG-C18 PEG with avg mol wt of 2000
  • PEG-cDMA PEG-carbamoyl-1,
  • the conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0 17.0, 18, 19.0 or 20.0 mol % of the total lipid present in the particle.
  • the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.
  • the nucleic acid-lipid particle further includes cholesterol at about 5 mol %, 10 mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol %.
  • the nucleic acid-lipid particle can include cholesterol at about 31.5 mol %, 34.4 mol %, 35 mol %, 38.5 mol %, or 40 mol % of the total lipid present in the particle.
  • the lipidoid ND98.4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is herein incorporated by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles).
  • Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml.
  • the ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio.
  • the combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM.
  • aqueous dsRNA e.g., in sodium acetate pH 5
  • Lipid-dsRNA nanoparticles typically form spontaneously upon mixing.
  • the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc).
  • a thermobarrel extruder such as Lipex Extruder (Northern Lipids, Inc).
  • the extrusion step can be omitted.
  • Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration.
  • Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.
  • PBS phosphate buffered saline
  • LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.
  • Additional exemplary lipid-dsRNA formulations arm as follows:
  • SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.
  • XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International patent application no. PCT/US10/22614, filed Jan. 29, 2010, which are hereby incorporated by reference.
  • MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, and U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, which are hereby incorporated by reference.
  • ALN100 i.e., ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.
  • any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention may be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.
  • Alkyl means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.
  • Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
  • saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.
  • Alkenyl means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.
  • Alkynyl means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons.
  • Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.
  • Acyl means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below.
  • —C( ⁇ O)alkyl, —C( ⁇ O)alkenyl, and —C( ⁇ O)alkynyl are acyl groups.
  • Heterocycle means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring.
  • the heterocycle may be attached via any heteroatom or carbon atom.
  • Heterocycles include heteroaryls as defined below.
  • Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
  • optionally substituted alkyl means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent ( ⁇ O) two hydrogen atoms are replaced.
  • substituents include oxo, halogen, heterocycle, —CN, —ORx, —NRxRy, —NRxC( ⁇ O)Ry, —NRxSO2Ry, —C( ⁇ O)Rx, —C( ⁇ O)ORx, —C( ⁇ O)NRxRy, —SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy, —NRxC( ⁇ O)Ry, —NRxSO2Ry, —C( ⁇ O)Rx, —C( ⁇ O)ORx, —C( ⁇ O)NRxRy, —SOnRx and —
  • Halogen means fluoro, chloro, bromo and iodo.
  • the methods of the invention may require the use of protecting groups.
  • protecting group methodology is well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, T. W. et al., Wiley-Interscience, New York City, 1999).
  • protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group.
  • a protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group.
  • an “alcohol protecting group” is used.
  • An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group.
  • Protecting groups can be added and removed using techniques well known in the art.
  • nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A;
  • XTC is a cationic lipid of formula A:
  • R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring.
  • the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.
  • Lipid A where R 1 and R 2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R 3 and R 4 are independently lower alkyl or R 3 and R 4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1.
  • Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.
  • the lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.
  • the ketone 1 starting material can be prepared according to Scheme 2.
  • Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.
  • the cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction ( ⁇ 3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature.
  • Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners.
  • formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay.
  • a sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100.
  • a formulation disrupting surfactant e.g. 0.5% Triton-X100.
  • the total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve.
  • the entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%.
  • the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm.
  • the suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.
  • compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators.
  • Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
  • Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate.
  • DCA chenodeoxycholic acid
  • UDCA ursodeoxychenodeoxycholic acid
  • cholic acid dehydrocholic acid
  • deoxycholic 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 acylcamitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium).
  • arachidonic acid 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, gly
  • 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 invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles.
  • DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches.
  • Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyomithine, 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).
  • TDAE polythiodiethylaminomethyl
  • compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.
  • the pharmaceutical formulations of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • compositions of the present invention may be prepared and formulated as emulsions.
  • Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 ⁇ m in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
  • Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other.
  • emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety.
  • aqueous phase 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.
  • oil-in-water (o/w) emulsion When an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion.
  • Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase.
  • compositions such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed.
  • Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.
  • Such complex formulations often provide certain advantages that simple binary emulsions do not.
  • Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion.
  • a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
  • Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion.
  • Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 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, LV., Popovich NG., and Ansel HC., 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.
  • HLB hydrophile/lipophile balance
  • Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 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.
  • 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.
  • 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.
  • polysaccharides for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth
  • cellulose derivatives for example, carboxymethylcellulose and carboxypropylcellulose
  • synthetic polymers for example, carbomers, cellulose ethers, and
  • emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives.
  • preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.
  • Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation.
  • Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite
  • antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • 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, LV., Popovich NG., and Ansel HC., 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.
  • compositions of iRNAs and nucleic acids are formulated as microemulsions.
  • a microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 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).
  • 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.
  • 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).
  • 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.
  • ionic surfactants non-ionic surfactants
  • Brij 96 polyoxyethylene oleyl ethers
  • polyglycerol fatty acid esters tetraglycerol monolaurate (ML310),
  • the cosurfactant usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.
  • Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art.
  • the aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol.
  • the oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs.
  • Lipid based microemulsions both o/w and w/o have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205).
  • Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature.
  • thermolabile drugs, peptides or iRNAs may be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs.
  • Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.
  • Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention.
  • Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
  • the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals.
  • nucleic acids particularly iRNAs
  • Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
  • Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, 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 are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced.
  • these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M.
  • Fatty acids Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcamitines, 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
  • Bile salts The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, 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).
  • 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.
  • POE polyoxyethylene-9-lauryl ether
  • Chelating agents can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, 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 (enaminesxsee e.g., Katdare, A.
  • EDTA disodium ethylenediaminetetraacetate
  • citric acid e.g., citric acid
  • salicylates e.g., sodium salicylate, 5-methoxysalicylate and homovanilate
  • N-acyl derivatives of collagen e.g., laureth-9
  • N-amino acyl derivatives of beta-diketones enaminesxsee e.g., Katdare, A.
  • Non-chelating non-surfactants As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33).
  • This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
  • Agents that enhance uptake of iRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention.
  • cationic lipids such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.
  • transfection reagents examples include, for example LipofectamineTM (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000TM (Invitrogen; Carlsbad, Calif.), 293FectinTM (Invitrogen; Carlsbad, Calif.), CellfectinTM (Invitrogen; Carlsbad, Calif.), DMRIE-CTTM (Invitrogen; Carlsbad, Calif.), FreeStyleTM MAX (Invitrogen; Carlsbad, Calif.), LipofectamineTM 2000 CD (Invitrogen; Carlsbad, Calif.), LipofectamineTM (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), OligofectamineTM (Invitrogen; Carlsbad, Calif.), OptifectTM (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection
  • agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
  • glycols such as ethylene glycol and propylene glycol
  • pyrrols such as 2-pyrrol
  • azones such as 2-pyrrol
  • terpenes such as limonene and menthone.
  • compositions of the present invention also incorporate carrier compounds in the formulation.
  • carrier compound or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation.
  • a nucleic acid and a carrier compound can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor.
  • the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.
  • 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 may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition.
  • Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
  • binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropy
  • compositions of the present invention can also be used to formulate the compositions of the present invention.
  • suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases.
  • the solutions may also contain buffers, diluents and other suitable additives.
  • Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
  • Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • Aqueous suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more biologic agents which function by a non-RNAi mechanism.
  • biologics include, biologics that target one or more of PD-1, PD-L1, or B7-H1 (CD80) (e.g., monoclonal antibodies against PD-1, PD-L1, or B7-H1), or one or more recombinant cytokines (e.g., IL6, IFN- ⁇ , and TNF).
  • 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 in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • a target sequence e.g., achieving a decreased concentration of the polypeptide
  • the IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the dual targeting siRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by PCSK9 expression.
  • the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
  • the invention provides use of a dual targeting siRNA agent for inhibiting the expression of the PCSK9 gene in a mammal.
  • the method includes administering a composition of the invention to the mammal such that expression of the target PCSK9 gene is decreased.
  • PCSK9 expression is decreased for an extended duration, e.g., at least one week, two weeks, three weeks, or four weeks or longer.
  • expression of the PCSK9 gene is suppressed by at least about 5%, 10%, 15%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a dual targeting siRNA agent described herein.
  • the PCSK9 gene is suppressed by at least about 60%, 70%, or 80% by administration of the dual targeting siRNA agent. In some embodiments, the is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide.
  • compositions described herein can be used to treat diseases and conditions that can be modulated by down regulating PCSK9 gene expression.
  • the compositions described herein can be used to treat hyperlipidemia and other forms of lipid imbalance such as hypercholesterolemia, hypertriglyceridemia and the pathological conditions associated with these disorders such as heart and circulatory diseases
  • the invention also relates to the use of a dual targeting siRNA agent for the treatment of a PCSK9-mediated disorder or disease.
  • a dual targeting siRNA agent is used for treatment of a hyperlipidemia.
  • the effect of the decreased PCSK9 gene preferably results in a decrease in LDLc (low density lipoprotein cholesterol) levels in the blood, and more particularly in the serum, of the mammal.
  • LDLc levels are decreased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.
  • the method includes administering a dual targeting siRNA agent to the subject to be treated.
  • the composition can be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, and airway (aerosol) administration.
  • the compositions are administered by intravenous infusion or injection.
  • the method includes administering a dual targeting siRNA agent, e.g., a dose sufficient to depress levels of PCSK9 mRNA for at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days; and optionally, administering a second single dose of dsRNA, wherein the second single dose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the first single dose is administered, thereby inhibiting the expression of the PCSK9 gene in a subject.
  • a dual targeting siRNA agent e.g., a dose sufficient to depress levels of PCSK9 mRNA for at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days
  • a second single dose of dsRNA wherein the second single dose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the first single dose is administered, thereby inhibiting the expression of the PCSK9 gene in a subject.
  • doses of dual targeting siRNA agent are administered not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week.
  • the administrations can be maintained for one, two, three, or six months, or one year or longer.
  • administration can be provided when Low Density Lipoprotein cholesterol (LDLc) levels reach or surpass a predetermined minimal level, such as greater than 70 mg/dL, 130 mg/dL, 150 mg/dL, 200 mVdL, 300 mg/dL, or 400 mg/dL.
  • LDLc Low Density Lipoprotein cholesterol
  • a subject can be administered a therapeutic amount of dual targeting siRNA agent, such as 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA.
  • the dual targeting siRNA agent can be administered by intravenous infusion over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
  • the administration is repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer.
  • the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer.
  • Administration of the dual targeting siRNA agent can reduce PCSK9 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least 10%, at least 15%, at least 20%, at least 25%., at least 30%, at least 40%., at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more.
  • patients Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction, or for elevated lipid levels or blood pressure.
  • a smaller dose such as a 5% infusion reaction
  • adverse effects such as an allergic reaction, or for elevated lipid levels or blood pressure.
  • the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.
  • cytokine e.g., TNF-alpha or INF-alpha
  • 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.
  • 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 dual targeting siRNA 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.
  • administration of a dual targeting siRNA agent is administered in combination an additional therapeutic agent.
  • the dual targeting siRNA agent and an additional therapeutic agent can be administered in combination in the same composition, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.
  • a dual targeting siRNA agent featured in the invention can be administered with, e.g., an HMG-CoA reductase inhibitor (e.g., a statin), a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist (e.g., losartan potassium, such as Merck & Co.'s Cozaar®), an acylCoA cholesterol acetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor, a cholesterol modulator, a bile acid modulator, a peroxisome proliferation activated receptor (PPAR) agonist, a gene
  • HMG-CoA reductase inhibitor e.g., a statin
  • a fibrate e.g., a bile
  • HMG-CoA reductase inhibitors include atorvastatin (Pfizer's Lipitor®/Tahor/Sortis/Torvast/Cardyl), pravastatin (Bristol-Myers Squibb's Pravachol, Sankyo's Mevalotin/Sanaprav), simvastatin (Merck's Zocor®/Sinvacor, Boehringer Ingelheim's Denan, Banyu's Lipovas), lovastatin (Merck's Mevacor/Mevinacor, Bexal's Lovastatina, Cepa; Schwarz Pharma's Liposcler), fluvastatin (Novartis' Lescol®/Locol/Lochol, Fujisawa's Cranoc, Solvay's Digaril), cerivastatin (Bayer's Lipobay/GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca's Crest
  • Exemplary fibrates include, e.g., bezafibrate (e.g., Roche's Befizal®/Cedur®/Bezalip®, Kissei's Bezatol), clofibrate (e.g., Wyeth's Atromid-S®), fenofibrate (e.g., Fournier's Lipidil/Lipantil, Abbott's Tricor®, Takeda's Lipantil, generics), gemfibrozil (e.g., Pfizer's Lopid/Lipur) and ciprofibrate (Sanofi-Synthelabo's Modalim®).
  • bezafibrate e.g., Roche's Befizal®/Cedur®/Bezalip®, Kissei's Bezatol
  • clofibrate e.g., Wyeth's Atromid-S®
  • fenofibrate e.g.,
  • Exemplary bile acid sequestrants include, e.g., cholestyramine (Bristol-Myers Squibb's Questran® and Questran LightTM), colestipol (e.g., Pharmacia's Colestid), and colesevelam (Genzyme/Sankyo's WelCholTM).
  • Exemplary niacin therapies include, e.g., immediate release formulations, such as Aventis' Nicobid, Upsher-Smith's Niacor, Aventis' Nicolar, and Sanwakagaku's Perycit.
  • Niacin extended release formulations include, e.g., Kos Pharmaceuticals' Niaspan and Upsher-Smith's SIo-Niacin.
  • antiplatelet agents include, e.g., aspirin (e.g., Bayer's aspirin), clopidogrel (Sanof ⁇ dot over ( ⁇ ) ⁇ -Synthelabo/Bristol-Myers Squibb's Plavix), and ticlopidine (e.g., Sanof ⁇ dot over ( ⁇ ) ⁇ -Synthelabo's Ticlid and Daiichi's Panaldine).
  • aspirin-like compounds useful in combination with a dsRNA targeting PCSK9 include, e.g., Asacard (slow-release aspirin, by Pharmacia) and Pamicogrel (Kanebo/Angelini Ricerche/CEPA).
  • Exemplary angiotensin-converting enzyme inhibitors include, e.g., ramipril (e.g., Aventis' Altace) and enalapril (e.g., Merck & Co.'s Vasotec).
  • Exemplary acyl CoA cholesterol acetyltransferase (ACAT) inhibitors include, e.g., avasimibe (Pfizer), eflucimibe (BioM ⁇ acute over ( ⁇ ) ⁇ rieux Pierre Fabre/Eli Lilly), CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito).
  • Exemplary cholesterol absorption inhibitors include, e.g., ezetimibe (Merck/Schering-Plough Pharmaceuticals Zetia®) and Pamaqueside (Pfizer).
  • Exemplary CETP inhibitors include, e.g., Torcetrapib (also called CP-529414, Pfizer), JTT-705 (Japan Tobacco), and CETi-I (Avant Immunotherapeutics).
  • Exemplary microsomal triglyceride transfer protein (MTTP) inhibitors include, e.g., implitapide (Bayer), R-103757 (Janssen), and CP-346086 (Pfizer).
  • exemplary cholesterol modulators include, e.g., NO-1886 (Otsuka/TAP Pharmaceutical), CI-1027 (Pfizer), and WAY-135433 (Wyeth-Ayerst).
  • exemplary bile acid modulators include, e.g., HBS-107 (Hisamitsu/Banyu), Btg-511 (British Technology Group), BARI-1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), and AZD-7806 (AstraZeneca).
  • Exemplary peroxisome proliferation activated receptor (PPAR) agonists include, e.g., tesaglitazar (AZ-242) (AstraZeneca), Netoglitazone (MCC-555) (Mitsubishi/Johnson & Johnson), GW-409544 (Ligand Pharmaceuticals/GlaxoSmithKline), GW-501516 (Ligand Pharmaceuticals/GlaxoSmithKline), LY-929 (Ligand Pharmaceuticals and Eli Lilly), LY-465608 (Ligand Pharmaceuticals and Eli Lilly), LY-518674 (Ligand Pharmaceuticals and Eli Lilly), and MK-767 (Merck and Kyorin).
  • Exemplary gene-based therapies include, e.g., AdGWEGF121.10 (GenVec), ApoAl (UCB Pharma/Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics), and ATP-binding cassette transporter-Al (ABCAl) (CV Therapeutics/Incyte, Aventis, Xenon).
  • Exemplary Glycoprotein Ilb/IIIa inhibitors include, e.g., roxifiban (also called DMP754, Bristol-Myers Squibb), Gantofiban (Merck KGaA/Yamanouchi), and Cromafiban (Millennium Pharmaceuticals).
  • Exemplary squalene synthase inhibitors include, e.g., BMS-1884941 (Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer), CP-294838 (Pfizer), and TAK-475 (Takeda).
  • An exemplary MCP-I inhibitor is, e.g., RS-504393 (Roche Bioscience).
  • the anti-atherosclerotic agent BO-653 (Chugai Pharmaceuticals), and the nicotinic acid derivative Nyclin (Yamanouchi Pharmaceuticals) are also appropriate for administering in combination with a dsRNA featured in the invention.
  • Exemplary combination therapies suitable for administration with a dsRNA targeting PCSK9 include, e.g., advicor (Niacin/lovastatin from Kos Pharmaceuticals), amlodipine/atorvastatin (Pfizer), and ezetimibe/simvastatin (e.g., Vytorin® 10/10, 10/20, 10/40, and 10/80 tablets by Merck/Schering-Plough Pharmaceuticals).
  • advicor Niacin/lovastatin from Kos Pharmaceuticals
  • Amlodipine/atorvastatin Pfizer
  • ezetimibe/simvastatin e.g., Vytorin® 10/10, 10/20, 10/40, and 10/80 tablets by Merck/Schering-Plough Pharmaceuticals.
  • Agents for treating hypercholesterolemia, and suitable for administration in combination with a dsRNA targeting PCSK9 include, e.g., lovastatin, niacin Altoprev® Extended-Release Tablets (Andrx Labs), lovastatin Caduet® Tablets (Pfizer), amlodipine besylate, atorvastatin calcium Crestor® Tablets (AstraZeneca), rosuvastatin calcium Lescol® Capsules (Novartis), fluvastatin sodium Lescol® (Reliant, Novartis), fluvastatin sodium Lipitor® Tablets (Parke-Davis), atorvastatin calcium Lofibra® Capsules (Gate), Niaspan Extended-Release Tablets (Kos), niacin Pravachol Tablets (Bristol-Myers Squibb), pravastatin sodium TriCor® Tablets (Abbott), fenofibrate Vytorin® 10
  • a dual targeting siRNA agent is administered in combination with an ezetimibe/simvastatin combination (e.g., Vytorin® (Merck/Schering-Plough Pharmaceuticals)).
  • an ezetimibe/simvastatin combination e.g., Vytorin® (Merck/Schering-Plough Pharmaceuticals)
  • the dual targeting siRNA agent is administered to the patient, and then the additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the dual targeting siRNA agent and the additional therapeutic agent are administered at the same time.
  • the invention features, a method of instructing an end user, e.g., a caregiver or a subject, on how to administer a dual targeting siRNA agent described herein.
  • the method includes, optionally, providing the end user with one or more doses of the dual targeting siRNA agent, and instructing the end user to administer the dual targeting siRNA agent on a regimen described herein, thereby instructing the end user.
  • the invention provides a method of treating a patient by selecting a patient on the basis that the patient is in need of LDL lowering, LDL lowering without lowering of HDL, ApoB lowering, or total cholesterol lowering.
  • the method includes administering to the patient a dual targeting siRNA agent in an amount sufficient to lower the patient's LDL levels or ApoB levels, e.g., without substantially lowering HDL levels.
  • a patient in need of a dual targeting siRNA agent can be identified by taking a family history, or, for example, screening for one or more genetic markers or variants.
  • a healthcare provider such as a doctor, nurse, or family member, can take a family history before prescribing or administering a dual targeting siRNA agent.
  • a DNA test may also be performed on the patient to identify a mutation in the PCSK9 gene, before a PCSK9 dsRNA is administered to the patient.
  • such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
  • oligonucleotides are synthesized on an AKTAoligopilot synthesizer.
  • Commercially available controlled pore glass solid support dT-CPG, 500 ⁇ acute over ( ⁇ ) ⁇ , Prime Synthesis
  • RNA phosphoramidites with standard protecting groups 5′-O-dimethoxytrityl N6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—
  • the 2′-F phosphoramidites, 5′-O-dimethoxytrityl-N4-acetyl-2′-fluro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite and 5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite are purchased from (Promega). All phosphoramidites are used at a concentration of 0.2M in acetonitrile (CH 3 CN) except for guanosine which is used at 0.2M concentration in 10% THF/ANC (v/v).
  • Coupling/recycling time of 16 minutes is used.
  • the activator is 5-ethyl thiotetrazole (0.75M, American International Chemicals); for the PO-oxidation iodine/water/pyridine is used and for the PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) is used.
  • 3′-ligand conjugated strands are synthesized using solid support containing the corresponding ligand.
  • the introduction of cholesterol unit in the sequence is performed from a hydroxyprolinol-cholesterol phosphoramidite.
  • Cholesterol is tethered to trans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain a hydroxyprolinol-cholesterol moiety.
  • 5′-end Cy-3 and Cy-5.5 (fluorophore) labeled iRNAs are synthesized from the corresponding Quasar-570 (Cy-3) phosphoramidite are purchased from Biosearch Technologies.
  • Conjugation of ligands to 5′-end and or internal position is achieved by using appropriately protected ligand-phosphoramidite building block.
  • Oxidation of the internucleotide phosphite to the phosphate is carried out using standard iodine-water as reported (1) or by treatment with tert-butyl hydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation wait time conjugated oligonucleotide.
  • Phosphorothioate is introduced by the oxidation of phosphite to phosphorothioate by using a sulfur transfer reagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucage reagent.
  • DDTT purchased from AM Chemicals
  • PADS PADS
  • Beaucage reagent The cholesterol phosphoramidite is synthesized in house and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite is 16 minutes.
  • the support is transferred to a 100 mL glass bottle (VWR).
  • the oligonucleotide is cleaved from the support with simultaneous deprotection of base and phosphate groups with 80 mL of a mixture of ethanolic ammonia [ammonia: ethanol (3:1)] for 6.5 h at 55° C.
  • the bottle is cooled briefly on ice and then the ethanolic ammonia mixture is filtered into a new 250-mL bottle.
  • the CPG is washed with 2 ⁇ 40 mL portions of ethanol/water (1:1 v/v).
  • the volume of the mixture is then reduced to ⁇ 30 mL by roto-vap.
  • the mixture is then frozen on dry ice and dried under vacuum on a speed vac.
  • the dried residue is resuspended in 26 mL of triethylamine, triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60° C. for 90 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′ position.
  • TDMS tert-butyldimethylsilyl
  • the oligonucleotides are analyzed by high-performance liquid chromatography (HPLC) prior to purification and selection of buffer and column depends on nature of the sequence and or conjugated ligand.
  • HPLC high-performance liquid chromatography
  • the ligand-conjugated oligonucleotides are purified by reverse-phase preparative HPLC.
  • the unconjugated oligonucleotides are purified by anion-exchange HPLC on a TSK gel column packed in house.
  • the buffers are 20 mM sodium phosphate (pH 8.5) in 10% CH 3 CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH 3 CN, 1M NaBr (buffer B). Fractions containing full-length oligonucleotides are pooled, desalted, and lyophilized.
  • oligonucleotides Approximately 0.15 OD of desalted oligonucleotides are diluted in water to 150 ⁇ L and then pipetted into special vials for CGE and LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.
  • equimolar amounts of sense and antisense strand are heated in 1 ⁇ PBS at 95° C. for 5 min and slowly cooled to room temperature. Integrity of the duplex is confirmed by HPLC analysis.
  • 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.
  • Nucleotide (s) A adenosine C cytidine G guanosine U uridine N any nucleotide (G, A, C, T or U) a 2′-O-methyladenosine c 2′-O-methylcytidine g 2′-O-methylguanosine u 2′-O-methyluridine dT , T 2′-deoxythymidine s phosphorothioate linkage
  • siRNA targeting a PCSK9 gene The sequences of siRNA targeting a PCSK9 gene are described in Table 1 and Table 2 above, and Tables 4-8 below.
  • a dual targeting siRNA agent was synthesized.
  • the sense and antisense strands for AD-10792 (target gene is PCSK9, see Table 2)) and AD-18038 (target gene is XBP-1, see Table 3) were synthesized.
  • the two sense strands were covalently bound using a disulfide linker “Q51” with the structure shown below.
  • the resulting dual sense strand was hybridized to the corresponding antisense strands to create a 42 mer dual targeting siRNA agent “AD-23426” (SEQ ID NOS 4162-4165, respectively, in order of appearance):
  • PCSK9, Xbp-1 and GAPDH transcripts were measured via bDNA in cell lysates prepared according to manufacturer's protocol. PCSK9 to GAPDH or Xbp-1 to GAPDH ratios were normalized to control (luciferase) and graphed.
  • the dual targeting siRNA was at least as effective at inhibiting their corresponding target gene as the single siRNAs.
  • the dual targeting AD-23426 was formulated in an LNP09 formulation: XTC/DSPC/Cholesterol/PEG-DMG in a % mol ratio of 50/10/38.5/1.5 with a lipid:siRNA ratio of about 10:1.
  • the LNP09-AD-23426 was administered by tail vein injection into C57B6 mice at 6.0 mg/kg, 2.0 mg/kg and 0.6 mg/kg.
  • LNP09 formulated single siRNAs (AD-10792 and AD-18038) were administered each at 3.0 mg/kg, 1.0 mg/kg and 0.3 mg/kg. Livers and plasma were harvested 72 hours post-injection (5 animals per group).
  • PCSK9, Xbp-1 and GAPDH transcript levels were measured via bDNA in livers prepared according to the manufacturer's protocol.
  • PCSK9 to GAPDH or Xbp-1 to GAPDH ratios were normalized to control (luciferase) and graphed. The results are shown in FIG. 2 .
  • Total cholesterol was measure in serum according to manufacturer's instructions using a cholesterol kit from WAKO Tex.
  • results demonstrate that the dual targeting siRNAs were at least as effective at inhibiting their corresponding target as single siRNAs in vivo.
  • results also show that the dual targeting construct has an additive effect compared to the single siRNAs at reducing total serum cholesterol.
  • PBMC Peripheral blood mononuclear cells
  • Isolated PBMC were seeded at 1 ⁇ 10 5 cells/well in 96 well plates and cultured in RPMI 1640 GlutaMax Medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic/antimycotic (Invitrogen).
  • siRNAs were transfected using DOTAP Transfection Reagent (Roche Applied Science).
  • DOTAP was first diluted in Opti-MEM (Invitrogen) for 5 minutes before mixing with an equal volume of Opti-MEM containing the siRNA.
  • siRNA/transfection reagent complexes were incubated for 15 minutes at room temperature prior to being added to PBMC.
  • siRNAs were transfected at final concentrations of 266 nM, 133 nM or 67 nM using 16 ⁇ g/ml, 8 ⁇ g/ml or 4 ⁇ g/ml DOTAP, respectively.
  • the ratio of siRNA to DOTAP is 16.5 ⁇ mol/ ⁇ g.
  • Transfected PBMC were incubated at 37° C., 5% CO 2 for 24 hrs after which supernatants were harvested and stored at ⁇ 80° C. until analysis. Quantitative cytokine analysis was done using commercially available Instant ELISA Kits for IFN- ⁇ , (BMS216INST) and TNF- ⁇ (BMS223INST); both from Bender MedSystems (
  • siRNAs LNP09 and DOTAP formulated siRNAs were administered. Control siRNAs were AD-1730, AD-1955, AD-6248, AD-18889, AD-5048, and AD-18221.
  • AD-10792 PCSK9 siRNA.
  • AD-18038 XBP-1 siRNA.
  • AD-23426 did not induce production of IFN- ⁇ and TNF- ⁇ , similar to the result obtained with the single target gene siRNAs.
  • unmodified siRNAs AD-5048 and AD-18889 induced production of both IFN- ⁇ and TNF- ⁇ .
  • a human subject is treated with a pharmaceutical composition, e.g., a nucleic acid-lipid particle having a dual targeting siRNA agent.
  • a pharmaceutical composition e.g., a nucleic acid-lipid particle having a dual targeting siRNA agent.
  • a suitable first dose of the pharmaceutical composition is subcutaneously administered to the subject.
  • the composition is formulated as described herein.
  • the subject's condition is evaluated, e.g., by measurement of total serum cholesterol. This measurement can be accompanied by a measurement of PCSK9 expression in said subject, and/or the products of the successful siRNA-targeting of PCSK9 mRNA. Other relevant criteria can also be measured.
  • the number and strength of doses are adjusted according to the subject's needs.
  • the subject's condition is compared to the condition existing prior to the treatment, or relative to the condition of a similarly afflicted but untreated subject.
  • dsRNA targeted to PCSK9 mismatches and modifications Strand: S/Sense; AS/Antisense; U, C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g: corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding 2′-deoxy-2′-fluoro ribonucleotide; Y1 corresponds to DFT difluorotoluyl ribo(or deoxyribo)nucleotide; where nucleotides are written in sequence, they are connected by 3′-5′ phospho- diester groups; nucleotides with interjected “s” are connected by 3′- O-5′-O phosphorothiodiester groups; unless denoted by prefix “P*”, oligonucleotides are devoid of a 5′-phosphate group on the 5′-most nucle
  • NM_001004210 is the gene for rat XBP-1.
  • XM_001103095 is the sequence for Macaca mulatta (rhesus monkey) XBP-1.
  • Target gene name and target sequence location for dsRNA targeting XBP-1 Duplex # Target gene and location of target sequence AD18027 NM_001004210_1128-1146 AD18028 NM_001004210_1129-1147 AD18029 NM_001004210_677-695 AD18030 NM_001004210_893-911 AD18031 NM_001004210_895-913 AD18032 NM_001004210_1127-1145 AD18033 NM_001004210_894-912 AD18034 NM_001004210_1760-1778 AD18035 NM_001004210_215-233 AD18036 NM_001004210_1759-1777 AD18037 NM_001004210_367-385 AD18038 NM_001004210_896-914 AD18039 NM_001004210_214-232 AD18040 NM_001004210_216-234 AD18041 XM_001103095_387-405 AD180

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Abstract

The invention relates to dual targeting siRNA agents targeting a PCSK9 gene and a second gene, and methods of using dual targeting siRNA agents to inhibit expression of PCSK9 and to treat PCSK9 related disorders, e.g., hyperlipidemia.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application is a continuation of U.S. application Ser. No. 16/803,738, filed Feb. 27, 2020, which is a continuation of U.S. application Ser. No. 16/521,422, filed Jul. 24, 2019 (abandoned), which is a continuation of U.S. application Ser. No. 15/724,175, filed Oct. 3, 2017 (abandoned), which is a continuation of U.S. application Ser. No. 14/885,342, filed Oct. 16, 2015 (abandoned), which is a continuation of U.S. application Ser. No. 13/497,226, with a 371(c) filing date of Oct. 10, 2012, now U.S. Pat. No. 9,187,746, issued Nov. 17, 2015, which is a National Stage of International Application No. PCT/US2010/049868, filed Sep. 22, 2010, which claims the benefit of U.S. Provisional Application No. 61/244,859, filed Sep. 22, 2009, and claims the benefit of U.S. Provisional Application No. 61/313,584, filed Mar. 12, 2010, all of which are hereby incorporated in their entirety by reference.
    • REFERENCE TO A SEQUENCE LISTING
  • This application includes a Sequence Listing with 4166 sequences submitted electronically as a text file named AYL108C4_sequencelisting.txt, created on Feb. 20, 2020, with a size of 1,351,680 bytes. The sequence listing is incorporated by reference.
    • FIELD OF THE INVENTION
  • The invention relates to a composition of two covalently linked siRNAs, e.g., a dual targeting siRNA agent. At least one siRNA is a dsRNA that targets a PCSK9 gene. The covalently linked siRNA agent is used in methods of inhibition of PCSK9 gene expression and methods of treatment of pathological conditions associated with PCSK9 gene expression, e.g., hyperlipidemia.
    • BACKGROUND OF THE INVENTION
  • Proprotein convertase subtilisin kexin 9 (PCSK9) is a member of the subtilisin serine protease family. The other eight mammalian subtilisin proteases, PCSK1-PCSK8 (also called PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, and SIP/SKI-1) are proprotein convertases that process a wide variety of proteins in the secretory pathway and play roles in diverse biological processes (Bergeron, F. (2000) J. Mol. Endocrinol. 24, 1-22, Gensberg, K., (1998) 30 Semin. Cell Dev. Biol. 9, 11-17, Seidah, N. G. (1999) Brain Res. 848, 45-62, Taylor, N. A., (2003) FASEB J. 17, 1215-1227, and Zhou, A., (1999) J. Biol. Chem. 274, 20745-20748). PCSK9 has been proposed to play a role in cholesterol metabolism. PCSK9 mRNA expression is down-regulated by dietary cholesterol feeding in mice (Maxwell, K. N., (2003) J. Lipid Res. 44, 2109-2119), up-regulated by statins in HepG2 cells (Dubuc, G., (2004) Arterioscler. Thromb. Vasc. Biol. 24, 1454-1459), and up-regulated in sterol regulatory element binding protein (SREBP) transgenic mice (Horton, J. D., (2003) Proc. Natl. Acad. Sci. USA 100, 12027-12032), similar to the cholesterol biosynthetic enzymes and the low-density lipoprotein receptor (LDLR). Furthermore, PCSK9 missense mutations have been found to be associated with a form of autosomal dominant hypercholesterolemia (Hchola3) (Abifadel, M., et al. (2003) Nat. Genet. 34, 154-156, Timms, K. M., (2004) Hum. Genet. 114, 349-353, Leren, T. P. (2004) Clin. Genet. 65, 419-422). PCSK9 may also play a role in determining LDL cholesterol levels in the general population, because single-nucleotide polymorphisms (SNPs) have been associated with cholesterol levels in a Japanese population (Shioji, K., (2004) J. Hum. Genet. 49, 109-114).
  • Autosomal dominant hypercholesterolemias (ADHs) are monogenic diseases in which patients exhibit elevated total and LDL cholesterol levels, tendon xanthomas, and premature atherosclerosis (Rader, D. J., (2003) J. Clin. Invest. 111, 1795-1803). The pathogenesis of ADHs and a recessive form, autosomal recessive hypercholesterolemia (ARH) (Cohen, J. C., (2003) Curr. Opin. Lipidol. 14, 121-127), is due to defects in LDL uptake by the liver. ADH may be caused by LDLR mutations, which prevent LDL uptake, or by mutations in the protein on LDL, apolipoprotein B, which binds to the LDLR. ARH is caused by mutations in the ARH protein that are necessary for endocytosis of the LDLR-LDL complex via its interaction with clathrin. Therefore, if PCSK9 mutations are causative in Hchola3 families, it seems likely that PCSK9 plays a role in receptor-mediated LDL uptake.
  • Overexpression studies point to a role for PCSK9 in controlling LDLR levels and, hence, LDL uptake by the liver (Maxwell, K. N. (2004) Proc. Natl. Acad. Sci. USA 101, 7100-7105, Benjannet, S., et al. (2004) J. Biol. Chem. 279, 48865-48875, Park, S. W., (2004) J. Biol. Chem. 279, 50630-50638). Adenoviral-mediated overexpression of mouse or human PCSK9 for 3 or 4 days in mice results in elevated total and LDL cholesterol levels; this effect is not seen in LDLR knockout animals (Maxwell, K. N. (2004) Proc. Natl. Acad. Sci. USA 101, 7100-7105, Benjannet, S., et al. (2004) J. Biol. Chem. 279, 48865-48875, Park, S. W., (2004) J. Biol. Chem. 279, 50630-50638). In addition, PCSK9 overexpression results in a severe reduction in hepatic LDLR protein, without affecting LDLR mRNA levels, SREBP protein levels, or SREBP protein nuclear to cytoplasmic ratio.
  • Loss of function mutations in PCSK9 have been designed in mouse models (Rashid et al., (2005) PNAS, 102, 5374-5379), and identified in human individuals (Cohen et al. (2005) Nature Genetics 37:161-165). In both cases loss of PCSK9 function lead to lowering of total and LDLc cholesterol. In a retrospective outcome study over 15 years, loss of one copy of PCSK9 was shown to shift LDLc levels lower and to lead to an increased risk-benefit protection from developing cardiovascular heart disease (Cohen et al., (2006) N. Engl. J. Med., 354:1264-1272).
  • X-box binding protein 1 (XBP-1) is a basic leucine zipper transcription factor that is involved in the cellular unfolded protein response (UPR). XBP-1 is known to be active in the endoplasmic reticulum (ER). The ER consists of a system of folded membranes and tubules in the cytoplasm of cells. Proteins and lipids are manufactured and processed in the ER. When unusual demands are placed on the ER, “ER stress” occurs. ER stress can be triggered by a viral infection, gene mutations, exposure to toxins, aggregation of improperly folded proteins or a shortage of intracellular nutrients. The result can be Type II diabetes, metabolic syndrome, a neurological disorder or cancer.
  • Two XBP-1 isoforms are known to exist in cells: spliced XBP-1S and unspliced XBP-1U. Both isoforms of XBP-1 bind to the 21-bp Tax-responsive element of the human T-lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR) in vitro and transactivate HTLV-1 transcription. HTLV-1 is associated with a rare form of blood dyscrasia known as Adult T-cell Leukemia/lymphoma (ATLL) and a myelopathy, tropical spastic paresis.
  • Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) disclosed the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.
  • A description of siRNA targeting PCSK9 can be found in U.S. patent application Ser. No. 11/746,864 filed on May 10, 2007 (now U.S. Pat. No. 7,605,251) and International Patent Application No. PCT/US2007/068655 filed May 10, 2007 (published as WO 2007/134161). Additional disclosure can be found in U.S. patent application Ser. No. 12/478,452 filed Jun. 4, 2009 (published as US 2010/0010066) and International Patent Application No. PCT/US2009/032743 filed Jan. 30, 2009 (published as WO 2009/134487).
  • A description of siRNA targeting XPB-1 can be found in U.S. patent application Ser. No. 12/425,811 filed on Apr. 17, 2009 and published as US 2009-0275638.
  • Dual targeting siRNAs can be found in International patent application publication no. WO/2007/091269.
    • SUMMARY OF THE INVENTION
  • Described herein are dual targeting siRNA agent in which a first siRNA targeting PCSK9 is covalently joined to a second siRNA targeting a gene implicated in cholesterol metabolism, e.g., XBP-1. The two siRNAs are covalently linked via, e.g., a disulfide linker.
  • Accordingly one aspect of the invention is a dual targeting siRNA agent having a first dsRNA targeting a PCSK9 gene and a second dsRNA targeting a second gene, wherein the first dsRNA and the second dsRNA are linked with a covalent linker. The second gene is can be, e.g., XBP-1, PCSK9, PCSK5, ApoC3, SCAP, or MIG12. In one embodiment, the second gene is XBP-1. Each dsRNA is 30 nucleotides or less in length. In general, each strand of each dsRNA is 19-23 bases in length.
  • In one embodiment, the dual targeting siRNA agent comprising a first dsRNA AD-10792 targeting a PCSK9 gene and a second dsRNA AD-18038 targeting an XBP-1 gene, wherein AD-10792 sense strand and AD-18038 sense strand are covalently linked with a disulfide linker.
  • The first dsRNA of the dual targeting siRNA agent targets a PCSK9 gene. In one aspect, the first dsRNA includes at least 15 contiguous nucleotides of an antisense strand of one of Tables 1, 2, or 4-8, or includes an antisense strand of one of Tables 1, 2, or 4-8, or includes a sense strand and an antisense strand of one of Tables 1, 2, or 4-8. The first dsRNA can be AD-9680 or AD-10792.
  • In some embodiments, the second dsRNA target XBP-1. In one aspect, the second dsRNA includes at least 15 contiguous nucleotides of an antisense strand of one of Tables 3 or 9-13, or includes an antisense strand of one of Tables 3 or 9-13, or includes a sense strand and an antisense strand of one of Tables 3 or 9-13. For example, the second dsRNA can be AD-18038.
  • Either the first and second dsRNA can include at least one modified nucleotide, e.g., a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. In some embodiments, the first and second dsRNAs include “endo-light” modification with 2′-O-methyl modified nucleotides and nucleotides comprising a 5′-phosphorothioate group.
  • The first and second dsRNAs are linked with a covalent linker. In some embodiments, the linker is a disulfide linker. Various combinations of strands can be linked, e.g., the first and second dsRNA sense strands are covalently linked or, e.g., the first and second dsRNA antisense strands are covalently linked. In some embodiments, any of the dual targeting siRNA agents of the invention include a ligand.
  • Also included in the invention are isolated cells having and vectors encoding the dual targeting siRNA agent described herein.
  • In one aspect, administration of the dual targeting siRNA agent to a cell inhibits expression of the PCSK9 gene and the second gene at a level equivalent to inhibition of expression of both genes using administration of each siRNA individually. In another aspect, administration of the dual targeting siRNA agent to a subject results in a greater reduction of total serum cholesterol that that obtained by administration of each siRNA alone.
  • The invention also includes a pharmaceutical composition comprising the dual targeting siRNA agents described herein and a pharmaceutical carrier. In one embodiment, the pharmaceutical carrier is a lipid formulation, e.g., a lipid formulation including cationic lipid DLinDMA or cationic lipid XTC. Examples of lipid formulations described in (but not limited to) Table A, below. The lipid formulation can be XTC/DSPC/Cholesterol/PEG-DMG at % mol ratios of 50/10/38.5/1.5.
  • Another aspect of the invention includes methods of using the dual targeting siRNA agents described herein. In one embodiment, the invention is a method of inhibiting expression of the PCSK9 gene and a second gene in a cell, the method comprising (a) introducing into the cell the any of the dual targeting siRNA agents and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the PCSK9 gene and the second gene, thereby inhibiting expression of the PCSK9 gene and the second gene in the cell.
  • In another embodiment, the invention includes methods of treating a disorder mediated by PCSK9 expression with the step of administering to a subject in need of such treatment a therapeutically effective amount of the pharmaceutical compositions described herein. In one aspect, the disorder is hyperlipidemia. In still another embodiment, the invention includes methods of reducing total serum cholesterol in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical compositions described herein.
  • The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.
    • DESCRIPTION OF THE DRAWINGS
  • FIG. 1A is a graph showing the effect on PCSK9 mRNA levels in primary mouse hepatocytes following treatment with a dual targeting siRNA, AD-23426. AD-23426 was as effective at reducing mRNA expression as each single gene target siRNA. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA. Lipo2000: control transfection agent only FIG. 1Bis a graph showing the effect on XBP-1 mRNA levels in primary mouse hepatocytes following treatment with a dual targeting siRNA, AD-23426. AD-23426 was as effective at reducing mRNA expression as each single gene target siRNA. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA. Lipo2000: control transfection agent only.
  • FIG. 2 is a graph showing the effect on PCSK9 and XBP-1 mRNA levels in mice following treatment with a dual targeting siRNA, AD-23426. LNP09 (lipid) formulated siRNA was administered to mice as described. AD-23426 was as effective at reducing mRNA expression as each single gene target siRNA. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA.
  • FIG. 3 is a graph showing the effect on serum cholesterol levels in mice following treatment with a dual targeting siRNA, AD-23426. LNP09 (lipid) formulated siRNA was administered to mice as described. AD-23426 was more effective at reducing serum cholesterol compared to each single gene target siRNA. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA.
  • FIG. 4A is a graph showing the effect on IFN-α, in human PBMC following treatment with a dual targeting siRNA, AD-23426. FIG. 4B is a graph showing the effect on TNF-α in human PBMC following treatment with a dual targeting siRNA, AD-23426. DOTAP and LNP09 (lipid) formulated siRNAs was administered huPBMC as described below. AD-23426 did not induce IFN-α or TNF-α.
    •  DETAILED DESCRIPTION OF THE INVENTION
  • The invention provides a solution to the problem of treating diseases that can be modulated by the down regulation of the PCSK9 gene, such as hyperlipidemia, by using dual targeting siRNA to silence the PCSK9 gene.
  • The invention provides compositions and methods for inhibiting the expression of the PCSK9 gene in a subject using two siRNA, e.g., a dual targeting siRNA. The invention also provides compositions and methods for treating pathological conditions and diseases, such as hyperlipidemia, that can be modulated by down regulating the expression of the PCSK9 gene.
  • The dual targeting siRNA agents target a PCSK9 gene and at least one other gene. The other gene can be another region of the PCSK9 gene, or can be another gene, e.g., XBP-1.
  • The dual targeting siRNA agents have the advantage of lower toxicity, lower off-target effects, and lower effective concentration compared to individual siRNAs.
  • The use of the dual targeting siRNA dsRNAs enables the targeted degradation of an mRNA that is involved in the regulation of the LDL receptor and circulating cholesterol levels. Using cell-based and animal assays it was demonstrated that inhibiting both a PCSK9 gene and an XBP-1 gene using a dual targeting siRNA is at least as effective at inhibiting their corresponding targets as the use of single siRNAs. It was also demonstrated that administration of a dual targeting siRNA results in a synergistic lowering of total serum cholesterol. Thus, reduction of total serum cholesterol is enhanced with a dual targeting siRNA compared to a single target siRNA.
    • Definitions
  • For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.
  • “G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. “T” and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine. 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. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of dsRNA featured in the invention 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 invention.
  • The term “PCSK9” refers to the proprotein convertase subtilisin kexin 9 gene or protein (also known as FH3, HCHOLA3, NARC-1, NARC1). Examples of mRNA sequences to PCSK9 include but are not limited to the following: human: NM_174936; mouse: NM_153565, and rat: NM_199253. Additional examples of PCSK9 mRNA sequences are readily available using, e.g., GenBank.
  • The term “XBP-1” refers to -Box Protein 1, which is also known as Tax-responsive element-binding protein 5, TREB5, and XBP2. XBP-1 sequence can be found as NCBI GeneID:7494 and RefSeq ID number: NM_005080 (human) and NM_013842 (mouse). A dsRNA featured in the invention can target a specific XBP-1 isoform, e.g., the spliced form (XBP-1S) or the unspliced form (XBP-1U), or a dsRNA featured in the invention can target both isoforms by binding to a common region of the mRNA transcript.
  • The term “PCSK5” refers to the Proprotein convertase subtilisin/kexin type 5 gene, mRNA or protein belonging to the subtilisin-like proprotein convertase family.
  • The term “ApoC3” refers to the Apolipoprotein C-III protein gene, mRNA or protein, and is a very low density lipoprotein (VLDL).
  • The term “SCAP” refers to the SREBP cleavage-activating protein gene, mRNA or protein. SCAP is a regulatory protein that is required for the proteolytic cleavage of the sterol regulatory element binding protein (SREBP). Example of siRNA targeting SCAP are described in U.S. patent application Ser. No. 11/857,120, filed on Sep. 18, 2007, published as US 20090093426. This application and the siRNA sequences described therein are incorporated by reference for all purposes.
  • The term “MIG12” is a gene also known as TMSB10 and TB10 refers to the thymosin beta 10 gene. Example of siRNA targeting MIG12 are described International patent application no. PCT/US10/25444, filed on Feb. 25, 2010, published as WO/20XX/XXXXXX This application and the siRNA sequences described therein are incorporated by reference for all purposes.
  • As used herein, the term “iRNA” refers to an agent that contains RNA and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. The term iRNA includes siRNA.
  • As described in more detail below, the term “siRNA” and “siRNA agent” refers to a dsRNA that mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway.
  • A “double-stranded RNA” or “dsRNA,” as used herein, refers to an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having “sense” and “antisense” orientations with respect to a target RNA.
  • The term “dual targeting siRNA agent” refers to a composition of two siRNAs, e.g., two dsRNAs. One dsRNA includes an antisense strand with a first region of complementarity to a first target gene, e.g., PCSK9. The second dsRNA include an antisense strand with a second region of complementarity to a second target gene. In some embodiments, the first and second target genes are identical, e.g., both are PCSK9 and each dsRNA targets a different region of PCSK9. In other embodiments, the first and second target genes are different, e.g., the first dsRNA targets PCSK9 and the second dsRNA targets a different gene, e.g., XBP-1.
  • “Covalent linker” refers to a molecule for covalently joining two molecules, e.g., two dsRNAs. As described in more detail below, the term includes, e.g., a nucleic acid linker, a peptide linker, and the like and includes disulfide linkers.
  • The term “target gene” refers to a gene of interest, e.g., PCSK9 or a second gene, e.g., XBP-1, targeted by an siRNA of the invention for inhibition of expression.
  • As described in more detail below, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all sub-ranges therebetween.
  • As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
  • As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
  • Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 5, 4, 1 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.
  • “Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but are not limited to, G:U Wobble or Hoogstein base pairing.
  • The terms “complementary,” “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.
  • As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of the target gene (e.g., an mRNA encoding PCSK9 or a second gene, e.g., XBP-1). For example, a polynucleotide is complementary to at least a part of a PCSK9 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding PCSK9.
  • The skilled artisan will recognize that the term “RNA molecule” or “ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, a “ribonucleoside” includes a nucleoside base and a ribose sugar, and a “ribonucleotide” is a ribonucleoside with one, two or three phosphate moieties. However, the terms “ribonucleoside” and “ribonucleotide” can be considered to be equivalent as used herein. The RNA can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described herein below. However, the molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex. As non-limiting examples, an RNA molecule can also include at least one modified ribonucleoside including but not limited to a 2′-O-methyl modified nucleotide, a nucleoside comprising a 5′ phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2′-deoxy-2′-fluoro modified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, an RNA molecule can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the dsRNA molecule. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule. In one embodiment, modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA via a RISC pathway.
  • In one aspect, a modified ribonucleoside includes a deoxyribonucleoside. In such an instance, an iRNA agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double stranded portion of a dsRNA. However, it is self evident that under no circumstances is a double stranded DNA molecule encompassed by the term “iRNA.”
  • As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) may be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′ end, 3′ end or both ends of either an antisense or sense strand of a dsRNA. One or more of the nucleotides in the overhang can be replaced with a nucleoside thiophosphate.
  • 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 an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.
  • The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.
  • As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in International Application No. WO 2009082817. These applications are incorporated herein by reference in their entirety.
  • “Introducing into a cell,” when referring to an iRNA, means facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; an iRNA may also be “introduced into a cell,” wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or known in the art.
  • As used herein, the term “modulate the expression of,” refers to at an least partial “inhibition” or partial “activation” of target gene expression in a cell treated with an iRNA composition as described herein compared to the expression of the target gene in an untreated cell.
  • The terms “activate,” “enhance,” “up-regulate the expression of,” “increase the expression of,” and the like, in so far as they refer to a target gene, herein refer to the at least partial activation of the expression of a target gene, as manifested by an increase in the amount of target mRNA, which may be isolated from or detected in a first cell or group of cells in which a target gene is transcribed and which has or have been treated such that the expression of a target gene is increased, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).
  • In one embodiment, expression of a target gene is activated by at least about 10%, 15%,
    Figure US20220213475A1-20220707-P00001
    25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA as described herein. In some embodiments, a target gene is activated by at least about 60%, 70%, or 80% by administration of an iRNA featured in the invention. In some embodiments, expression of a target gene is activated by at least about 85%, 90%, or 95% or more by administration of an iRNA as described herein. In some embodiments, the target gene expression is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000 fold or more in cells treated with an iRNA as described herein compared to the expression in an untreated cell. Activation of expression by small dsRNAs is described, for example, in Li et al., 2006 Proc. Natl. Acad. Sci. U.S.A. 103:17337-42, and in US20070111963 and US2005226848, each of which is incorporated herein by reference.
  • The terms “silence,” “inhibit the expression of,” “down-regulate the expression of,” “suppress the expression of,” and the like, in so far as they refer to a target gene, herein refer to the at least partial suppression of the expression of a target gene, as manifested by a reduction of the amount of target mRNA which may be isolated from or detected in a first cell or group of cells in which a target gene is transcribed and which has or have been treated such that the expression of target 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 is usually expressed in terms of
  • ( mRNA ⁢ ⁢ in ⁢ ⁢ control ⁢ ⁢ cells ) - ( mRNA ⁢ ⁢ in ⁢ ⁢ treated ⁢ ⁢ cells ) ( mRNA ⁢ ⁢ in ⁢ ⁢ control ⁢ ⁢ cells ) · 100 ⁢ %
  • Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to target gene expression, e.g., the amount of protein encoded by a target gene, or the number of cells displaying a certain phenotype, e.g., lack of or decreased cytokine production. In principle, target gene silencing may be determined in any cell expressing target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given iRNA inhibits the expression of the target gene by a certain degree and therefore is encompassed by the instant invention, the assays provided in the Examples below shall serve as such reference.
  • For example, in certain instances, expression of a target gene is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% by administration of an iRNA featured in the invention. In some embodiments, a target gene is suppressed by at least about 60%, 65%, 70%, 75%, or 80% by administration of an iRNA featured in the invention. In some embodiments, a target gene is suppressed by at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more by administration of an iRNA as described herein.
  • As used herein in the context of target gene expression, the terms “treat,” “treatment,” and the like, refer to relief from or alleviation of pathological processes mediated by target expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by target expression), the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression or anticipated progression of such condition.
  • By “lower” in the context of a disease marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without such disorder.
  • As used herein, the phrase “therapeutically effective amount” ” refers to an amount that provides a therapeutic benefit in the treatment or management of pathological processes mediated by target gene expression, e.g., PCSK9 and/or a second gene, e.g., XBP-1, or an overt symptom of pathological processes mediated target gene expression. The phrase “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the prevention of pathological processes mediated by target gene expression or an overt symptom of pathological processes mediated by target gene expression. The specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by target gene expression, the patient's history and age, the stage of pathological processes mediated by target gene expression, and the administration of other agents that inhibit pathological processes mediated by target gene expression.
  • As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of an iRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an iRNA effective to produce the intended pharmacological or therapeutic result. For example, if a given clinical treatment is considered effective when there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 10% reduction in that parameter.
  • The term “pharmaceutically carrier” refers to a carrier for administration of a therapeutic agent, e.g., a dual targeting siRNA agent. Carriers are described in more detail below, and include lipid formulations, e.g., LNP09 and SNALP formulations.
  • Double-Stranded Ribonucleic Acid (dsRNA)
  • Described herein are dual targeting siRNA agents, e.g., siRNAs that inhibit the expression of a PCSK9 gene and a second gene. The dual targeting siRNA agent includes two siRNA covalently linked via, e.g., a disulfide linker. The first siRNA targets a first region of a PCSK9 gene. The second siRNA targets a second gene, e.g., XBP-1, or, e.g., targets a second region of the PCSK9 gene.
  • The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Applied Biosystems, Inc. Further descriptions of synthesis are found below and in the examples.
  • Each siRNA is a dsRNA. A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of a target 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.
  • Where the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a “hairpin loop”) between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.
  • Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a “linker.”
  • Generally, the duplex structure of the siRNA, e.g., dsRNA, is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Considering a duplex between 9 and 36 base pairs, the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs.
  • The two siRNAs in the dual targeting siRNA agent can have duplex lengths that are identical or that differ.
  • The region of complementarity to the target sequence in an siRNA is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive. In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. The region of complementarity can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. As non-limiting examples, the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides. In some embodiments the target sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
  • The two siRNAs in the dual targeting siRNA agent can have regions of complementarity that are identical in length or that differ in length.
  • Any of the dsRNA, e.g., siRNA as described herein may include one or more single-stranded nucleotide overhangs. In one embodiment, at least one end of a dsRNA has a single-stranded nucleotide overhang of 1 to 4, or 1 or 2 or 3 or 4 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA can also have a blunt end, generally located at the 5′-end of the antisense strand. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. The two siRNAs in the dual targeting siRNA agent can have different or identical overhangs as described by location, length, and nucleotide.
  • The dual targeting siRNA agent includes at least a first siRNA targeting a first region of a PCSK9 gene. In one embodiment, a PCSK9 gene is a human PCSK9 gene. In another embodiment the PCSK9 gene is a mouse or a rat PCSK9 gene. Exemplary siRNA targeting PCSK9 are described in U.S. patent application Ser. No. 11/746,864 filed on May 10, 2007 (now U.S. Pat. No. 7,605,251) and International Patent Application No. PCT/US2007/068655 filed May 10, 2007 (published as WO 2007/134161). Additional disclosure can be found in U.S. patent application Ser. No. 12/478,452 filed Jun. 4, 2009 (published as US 2010/0010066) and International Patent Application No. PCT/US2009/032743 filed Jan. 30, 2009 (published as WO 2009/134487). The sequences of the target, sense, and antisense strands are incorporated by reference for all purposes.
  • Tables 1, 2, and 4-8 disclose sequences of the target, sense strands, and antisense strands of PCSK9 targeting siRNA.
  • In one embodiment the first siRNA is AD-9680. The dsRNA AD-9680 targets the human PCSK 9 gene at nucleotides 3530-3548 of a human PCSK9 gene (accession number NM_174936).
  • TABLE 1
    AD-9680 siRNA sequences
    SEQ
    ID
    Table 1: AD-9680 Sequence 5′ to 3′ NO:
    Target sequence UUCUAGACCUGUUUUGCUU 4142
    Sense strand UUCUAGACCUGUUUUGCUU 4143
    Sense strand, uucuAGAccuGuuuuGcuuTsT 4144
    modified
    Antisense strand AAGCAAAACAGGUCUAGAA 4145
    Antisense strand, AAGcAAAAcAGGUCuAGAATsT 4146
    modified
  • In another embodiment, the first siRNA is AD-10792. The dsRNA AD-10792 targets the PCSK9 gene at nucleotides 1091-1109 of a human PCSK9 gene (accession number NM 174936). AD-10792 is also complementary to rodent PCSK9.
  • TABLE 2
    AD-10792 siRNA sequences
    SEQ
    ID
    Table 2: AD-10792 Sequence 5′ to 3′ NO:
    Target sequence GCCUGGAGUUUAUUCGGAA 4147
    Sense strand GCCUGGAGUUUAUUCGGAA 4148
    Sense strand, GccuGGAGuuuAuucGGAATsT 4149
    modified
    Antisense strand UUCCGAAUAAACUCCAGGC 4150
    Antisense strand, UUCCGAAuAAACUCcAGGCTsT 4151
    modified
  • The second siRNA of the dual targeting siRNA agent targets a second gene. In one embodiment, the second gene is PCSK9, and the second siRNA target a region of PCSK9 that is different from the region targeted by the first siRNA.
  • Alternatively, the second siRNA targets a different second gene. Examples include genes that interact with PCSK9 and/or are involved with lipid metabolism or cholesterol metabolism. For example, the second target gene can be XBP-1, PCSK5, ApoC3, SCAP, MIG12, HMG CoA Reductase, or IDOL (Inducible Degrader of the LDLR) and the like. In one embodiment, the second gene is a human gene. In another embodiment the second gene is a mouse or a rat gene.
  • In one embodiment, the second siRNA targets the XBP-1 gene. Exemplary siRNA targeting XBP-1 can be found in U.S. patent application Ser. No. 12/425,811 filed Apr. 17, 2009 (published as US 2009-0275638). The sequences of the target, sense, and antisense strands are incorporated by reference for all purposes.
  • Tables 3 and 9-13 disclose sequences of the target, sense strands, and antisense strands of XBP-1 targeting siRNA.
  • In one embodiment the first siRNA is AD-18038. The dsRNA AD-18038 targets the human XBP-1 gene at nucleotides 896-914 of a human XBP-1 gene (accession number NM_001004210).
  • TABLE 3
    AD-18038 siRNA sequences
    SEQ
    ID
    Table 3: AD-18038 Sequence 5′ to 3′ NO:
    Target sequence CACCCUGAAUUCAUUGUCU 4153
    Sense strand CACCCUGAAUUCAUUGUCU 4154
    Sense strand, cAcccuGAAuucAuuGucudTsdT 4155
    modified
    Antisense strand AGACAAUGAAUUCAGGGUG 4156
    Antisense strand, AGAcAAUGAAUUcAGGGUGdTsdT 4157
    modified
  • Additional dsRNA
  • A dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences in Tables 1-13, and differing in their ability to inhibit the expression of a target gene by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated according to the invention.
  • In addition, the RNAs provided in Tables 1-13 identify a site in the target gene transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of such sequences. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least 15 contiguous nucleotides from one of the sequences provided herein coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a target gene.
  • While a target sequence is generally 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that may serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, above represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.
  • Further, it is contemplated that for any sequence identified, e.g., in Tables 1-13, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those and sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.
  • An iRNA as described in Tables 1-13 can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described in Tables 1-13 contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide iRNA agent RNA strand which is complementary to a region of a PCSK9 gene, the RNA strand generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a PCSK9 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of a PCSK9 gene is important, especially if the particular region of complementarity in a PCSK9 gene is known to have polymorphic sequence variation within the population.
  • Covalent Linkage
  • The dual targeting siRNA agents of the invention include two siRNAs joined via a covalent linker. Covalent linkers are well-known to one of skill in the art and include, e.g., a nucleic acid linker, a peptide linker, and the like.
  • The covalent linker joins the two siRNAs. The covalent linker can join two sense strands, two antisense strands, one sense and one antisense strand, two sense strands and one antisense strand, two antisense strands and one sense strand, or two sense and two antisense strands.
  • The covalent linker can include RNA and/or DNA and/or a peptide. The linker can be single stranded, double stranded, partially single strands, or partially double stranded. In some embodiments the linker includes a disulfide bond. The linker can be cleavable or non-cleavable.
  • The covalent linker can be, e.g., dTsdTuu=(5′-2′deoxythymidy1-3′-thiophosphate-5′-2′deoxythymidy 1-3′-phosphate-5′-uridy 1-3′-phosphate-5′-uridy 1-3′-phosphate); rUsrU (a thiophosphate linker: 5′-uridy1-3′-thiophosphate-5′-uridy1-3′-phosphate); an rUrU linker; dTsdTaa (aadTsdT, 5′-2′deoxythymidy1-3′-thiophosphate-5′-2′deoxythymidy1-3′-phosphate-5′-adeny1-3′-phosphate-5′-adeny1-3′-phosphate); dTsdT (5′-2′deoxythymidy1-3′-thiophosphate-5′-2′ deoxythymidy1-3′-phosphate); dTsdTuu=uudTsdT=5′-2′deoxythymidy1-3′-thiophosphate-5′-2′deoxythymidy 1-3′-phosphate-5′-uridy1-3′-phosphate-5′-uridy1-3′-phosphate.
  • The covalent linker can be a polyRNA, such as poly(5′-adenyl-3′-phosphate—AAAAAAAA) or poly(5′-cytidyl-3′-phosphate-5′-uridyl-3′-phosphate—CUCUCUCU)), e.g., Xn single stranded poly RNA linker wherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive. Modified nucleotides or a mixture of nucleotides can also be present in said polyRNA linker. The covalent linker can be a polyDNA, such as poly(5′-2′deoxythymidy1-3′-phosphate—TTTTTTTT), e.g., wherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive. Modified nucleotides or a mixture of nucleotides can also be present in said polyDNA linker. a single stranded polyDNA linker wherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive. Modified nucleotides or a mixture of nucleotides can also be present in said polyDNA linker.
  • The covalent linker can include a disulfide bond, optionally a bis-hexyl-disulfide linker. In one embodiment, the disulfide linker is
  • Figure US20220213475A1-20220707-C00001
  • The covalent linker can include a peptide bond, e.g., include amino acids. In one embodiment, the covalent linker is a 1-10 amino acid long linker, preferably comprising 4-5 amino acids, optionally X-Gly-Phe-Gly-Y wherein X and Y represent any amino acid.
  • The covalent linker can include HEG, a hexaethylenglycol linker.
  • Modifications
  • In yet another embodiment, at least one of the siRNA of the dual targeting siRNA agent is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in this invention include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.
  • Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.
  • Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. No. RE39464, each of which is herein incorporated by reference
  • Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
  • Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.
  • In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.
  • Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2—[known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2—[wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
  • Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, 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, also described in examples herein below.
  • Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.
  • An iRNA may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
  • Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.
  • The RNA of an iRNA can also be modified to include one or more 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).
  • Representative U.S. Patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is herein incorporated by reference in its entirety.
  • 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 U.S. Provisional Patent Application No. 61/223,665 (“the '665 application”), filed Jul. 7, 2009, entitled “Oligonucleotide End Caps” and International patent application no. PCT/US10/41214, filed Jul. 7, 2010.
  • Ligands
  • Another modification of the RNA of an iRNA featured in the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
  • In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred 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. Preferred 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 alpha 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-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.
  • Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.
  • Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as 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-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.
  • The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
  • In one ligand, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., 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, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
  • A lipid based ligand can be used to modulate, e.g., control 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 a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
  • In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. 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).
  • In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has 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:4158). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:4159)) 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:4160)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:4161)) 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). Preferably 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 moiety can be used to target 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). Preferably, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver a iRNA agent to a tumor cell expressing αvß3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).
  • A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).
  • Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which is herein incorporated by reference.
  • Chimeras
  • It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds. “Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
  • Non-Ligand Groups
  • 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 1,2d-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.
  • Delivery of iRNA
  • The delivery of an iRNA to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be performed directly by administering a composition comprising an iRNA, e.g. a dsRNA, to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.
  • Direct Delivery
  • In general, any method of delivering a nucleic acid molecule can be adapted for use with an iRNA (see e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). However, there are three factors that are important to consider in order to successfully deliver an iRNA molecule in vivo: (a) biological stability of the delivered molecule, (2) preventing non-specific effects, and (3) accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example by direct injection or implantation into a tissue (as a non-limiting example, a tumor) or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that may otherwise be harmed by the agent or that may degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, 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 (Dom, 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 an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O., et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim SH., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.
  • Vector Encoded dsRNAs
  • In another aspect, the dsRNAs of the invention can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillem, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).
  • The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
  • iRNA 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 an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
  • iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
  • Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picomavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.
  • Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.
  • Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.
  • In a specific embodiment, viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitates delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.
  • Adenoviruses are also contemplated for use in delivery of iRNAs. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing an iRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
  • Use of Adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
  • Another preferred viral vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.
  • The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
  • The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • Pharmaceutical Compositions Containing iRNA
  • In one embodiment, the invention provides pharmaceutical compositions containing a dual targeting siRNA agent, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the siRNA is useful for treating a disease or disorder associated with the expression or activity of a target gene, such as pathological processes mediated by PCSK9 expression. 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) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion.
  • The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of the target genes. In general, a suitable dose of siRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21 mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30 mg/kg, 31 mg/kg, 32 mg/kg, 33 mg/kg, 34 mg/kg, 35 mg/kg, 36 mg/kg, 37 mg/kg, 38 mg/kg, 39 mg/kg, 40 mg/kg, 41 mg/kg, 42 mg/kg, 43 mg/kg, 44 mg/kg, 45 mg/kg, 46 mg/kg, 47 mg/kg, 48 mg/kg, 49 mg/kg, or 50 mg/kg per single dose.
  • The pharmaceutical composition may be administered once daily, or the iRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
  • The effect of a single dose of siRNA on PCSK9 levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.
  • The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
  • Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by PCSK9 expression. Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a transgene expressing human PCSK9.
  • The present invention also includes pharmaceutical compositions and formulations that include the iRNA compounds featured in the invention. The pharmaceutical compositions of the present invention 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 (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 iRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).
  • Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or a C1-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.
  • Liposomal Formulations
  • There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
  • Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
  • In order to traverse intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
  • Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
  • Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
  • Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
  • Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis
  • Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
  • Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).
  • One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).
  • Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).
  • Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).
  • Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).
  • Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
  • A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.
  • Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
  • Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
  • If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
  • If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
  • If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
  • If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
  • The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
  • Nucleic Acid Lipid Particles
  • In one embodiment, a dual targeting siRNA agent featured in the invention is fully encapsulated in the lipid formulation, e.g., to form a nucleic acid-lipid particle, e.g., a SPLP, pSPLP, or SNALP. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. Nucleic acid-lipid particles, e.g., SNALPs, typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP”, which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683.
  • The particles of the present invention 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. For example, the mean diameter of the particles can be about 50 nm, 55 nm, 60 nn, 65 nn, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 140 nm, 145 nm, or 150 nm.
  • In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.
  • In 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. The lipid to dsRNA ratio can be about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 113:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, or 50:1.
  • The nucleic acid lipid particles include a cationic lipid. The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC), (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1, e.g., C12-200), or a mixture thereof. The cationic lipid may comprise from about 10 mol % to about 70 mol % or about 40 mol % of the total lipid present in the particle. The cationic lipid may comprise 10 mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol %, 70 mol %, 75 mol %, 80 mol %, 85 mol %, 90 mol %, or 95 mol % of the total lipid present in the particle. The cationic lipid may comprise 57.1 mol % or 57.5 mol % of the total lipid present in the particle.
  • In one embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC) can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.
  • In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.
  • The nucleic acid lipid particle generally includes a non-cationic lipid. The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof.
  • The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle. The non-cationic lipid may be about 5 mol %, 6 mol %, 7 mol %, 7.5 mol %, 7.7 mol %, 8 mol %. 9 mol %, 10 mol %, 11 mol %, 12 mol %, 13 mol %, 14 mol %, 15 mol %, 16 mol %, 17 mol %, 18 mol %, 19 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol %, 70 mol %, 75 mol %, 80 mol %, 85 mol %, 90 mol %, or 95 mol %.
  • The nucleic acid lipid particle generally includes a conjugated lipid. The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or a PEG-distearyloxypropyl (C]8). The conjugated lipid can be 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); or PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000).
  • The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0 17.0, 18, 19.0 or 20.0 mol % of the total lipid present in the particle.
  • In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle. For example, the nucleic acid-lipid particle further includes cholesterol at about 5 mol %, 10 mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol %. The nucleic acid-lipid particle can include cholesterol at about 31.5 mol %, 34.4 mol %, 35 mol %, 38.5 mol %, or 40 mol % of the total lipid present in the particle.
  • LNP01
  • In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is herein incorporated by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.
  • Figure US20220213475A1-20220707-C00002
  • LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.
  • Exemplary Nucleic Acid Lipid Particles
  • Additional exemplary lipid-dsRNA formulations arm as follows:
  • TABLE A
    cationic lipid/non-cationic
    lipid/cholesterol/PEG-lipid conjugate
    Cationic Mol % ratios
    Lipid Lipid:siRNA ratio
    SNALP DLinDMA DLinDMA/DPPC/Cholesterol/PEG-cDMA
    (57.1/7.1/34.4/1.4)
    lipid:siRNA ~ 7:1
    S-XTC XTC XTC/DPPC/Cholesterol/PEG-cDMA
    57.1/7.1/34.4/1.4
    lipid:siRNA ~ 7:1
    LNP05 XTC XTC/DSPC/Cholesterol/PEG-DMG
    57.5/7.5/31.5/3.5
    lipid:siRNA ~ 6:1
    LNP06 XTC XTC/DSPC/Cholesterol/PEG-DMG
    57.5/7.5/31.5/3.5
    lipid:siRNA ~ 11:1
    LNP07 XTC XTC/DSPC/Cholesterol/PEG-DMG
    60/7.5/31/1.5,
    lipid:siRNA ~ 6:1
    LNP08 XTC XTC/DSPC/Cholesterol/PEG-DMG
    60/7.5/31/1.5,
    lipid:siRNA ~ 11:1
    LNP09 XTC XTC/DSPC/Cholesterol/PEG-DMG
    50/10/38.5/1.5
    Lipid:siRNA 10:1
    LNP10 ALN100 ALN100/DSPC/Cholesterol/PEG-DMG
    50/10/38.5/1.5
    Lipid:siRNA 10:1
    LNP11 MC3 MC-3/DSPC/Cholesterol/PEG-DMG
    50/10/38.5/1.5
    Lipid:siRNA 10:1
    LNP12 C12-200 C12-200/DSPC/Cholesterol/PEG-DMG
    50/10/38.5/1.5
    Lipid:siRNA 10:1
    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
  • SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.
  • XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International patent application no. PCT/US10/22614, filed Jan. 29, 2010, which are hereby incorporated by reference.
  • MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, and U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, which are hereby incorporated by reference.
  • ALN100, i.e., ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.
  • C12-200, i.e., Tech G1 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009, which is hereby incorporated by reference.
  • Synthesis of Cationic Lipids.
  • Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention may be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.
  • “Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.
  • “Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.
  • “Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.
  • “Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.
  • “Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
  • The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy.
  • “Halogen” means fluoro, chloro, bromo and iodo.
  • In some embodiments, the methods of the invention may require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.
  • Synthesis of Formula A
  • In one embodiments, nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A; XTC is a cationic lipid of formula A:
  • Figure US20220213475A1-20220707-C00003
  • where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring.
  • In general, the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.
  • Figure US20220213475A1-20220707-C00004
  • Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.
  • Figure US20220213475A1-20220707-C00005
  • Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.
  • Synthesis of MC3
  • Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).
  • Synthesis of ALNY-100
  • Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:
  • Figure US20220213475A1-20220707-C00006
  • Synthesis of 515:
  • To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0 0C under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0 0C and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).
  • Synthesis of 516:
  • To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0 0C under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO3solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H] −232.3 (96.94%).
  • Synthesis of 517A and 517B:
  • The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (˜3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (lx 50 mL). Organic phase was dried over an. Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: −6 g crude
  • 517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS—[M+H]-266.3, [M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.
  • Synthesis of 518:
  • Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.
  • General Procedure for the Synthesis of Compound 519:
  • A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR □=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6, Found 654.6.
  • General Synthesis of Nucleic Add Lipid Particles
  • Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.
  • Other Formulations
  • Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcamitine, 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 invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyomithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.
  • Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.
  • The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
  • Additional Formulations
  • Emulsions
  • The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, 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 may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
  • Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 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, LV., Popovich NG., and Ansel HC., 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 may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 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 may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 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, LV., Popovich NG., and Ansel HC., 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.
  • In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 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, LV., Popovich NG., and Ansel HC., 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 may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.
  • Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
  • Penetration Enhancers
  • In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
  • Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, 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: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, 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).
  • Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcamitines, 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).
  • Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, 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: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, 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 (enaminesxsee 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 Rel., 1990, 14, 43-51).
  • Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
  • Agents that enhance uptake of iRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-CT™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invivogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.
  • Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
  • Carriers
  • Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.
  • 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 may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
  • Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
  • Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • Other Components
  • The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, 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 invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • Aqueous suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
  • In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more biologic agents which function by a non-RNAi mechanism. Examples of such biologics include, biologics that target one or more of PD-1, PD-L1, or B7-H1 (CD80) (e.g., monoclonal antibodies against PD-1, PD-L1, or B7-H1), or one or more recombinant cytokines (e.g., IL6, IFN-γ, and TNF).
  • 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 in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • In addition to their administration, as discussed above, the dual targeting siRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by PCSK9 expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
  • Method Using Dual Targeting siRNAs
  • In one aspect, the invention provides use of a dual targeting siRNA agent for inhibiting the expression of the PCSK9 gene in a mammal. The method includes administering a composition of the invention to the mammal such that expression of the target PCSK9 gene is decreased. In some embodiments, PCSK9 expression is decreased for an extended duration, e.g., at least one week, two weeks, three weeks, or four weeks or longer. For example, in certain instances, expression of the PCSK9 gene is suppressed by at least about 5%, 10%, 15%,
    Figure US20220213475A1-20220707-P00002
    25%, 30%, 35%, 40%, 45%, or 50% by administration of a dual targeting siRNA agent described herein. In some embodiments, the PCSK9 gene is suppressed by at least about 60%, 70%, or 80% by administration of the dual targeting siRNA agent. In some embodiments, the
    Figure US20220213475A1-20220707-P00003
    is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide.
  • The methods and compositions described herein can be used to treat diseases and conditions that can be modulated by down regulating PCSK9 gene expression. For example, the compositions described herein can be used to treat hyperlipidemia and other forms of lipid imbalance such as hypercholesterolemia, hypertriglyceridemia and the pathological conditions associated with these disorders such as heart and circulatory diseases
  • Therefore, the invention also relates to the use of a dual targeting siRNA agent for the treatment of a PCSK9-mediated disorder or disease. For example, a dual targeting siRNA agent is used for treatment of a hyperlipidemia.
  • The effect of the decreased PCSK9 gene preferably results in a decrease in LDLc (low density lipoprotein cholesterol) levels in the blood, and more particularly in the serum, of the mammal. In some embodiments, LDLc levels are decreased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.
  • The method includes administering a dual targeting siRNA agent to the subject 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 or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, and airway (aerosol) administration. In some embodiments, the compositions are administered by intravenous infusion or injection.
  • The method includes administering a dual targeting siRNA agent, e.g., a dose sufficient to depress levels of PCSK9 mRNA for at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days; and optionally, administering a second single dose of dsRNA, wherein the second single dose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the first single dose is administered, thereby inhibiting the expression of the PCSK9 gene in a subject.
  • In one embodiment, doses of dual targeting siRNA agent are administered not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week. In another embodiment, the administrations can be maintained for one, two, three, or six months, or one year or longer.
  • In another embodiment, administration can be provided when Low Density Lipoprotein cholesterol (LDLc) levels reach or surpass a predetermined minimal level, such as greater than 70 mg/dL, 130 mg/dL, 150 mg/dL, 200 mVdL, 300 mg/dL, or 400 mg/dL.
  • Figure US20220213475A1-20220707-P00004
    dual targeting siRNA agent
    Figure US20220213475A1-20220707-P00005
    Figure US20220213475A1-20220707-P00006
    Figure US20220213475A1-20220707-P00007
    Figure US20220213475A1-20220707-P00008
    Figure US20220213475A1-20220707-P00009
    Figure US20220213475A1-20220707-P00010
    Figure US20220213475A1-20220707-P00011
    Figure US20220213475A1-20220707-P00012
    Figure US20220213475A1-20220707-P00013
    Figure US20220213475A1-20220707-P00014
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    Figure US20220213475A1-20220707-P00016
    Figure US20220213475A1-20220707-P00017
    Figure US20220213475A1-20220707-P00018
  • For example, a subject can be administered a therapeutic amount of dual targeting siRNA agent, such as 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The dual targeting siRNA agent can be administered by intravenous infusion over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. The administration is repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. Administration of the dual targeting siRNA agent can reduce PCSK9 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least 10%, at least 15%, at least 20%, at least 25%., at least 30%, at least 40%., at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more.
  • Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction, or for elevated lipid levels or blood pressure. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.
  • 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 dual targeting siRNA 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.
  • Additional Agents
  • In further embodiments, administration of a dual targeting siRNA agent is administered in combination an additional therapeutic agent. The dual targeting siRNA agent and an additional therapeutic agent can be administered in combination in the same composition, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.
  • Examples of additional therapeutic agents include those known to treat an agent known to treat a lipid disorders, such as hypercholesterolemia, atherosclerosis or dyslipidemia. For example, a dual targeting siRNA agent featured in the invention can be administered with, e.g., an HMG-CoA reductase inhibitor (e.g., a statin), a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist (e.g., losartan potassium, such as Merck & Co.'s Cozaar®), an acylCoA cholesterol acetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor, a cholesterol modulator, a bile acid modulator, a peroxisome proliferation activated receptor (PPAR) agonist, a gene-based therapy, a composite vascular protectant (e.g., AGI-1067, from Atherogenics), a glycoprotein IIb/IIIa inhibitor, aspirin or an aspirin-like compound, an IBAT inhibitor (e.g., S-8921, from Shionogi), a squalene synthase inhibitor, or a monocyte chemoattractant protein (MCP)-I inhibitor. Exemplary HMG-CoA reductase inhibitors include atorvastatin (Pfizer's Lipitor®/Tahor/Sortis/Torvast/Cardyl), pravastatin (Bristol-Myers Squibb's Pravachol, Sankyo's Mevalotin/Sanaprav), simvastatin (Merck's Zocor®/Sinvacor, Boehringer Ingelheim's Denan, Banyu's Lipovas), lovastatin (Merck's Mevacor/Mevinacor, Bexal's Lovastatina, Cepa; Schwarz Pharma's Liposcler), fluvastatin (Novartis' Lescol®/Locol/Lochol, Fujisawa's Cranoc, Solvay's Digaril), cerivastatin (Bayer's Lipobay/GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca's Crestor®), and pitivastatin (itavastatin/risivastatin) (Nissan Chemical, Kowa Kogyo, Sankyo, and Novartis). Exemplary fibrates include, e.g., bezafibrate (e.g., Roche's Befizal®/Cedur®/Bezalip®, Kissei's Bezatol), clofibrate (e.g., Wyeth's Atromid-S®), fenofibrate (e.g., Fournier's Lipidil/Lipantil, Abbott's Tricor®, Takeda's Lipantil, generics), gemfibrozil (e.g., Pfizer's Lopid/Lipur) and ciprofibrate (Sanofi-Synthelabo's Modalim®). Exemplary bile acid sequestrants include, e.g., cholestyramine (Bristol-Myers Squibb's Questran® and Questran Light™), colestipol (e.g., Pharmacia's Colestid), and colesevelam (Genzyme/Sankyo's WelChol™). Exemplary niacin therapies include, e.g., immediate release formulations, such as Aventis' Nicobid, Upsher-Smith's Niacor, Aventis' Nicolar, and Sanwakagaku's Perycit. Niacin extended release formulations include, e.g., Kos Pharmaceuticals' Niaspan and Upsher-Smith's SIo-Niacin. Exemplary antiplatelet agents include, e.g., aspirin (e.g., Bayer's aspirin), clopidogrel (Sanof{dot over (ι)}-Synthelabo/Bristol-Myers Squibb's Plavix), and ticlopidine (e.g., Sanof{dot over (ι)}-Synthelabo's Ticlid and Daiichi's Panaldine). Other aspirin-like compounds useful in combination with a dsRNA targeting PCSK9 include, e.g., Asacard (slow-release aspirin, by Pharmacia) and Pamicogrel (Kanebo/Angelini Ricerche/CEPA). Exemplary angiotensin-converting enzyme inhibitors include, e.g., ramipril (e.g., Aventis' Altace) and enalapril (e.g., Merck & Co.'s Vasotec). Exemplary acyl CoA cholesterol acetyltransferase (ACAT) inhibitors include, e.g., avasimibe (Pfizer), eflucimibe (BioM{acute over (ε)}rieux Pierre Fabre/Eli Lilly), CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito). Exemplary cholesterol absorption inhibitors include, e.g., ezetimibe (Merck/Schering-Plough Pharmaceuticals Zetia®) and Pamaqueside (Pfizer). Exemplary CETP inhibitors include, e.g., Torcetrapib (also called CP-529414, Pfizer), JTT-705 (Japan Tobacco), and CETi-I (Avant Immunotherapeutics). Exemplary microsomal triglyceride transfer protein (MTTP) inhibitors include, e.g., implitapide (Bayer), R-103757 (Janssen), and CP-346086 (Pfizer). Other exemplary cholesterol modulators include, e.g., NO-1886 (Otsuka/TAP Pharmaceutical), CI-1027 (Pfizer), and WAY-135433 (Wyeth-Ayerst). Exemplary bile acid modulators include, e.g., HBS-107 (Hisamitsu/Banyu), Btg-511 (British Technology Group), BARI-1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), and AZD-7806 (AstraZeneca). Exemplary peroxisome proliferation activated receptor (PPAR) agonists include, e.g., tesaglitazar (AZ-242) (AstraZeneca), Netoglitazone (MCC-555) (Mitsubishi/Johnson & Johnson), GW-409544 (Ligand Pharmaceuticals/GlaxoSmithKline), GW-501516 (Ligand Pharmaceuticals/GlaxoSmithKline), LY-929 (Ligand Pharmaceuticals and Eli Lilly), LY-465608 (Ligand Pharmaceuticals and Eli Lilly), LY-518674 (Ligand Pharmaceuticals and Eli Lilly), and MK-767 (Merck and Kyorin). Exemplary gene-based therapies include, e.g., AdGWEGF121.10 (GenVec), ApoAl (UCB Pharma/Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics), and ATP-binding cassette transporter-Al (ABCAl) (CV Therapeutics/Incyte, Aventis, Xenon). Exemplary Glycoprotein Ilb/IIIa inhibitors include, e.g., roxifiban (also called DMP754, Bristol-Myers Squibb), Gantofiban (Merck KGaA/Yamanouchi), and Cromafiban (Millennium Pharmaceuticals). Exemplary squalene synthase inhibitors include, e.g., BMS-1884941 (Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer), CP-294838 (Pfizer), and TAK-475 (Takeda). An exemplary MCP-I inhibitor is, e.g., RS-504393 (Roche Bioscience). The anti-atherosclerotic agent BO-653 (Chugai Pharmaceuticals), and the nicotinic acid derivative Nyclin (Yamanouchi Pharmaceuticals) are also appropriate for administering in combination with a dsRNA featured in the invention. Exemplary combination therapies suitable for administration with a dsRNA targeting PCSK9 include, e.g., advicor (Niacin/lovastatin from Kos Pharmaceuticals), amlodipine/atorvastatin (Pfizer), and ezetimibe/simvastatin (e.g., Vytorin® 10/10, 10/20, 10/40, and 10/80 tablets by Merck/Schering-Plough Pharmaceuticals). Agents for treating hypercholesterolemia, and suitable for administration in combination with a dsRNA targeting PCSK9 include, e.g., lovastatin, niacin Altoprev® Extended-Release Tablets (Andrx Labs), lovastatin Caduet® Tablets (Pfizer), amlodipine besylate, atorvastatin calcium Crestor® Tablets (AstraZeneca), rosuvastatin calcium Lescol® Capsules (Novartis), fluvastatin sodium Lescol® (Reliant, Novartis), fluvastatin sodium Lipitor® Tablets (Parke-Davis), atorvastatin calcium Lofibra® Capsules (Gate), Niaspan Extended-Release Tablets (Kos), niacin Pravachol Tablets (Bristol-Myers Squibb), pravastatin sodium TriCor® Tablets (Abbott), fenofibrate Vytorin® 10/10 Tablets (Merck/Schering-Plough Pharmaceuticals), ezetimibe, simvastatin WelChol™ Tablets (Sankyo), colesevelam hydrochloride Zetia® Tablets (Schering), ezetimibe Zetia® Tablets (Merck/Schering-Plough Pharmaceuticals), and ezetimibe Zocor® Tablets (Merck).
  • In one embodiment, a dual targeting siRNA agent is administered in combination with an ezetimibe/simvastatin combination (e.g., Vytorin® (Merck/Schering-Plough Pharmaceuticals)).
  • In one embodiment, the dual targeting siRNA agent is administered to the patient, and then the additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the dual targeting siRNA agent and the additional therapeutic agent are administered at the same time.
  • In another aspect, the invention features, a method of instructing an end user, e.g., a caregiver or a subject, on how to administer a dual targeting siRNA agent described herein. The method includes, optionally, providing the end user with one or more doses of the dual targeting siRNA agent, and instructing the end user to administer the dual targeting siRNA agent on a regimen described herein, thereby instructing the end user.
  • Identification of Patients
  • In one aspect, the invention provides a method of treating a patient by selecting a patient on the basis that the patient is in need of LDL lowering, LDL lowering without lowering of HDL, ApoB lowering, or total cholesterol lowering. The method includes administering to the patient a dual targeting siRNA agent in an amount sufficient to lower the patient's LDL levels or ApoB levels, e.g., without substantially lowering HDL levels.
  • Genetic predisposition plays a role in the development of target gene associated diseases, e.g., hyperlipidemia. Therefore, a patient in need of a dual targeting siRNA agent can be identified by taking a family history, or, for example, screening for one or more genetic markers or variants. A healthcare provider, such as a doctor, nurse, or family member, can take a family history before prescribing or administering a dual targeting siRNA agent. For example, a DNA test may also be performed on the patient to identify a mutation in the PCSK9 gene, before a PCSK9 dsRNA is administered to the patient.
  • 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 iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein am incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples am illustrative only and not intended to be limiting.
    • EXAMPLES Example 1. iRNA Synthesis
  • Source of Reagents
  • Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
  • Oligonucleotide Synthesis.
  • All oligonucleotides are synthesized on an AKTAoligopilot synthesizer. Commercially available controlled pore glass solid support (dT-CPG, 500{acute over (Å)}, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5′-O-dimethoxytrityl N6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, and 5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) were used for the oligonucleotide synthesis. The 2′-F phosphoramidites, 5′-O-dimethoxytrityl-N4-acetyl-2′-fluro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite and 5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite are purchased from (Promega). All phosphoramidites are used at a concentration of 0.2M in acetonitrile (CH3CN) except for guanosine which is used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of 16 minutes is used. The activator is 5-ethyl thiotetrazole (0.75M, American International Chemicals); for the PO-oxidation iodine/water/pyridine is used and for the PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) is used.
  • 3′-ligand conjugated strands are synthesized using solid support containing the corresponding ligand. For example, the introduction of cholesterol unit in the sequence is performed from a hydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered to trans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain a hydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore) labeled iRNAs are synthesized from the corresponding Quasar-570 (Cy-3) phosphoramidite are purchased from Biosearch Technologies. Conjugation of ligands to 5′-end and or internal position is achieved by using appropriately protected ligand-phosphoramidite building block. An extended 15 min coupling of 0.1 M solution of phosphoramidite in anhydrous CH3CN in the presence of 5-(ethylthio)-1H-tetrazole activator to a solid-support-bound oligonucleotide. Oxidation of the internucleotide phosphite to the phosphate is carried out using standard iodine-water as reported (1) or by treatment with tert-butyl hydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation wait time conjugated oligonucleotide. Phosphorothioate is introduced by the oxidation of phosphite to phosphorothioate by using a sulfur transfer reagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucage reagent. The cholesterol phosphoramidite is synthesized in house and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite is 16 minutes.
  • Deprotection I (Nucleobase Deprotection)
  • After completion of synthesis, the support is transferred to a 100 mL glass bottle (VWR). The oligonucleotide is cleaved from the support with simultaneous deprotection of base and phosphate groups with 80 mL of a mixture of ethanolic ammonia [ammonia: ethanol (3:1)] for 6.5 h at 55° C. The bottle is cooled briefly on ice and then the ethanolic ammonia mixture is filtered into a new 250-mL bottle. The CPG is washed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixture is then reduced to ˜30 mL by roto-vap. The mixture is then frozen on dry ice and dried under vacuum on a speed vac.
  • Deprotection II (Removal of 2′-TBDMS Group)
  • The dried residue is resuspended in 26 mL of triethylamine, triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60° C. for 90 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reaction is then quenched with 50 mL of 20 mM sodium acetate and the pH is adjusted to 6.5. Oligonucleotide is stored in a freezer until purification.
  • Analysis
  • The oligonucleotides are analyzed by high-performance liquid chromatography (HPLC) prior to purification and selection of buffer and column depends on nature of the sequence and or conjugated ligand.
  • HPLC Purification
  • The ligand-conjugated oligonucleotides are purified by reverse-phase preparative HPLC. The unconjugated oligonucleotides are purified by anion-exchange HPLC on a TSK gel column packed in house. The buffers are 20 mM sodium phosphate (pH 8.5) in 10% CH3CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH3CN, 1M NaBr (buffer B). Fractions containing full-length oligonucleotides are pooled, desalted, and lyophilized. Approximately 0.15 OD of desalted oligonucleotides are diluted in water to 150 μL and then pipetted into special vials for CGE and LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.
  • iRNA Preparation
  • For the general preparation of iRNA, equimolar amounts of sense and antisense strand are heated in 1×PBS at 95° C. for 5 min and slowly cooled to room temperature. Integrity of the duplex is confirmed by HPLC analysis.
  • Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table B.
  • TABLE B
    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
    C cytidine
    G guanosine
    U uridine
    N any nucleotide (G, A, C, T or U)
    a 2′-O-methyladenosine
    c
    2′-O-methylcytidine
    g
    2′-O-methylguanosine
    u
    2′-O-methyluridine
    dT , T 2′-deoxythymidine
    s phosphorothioate linkage
    • Example 2. PCSK9 siRNA Design, Synthesis, and Screening
  • A description of the design, synthesis, and assays using PCSK9 siRNA can be found in detail in U.S. patent application Ser. No. 11/746,864 filed on May 10, 2007 (now U.S. Pat. No. 7,605,251) and International Patent Application No. PCT/US2007/068655 filed May 10, 2007 (published as WO 2007/134161) and in U.S. patent application Ser. No. 12/478,452 filed Jun. 4, 2009 (published as US 2010/0010066) and International Patent Application No. PCT/US2009/032743 filed Jan. 30, 2009 (published as WO 2009/134487). All are incorporated by reference in their entirety for all purposes.
  • The sequences of siRNA targeting a PCSK9 gene are described in Table 1 and Table 2 above, and Tables 4-8 below.
    • Example 3. XBP-1 siRNA Design, Synthesis, and Screening
  • A description of the design, synthesis, and assays using XBP-1 siRNA can be found in detail in U.S. patent application Ser. No. 12/425,811 filed on Apr. 17, 2009 and published as US 2009-0275638. This application is incorporated by reference in its entirety for all purposes. The sequences of siRNA targeting a XBP-1 gene are described in Table 3 above, and Tables 9-13 below.
    • Example 4. A Dual Targeting siRNA Agent
  • A dual targeting siRNA agent was synthesized. The sense and antisense strands for AD-10792 (target gene is PCSK9, see Table 2)) and AD-18038 (target gene is XBP-1, see Table 3) were synthesized. The two sense strands were covalently bound using a disulfide linker “Q51” with the structure shown below.
  • Figure US20220213475A1-20220707-C00007
  • The resulting dual sense strand was hybridized to the corresponding antisense strands to create a 42 mer dual targeting siRNA agent “AD-23426” (SEQ ID NOS 4162-4165, respectively, in order of appearance):
  • GccuGGAGuuuAuucGGAAdTsdTQ51cAcccuGAAuucAuuGucudTsdT
    dTsdTCGGAcCUCAAAuAAGCCUU dTsdTGUGGGAcUUAAGUAAcAGA
    • Example 5. Inhibition of PCSK9 and Xbp-1 mRNA Levels by the PCSK9-Xbp1 Dual Targeting siRNA in Primary Mouse Hepatocytes
  • Primary mouse hepatocytes were transfected with dual targeting AD-23426 or individual siRNAs (AD-10792 and AD-18038) in lipofectamine 2000 (Invitrogen protocol). 48 hours after transfection cells were harvested and lysed. PCSK9, Xbp-1 and GAPDH transcripts were measured via bDNA in cell lysates prepared according to manufacturer's protocol. PCSK9 to GAPDH or Xbp-1 to GAPDH ratios were normalized to control (luciferase) and graphed.
  • As shown in FIG. 1, the dual targeting siRNA was at least as effective at inhibiting their corresponding target gene as the single siRNAs.
    • Example 6. Inhibition of PCSK9 and Xbp-1 mRNA Levels and Reduction of Total Serum Cholesterol by the PCSK9-Xbp1 Dual Targeting siRNA in Mice
  • The dual targeting AD-23426 was formulated in an LNP09 formulation: XTC/DSPC/Cholesterol/PEG-DMG in a % mol ratio of 50/10/38.5/1.5 with a lipid:siRNA ratio of about 10:1. The LNP09-AD-23426 was administered by tail vein injection into C57B6 mice at 6.0 mg/kg, 2.0 mg/kg and 0.6 mg/kg. LNP09 formulated single siRNAs (AD-10792 and AD-18038) were administered each at 3.0 mg/kg, 1.0 mg/kg and 0.3 mg/kg. Livers and plasma were harvested 72 hours post-injection (5 animals per group).
  • PCSK9, Xbp-1 and GAPDH transcript levels were measured via bDNA in livers prepared according to the manufacturer's protocol. PCSK9 to GAPDH or Xbp-1 to GAPDH ratios were normalized to control (luciferase) and graphed. The results are shown in FIG. 2.
  • Total cholesterol was measure in serum according to manufacturer's instructions using a cholesterol kit from WAKO Tex.
  • The results demonstrate that the dual targeting siRNAs were at least as effective at inhibiting their corresponding target as single siRNAs in vivo. The results also show that the dual targeting construct has an additive effect compared to the single siRNAs at reducing total serum cholesterol.
    • Example 7: No Induction of IFN-α and TNF-α in HuPBMC
  • The effect of a dual targeting siRNA, AD-23426, on IFN-α and TNF-α in human PBMC was investigated.
  • Whole Blood anti-coagulated with Sodium Heparin was obtained from healthy donors at Research Blood Components, Inc (Boston, Mass.). Peripheral blood mononuclear cells (PBMC) were isolated by standard Ficoll-Hypaque density centrifugation. Isolated PBMC were seeded at 1×105cells/well in 96 well plates and cultured in RPMI 1640 GlutaMax Medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic/antimycotic (Invitrogen). siRNAs were transfected using DOTAP Transfection Reagent (Roche Applied Science). DOTAP was first diluted in Opti-MEM (Invitrogen) for 5 minutes before mixing with an equal volume of Opti-MEM containing the siRNA. siRNA/transfection reagent complexes were incubated for 15 minutes at room temperature prior to being added to PBMC. siRNAs were transfected at final concentrations of 266 nM, 133 nM or 67 nM using 16 μg/ml, 8 μg/ml or 4 μg/ml DOTAP, respectively. The ratio of siRNA to DOTAP is 16.5 μmol/μg. Transfected PBMC were incubated at 37° C., 5% CO2 for 24 hrs after which supernatants were harvested and stored at −80° C. until analysis. Quantitative cytokine analysis was done using commercially available Instant ELISA Kits for IFN-α, (BMS216INST) and TNF-α (BMS223INST); both from Bender MedSystems (Vienna, Austria).
  • LNP09 and DOTAP formulated siRNAs were administered. Control siRNAs were AD-1730, AD-1955, AD-6248, AD-18889, AD-5048, and AD-18221. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA.
  • The results are shown in FIG. 4. AD-23426 did not induce production of IFN-α and TNF-α, similar to the result obtained with the single target gene siRNAs. As expected, unmodified siRNAs (AD-5048 and AD-18889) induced production of both IFN-α and TNF-α. These results demonstrate that a dual targeting siRNA does not induce an immune response.
    • Example 8. Reduction of Total Serum Cholesterol with PCSK9-Xbp1 Dual Targeting siRNA Humans
  • A human subject is treated with a pharmaceutical composition, e.g., a nucleic acid-lipid particle having a dual targeting siRNA agent.
  • At time zero, a suitable first dose of the pharmaceutical composition is subcutaneously administered to the subject. The composition is formulated as described herein. After a period of time, the subject's condition is evaluated, e.g., by measurement of total serum cholesterol. This measurement can be accompanied by a measurement of PCSK9 expression in said subject, and/or the products of the successful siRNA-targeting of PCSK9 mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
  • After treatment, the subject's condition is compared to the condition existing prior to the treatment, or relative to the condition of a similarly afflicted but untreated subject.
  • Those skilled in the art are familiar with methods and compositions in addition to those specifically set out in the present disclosure which will allow them to practice this invention to the full scope of the claims hereinafter appended.
  • TABLE 4
    Sequences of siRNA targeted to PCSK9
    SEQ Antisense SEQ
    Sense strand ID strand ID
    *Target (5′-3′)1 NO: (5′-3′)1 NO: Duplex #
     2-20 AGCGACGUCGAGGCGCUCATT 1 UGAGCGCCUCGAC 2 AD-15220
    GUCGCUTT
    15-33 CGCUCAUGGUUGCAGGCGGTT 3 CCGCCUGCAACCA 4 AD-15275
    UGAGCGTT
    16-34 GCUCAUGGUUGCAGGCGGGTT 5 CCCGCCUGCAACC 6 AD-15301
    AUGAGCTT
    30-48 GCGGGCGCCGCCGUUCAGUTT 7 ACUGAACGGCGGC 8 AD-15276
    GCCCGCTT
    31-49 CGGGCGCCGCCGUUCAGUUTT 9 AACUGAACGGCGG 10 AD-15302
    CGCCCGTT
    32-50 GGGCGCCGCCGUUCAGUUCTT 11 GAACUGAACGGCG 12 AD-15303
    GCGCCCTT
    40-58 CCGUUCAGUUCAGGGUCUGTT 13 CAGACCCUGAACU 14 AD-15221
    GAACGGTT
    43-61 UUCAGUUCAGGGUCUGAGCTT 15 GCUCAGACCCUGA 16 AD-15413
    ACUGAATT
     82-100 GUGAGACUGGCUCGGGCGGTT 17 CCGCCCGAGCCAG 18 AD-15304
    UCUCACTT
    100-118 GGCCGGGACGCGUCGUUGCTT 19 GCAACGACGCGUC 20 AD-15305
    CCGGCCTT
    101-119 GCCGGGACGCGUCGUUGCATT 21 UGCAACGACGCGU 22 AD-15306
    CCCGGCTT
    102-120 CCGGGACGCGUCGUUGCAGTT 23 CUGCAACGACGCG 24 AD-15307
    UCCCGGTT
    105-123 GGACGCGUCGUUGCAGCAGTT 25 CUGCUGCAACGAC 26 AD-15277
    GCGUCCTT
    135-153 UCCCAGCCAGGAUUCCGCGTsT 27 CGCGGAAUCCUGG 28 AD-9526
    CUGGGATsT
    135-153 ucccAGccAGGAuuccGcGTsT 29 CGCGGAAUCCUGG 30 AD-9652
    CUGGGATsT
    136-154 CCCAGCCAGGAUUCCGCGCTsT 31 GCGCGGAAUCCUG 32 AD-9519
    GCUGGGTsT
    136-154 cccAGccAGGAuuccGcGcTsT 33 GCGCGGAAUCCUG 34 AD-9645
    GCUGGGTsT
    138-156 CAGCCAGGAUUCCGCGCGCTsT 35 GCGCGCGGAAUCC 36 AD-9523
    UGGCUGTsT
    138-156 cAGccAGGAuuccGcGcGcTsT 37 GCGCGCGGAAUCC 38 AD-9649
    UGGCUGTsT
    185-203 AGCUCCUGCACAGUCCUCCTsT 39 GGAGGACUGUGCA 40 AD-9569
    GGAGCUTsT
    185-203 AGcuccuGcAcAGuccuccTsT 41 GGAGGACUGUGcA 42 AD-9695
    GGAGCUTsT
    205-223 CACCGCAAGGCUCAAGGCGTT 43 CGCCUUGAGCCUU 44 AD-15222
    GCGGUGTT
    208-226 CGCAAGGCUCAAGGCGCCGTT 45 CGGCGCCUUGAGC 46 AD-15278
    CUUGCGTT
    210-228 CAAGGCUCAAGGCGCCGCCTT 47 GGCGGCGCCUUGA 48 AD-15178
    GCCUUGTT
    232-250 GUGGACCGCGCACGGCCUCTT 49 GAGGCCGUGCGCG 50 AD-15308
    GUCCACTT
    233-251 UGGACCGCGCACGGCCUCUTT 51 AGAGGCCGUGCGC 52 AD-15223
    GGUCCATT
    234-252 GGACCGCGCACGGCCUCUATT 53 UAGAGGCCGUGCG 54 AD-15309
    CGGUCCTT
    235-253 GACCGCGCACGGCCUCUAGTT 55 CUAGAGGCCGUGC 56 AD-15279
    GCGGUCTT
    236-254 ACCGCGCACGGCCUCUAGGTT 57 CCUAGAGGCCGUG 58 AD-15194
    CGCGGUTT
    237-255 CCGCGCACGGCCUCUAGGUTT 59 ACCUAGAGGCCGU 60 AD-15310
    GCGCGGTT
    238-256 CGCGCACGGCCUCUAGGUCTT 61 GACCUAGAGGCCG 62 AD-15311
    UGCGCGTT
    239-257 GCGCACGGCCUCUAGGUCUTT 63 AGACCUAGAGGCC 64 AD-15392
    GUGCGCTT
    240-258 CGCACGGCCUCUAGGUCUCTT 65 GAGACCUAGAGGC 66 AD-15312
    CGUGCGTT
    248-266 CUCUAGGUCUCCUCGCCAGTT 67 CUGGCGAGGAGAC 68 AD-15313
    CUAGAGTT
    249-267 UCUAGGUCUCCUCGCCAGGTT 69 CCUGGCGAGGAGA 70 AD-15280
    CCUAGATT
    250-268 CUAGGUCUCCUCGCCAGGATT 71 UCCUGGCGAGGAG 72 AD-15267
    ACCUAGTT
    252-270 AGGUCUCCUCGCCAGGACATT 73 UGUCCUGGCGAGG 74 AD-15314
    AGACCUTT
    258-276 CCUCGCCAGGACAGCAACCTT 75 GGUUGCUGUCCUG 76 AD-15315
    GCGAGGTT
    300-318 CGUCAGCUCCAGGCGGUCCTsT 77 GGACCGCCUGGAG 78 AD-9624
    CUGACGTsT
    300-318 cGucAGcuccAGGcGGuccTsT 79 GGACCGCCUGGAG 80 AD-9750
    CUGACGTsT
    301-319 GUCAGCUCCAGGCGGUCCUTsT 81 AGGACCGCCUGGA 82 AD-9623
    GCUGACTsT
    301-319 GucAGcuccAGGcGGuccuTsT 83 AGGACCGCCUGGA 84 AD-9749
    GCUGACTsT
    370-388 GGCGCCCGUGCGCAGGAGGTT 85 CCUCCUGCGCACG 86 AD-15384
    GGCGCCTT
    408-426 GGAGCUGGUGCUAGCCUUGTsT 87 CAAGGCUAGCACC 88 AD-9607
    AGCUCCTsT
    408-426 GGAGcuGGuGcuAGccuuGTsT 89 cAAGGCuAGcACc 90 AD-9733
    AGCUCCTsT
    411-429 GCUGGUGCUAGCCUUGCGUTsT 91 ACGCAAGGCUAGC 92 AD-9524
    ACCAGCTsT
    411-429 GcuGGuGcuAGccuuGcGuTsT 93 ACGcAAGGCuAGc 94 AD-9650
    ACcAGCTsT
    412-430 CUGGUGCUAGCCUUGCGUUTsT 95 AACGCAAGGCUAG 96 AD-9520
    CACCAGTsT
    412-430 CUGGUGCUAGCCUUGCGUUTsT 97 AACGCAAGGCUAG 98 AD-9520
    CACCAGTsT
    412-430 cuGGuGcuAGccuuGcGuuTsT 99 AACGcAAGGCuAG 100 AD-9646
    cACcAGTsT
    416-434 UGCUAGCCUUGCGUUCCGATsT 101 UCGGAACGCAAGG 102 AD-9608
    CUAGCATsT
    416-434 uGcuAGccuuGcGuuccGATsT 103 UCGGAACGcAAGG 104 AD-9734
    CuAGcATsT
    419-437 UAGCCUUGCGUUCCGAGGATsT 105 UCCUCGGAACGCA 106 AD-9546
    AGGCUATsT
    419-437 uAGccuuGcGuuccGAGGATsT 107 UCCUCGGAACGcA 108 AD-9672
    AGGCuATsT
    439-457 GACGGCCUGGCCGAAGCACTT 109 GUGCUUCGGCCAG 110 AD-15385
    GCCGUCTT
    447-465 GGCCGAAGCACCCGAGCACTT 111 GUGCUCGGGUGCU 112 AD-15393
    UCGGCCTT
    448-466 GCCGAAGCACCCGAGCACGTT 113 CGUGCUCGGGUGC 114 AD-15316
    UUCGGCTT
    449-467 CCGAAGCACCCGAGCACGGTT 115 CCGUGCUCGGGUG 116 AD-15317
    CUUCGGTT
    458-476 CCGAGCACGGAACCACAGCTT 117 GCUGUGGUUCCGU 118 AD-15318
    GCUCGGTT
    484-502 CACCGCUGCGCCAAGGAUCTT 119 GAUCCUUGGCGCA 120 AD-15195
    GCGGUGTT
    486-504 CCGCUGCGCCAAGGAUCCGTT 121 CGGAUCCUUGGCG 122 AD-15224
    CAGCGGTT
    487-505 CGCUGCGCCAAGGAUCCGUTT 123 ACGGAUCCUUGGC 124 AD-15188
    GCAGCGTT
    489-507 CUGCGCCAAGGAUCCGUGGTT 125 CCACGGAUCCUUG 126 AD-15225
    GCGCAGTT
    500-518 AUCCGUGGAGGUUGCCUGGTT 127 CCAGGCAACCUCC 128 AD-15281
    ACGGAUTT
    509-527 GGUUGCCUGGCACCUACGUTT 129 ACGUAGGUGCCAG 130 AD-15282
    GCAACCTT
    542-560 AGGAGACCCACCUCUCGCATT 131 UGCGAGAGGUGGG 132 AD-15319
    UCUCCUTT
    543-561 GGAGACCCACCUCUCGCAGTT 133 CUGCGAGAGGUGG 134 AD-15226
    GUCUCCTT
    544-562 GAGACCCACCUCUCGCAGUTT 135 ACUGCGAGAGGUG 136 AD-15271
    GGUCUCTT
    549-567 CCACCUCUCGCAGUCAGAGTT 137 CUCUGACUGCGAG 138 AD-15283
    AGGUGGTT
    552-570 CCUCUCGCAGUCAGAGCGCTT 139 GCGCUCUGACUGC 140 AD-15284
    GAGAGGTT
    553-571 CUCUCGCAGUCAGAGCGCATT 141 UGCGCUCUGACUG 142 AD-15189
    CGAGAGTT
    554-572 UCUCGCAGUCAGAGCGCACTT 143 GUGCGCUCUGACU 144 AD-15227
    GCGAGATT
    555-573 CUCGCAGUCAGAGCGCACUTsT 145 AGUGCGCUCUGAC 146 AD-9547
    UGCGAGTsT
    555-573 cucGcAGucAGAGcGcAcuTsT 147 AGUGCGCUCUGAC 148 AD-9673
    UGCGAGTsT
    558-576 GCAGUCAGAGCGCACUGCCTsT 149 GGCAGUGCGCUCU 150 AD-9548
    GACUGCTsT
    558-576 GcAGucAGAGcGcAcuGccTsT 151 GGcAGUGCGCUCU 152 AD-9674
    GACUGCTsT
    606-624 GGGAUACCUCACCAAGAUCTsT 153 GAUCUUGGUGAGG 154 AD-9529
    UAUCCCTsT
    606-624 GGGAuAccucAccAAGAucTsT 155 GAUCUUGGUGAGG 156 AD-9655
    uAUCCCTsT
    659-677 UGGUGAAGAUGAGUGGCGATsT 157 UCGCCACUCAUCU 158 AD-9605
    UCACCATsT
    659-677 uGGuGAAGAuGAGuGGcGATsT 159 UCGCcACUcAUCU 160 AD-9731
    UcACcATsT
    663-681 GAAGAUGAGUGGCGACCUGTsT 161 CAGGUCGCCACUC 162 AD-9596
    AUCUUCTsT
    663-681 GAAGAuGAGuGGcGAccuGTsT 163 cAGGUCGCcACUc 164 AD-9722
    AUCUUCTsT
    704-722 CCCAUGUCGACUACAUCGATsT 165 UCGAUGUAGUCGA 166 AD-9583
    CAUGGGTsT
    704-722 cccAuGucGAcuAcAucGATsT 167 UCGAUGuAGUCGA 168 AD-9709
    cAUGGGTsT
    718-736 AUCGAGGAGGACUCCUCUGTsT 169 CAGAGGAGUCCUC 170 AD-9579
    CUCGAUTsT
    718-736 AucGAGGAGGAcuccucuGTsT 171 cAGAGGAGUCCUC 172 AD-9705
    CUCGAUTsT
    758-776 GGAACCUGGAGCGGAUUACTT 173 GUAAUCCGCUCCA 174 AD-15394
    GGUUCCTT
    759-777 GAACCUGGAGCGGAUUACCTT 175 GGUAAUCCGCUCC 176 AD-15196
    AGGUUCTT
    760-778 AACCUGGAGCGGAUUACCCTT 177 GGGUAAUCCGCUC 178 AD-15197
    CAGGUUTT
    777-795 CCCUCCACGGUACCGGGCGTT 179 CGCCCGGUACCGU 180 AD-15198
    GGAGGGTT
    782-800 CACGGUACCGGGCGGAUGATsT 181 UCAUCCGCCCGGU 182 AD-9609
    ACCGUGTsT
    782-800 cAcGGuAccGGGcGGAuGATsT 183 UcAUCCGCCCGGu 184 AD-9735
    ACCGUGTsT
    783-801 ACGGUACCGGGCGGAUGAATsT 185 UUCAUCCGCCCGG 186 AD-9537
    UACCGUTsT
    783-801 AcGGuAccGGGcGGAuGAATsT 187 UUcAUCCGCCCGG 188 AD-9663
    uACCGUTsT
    784-802 CGGUACCGGGCGGAUGAAUTsT 189 AUUCAUCCGCCCG 190 AD-9528
    GUACCGTsT
    784-802 cGGuAccGGGcGGAuGAAuTsT 191 AUUcAUCCGCCCG 192 AD-9654
    GuACCGTsT
    785-803 GGUACCGGGCGGAUGAAUATsT 193 UAUUCAUCCGCCC 194 AD-9515
    GGUACCTsT
    785-803 GGuAccGGGcGGAuGAAuATsT 195 uAUUcAUCCGCCC 196 AD-9641
    GGuACCTsT
    786-804 GUACCGGGCGGAUGAAUACTsT 197 GUAUUCAUCCGCC 198 AD-9514
    CGGUACTsT
    786-804 GuAccGGGcGGAuGAAuAcTsT 199 GuAUUcAUCCGCC 200 AD-9640
    CGGuACTsT
    788-806 ACCGGGCGGAUGAAUACCATsT 201 UGGUAUUCAUCCG 202 AD-9530
    CCCGGUTsT
    788-806 AccGGGcGGAuGAAuAccATsT 203 UGGuAUUcAUCCG 204 AD-9656
    CCCGGUTsT
    789-807 CCGGGCGGAUGAAUACCAGTsT 205 CUGGUAUUCAUCC 206 AD-9538
    GCCCGGTsT
    789-807 ccGGGcGGAuGAAuAccAGTsT 207 CUGGuAUUcAUCC 208 AD-9664
    GCCCGGTsT
    825-843 CCUGGUGGAGGUGUAUCUCTsT 209 GAGAUACACCUCC 210 AD-9598
    ACCAGGTsT
    825-843 ccuGGuGGAGGuGuAucucTsT 211 GAGAuAcACCUCc 212 AD-9724
    ACcAGGTsT
    826-844 CUGGUGGAGGUGUAUCUCCTsT 213 GGAGAUACACCUC 214 AD-9625
    CACCAGTsT
    826-844 cuGGuGGAGGuGuAucuccTsT 215 GGAGAuAcACCUC 216 AD-9751
    cACcAGTsT
    827-845 UGGUGGAGGUGUAUCUCCUTsT 217 AGGAGAUACACCU 218 AD-9556
    CCACCATsT
    827-845 uGGuGGAGGuGuAucuccuTsT 219 AGGAGAuAcACCU 220 AD-9682
    CcACcATsT
    828-846 GGUGGAGGUGUAUCUCCUATsT 221 UAGGAGAUACACC 222 AD-9539
    UCCACCTsT
    828-846 GGuGGAGGuGuAucuccuATsT 223 uAGGAGAuAcACC 224 AD-9665
    UCcACCTsT
    831-849 GGAGGUGUAUCUCCUAGACTsT 225 GUCUAGGAGAUAC 226 AD-9517
    ACCUCCTsT
    831-849 GGAGGuGuAucuccuAGAcTsT 227 GUCuAGGAGAuAc 228 AD-9643
    ACCUCCTsT
    833-851 AGGUGUAUCUCCUAGACACTsT 229 GUGUCUAGGAGAU 230 AD-9610
    ACACCUTsT
    833-851 AGGuGuAucuccuAGAcAcTsT 231 GUGUCuAGGAGAu 232 AD-9736
    AcACCUTsT
    833-851 AfgGfuGfuAfuCfuCfcUfaG 233 P*gUfgUfcUfaG 234 AD-14681
    faCfaCfTsT fgAfgAfuAfcAf
    cCfuTsT
    833-851 AGGUfGUfAUfCfUfCfCfUfA 235 GUfGUfCfUfAGG 236 AD-14691
    GACfACfTsT AGAUfACfACfCf
    UfTsT
    833-851 AgGuGuAuCuCcUaGaCaCTsT 237 P*gUfgUfcUfaG 238 AD-14701
    fgAfgAfuAfcAf
    cCfuTsT
    833-851 AgGuGuAuCuCcUaGaCaCTsT 239 GUfGUfCfUfAGG 240 AD-14711
    AGAUfACfACfCf
    UfTsT
    833-851 AfgGfuGfuAfuCfuCfcUfaG 241 GUGUCuaGGagAU 242 AD-14721
    faCfaCfTsT ACAccuTsT
    833-851 AGGUfGUfAUfCfUfCfCfUfA 243 GUGUCuaGGagAU 244 AD-14731
    GACfACfTsT ACAccuTsT
    833-851 AgGuGuAuCuCcUaGaCaCTsT 245 GUGUCuaGGagAU 246 AD-14741
    ACAccuTsT
    833-851 GfcAfcCfcUfcAfuAfgGfcC 247 P*uCfcAfgGfcC 248 AD-15087
    fuGfgAfTsT fuAfuGfaGfgGf
    uGfcTsT
    833-851 GCfACfCfCfUfCfAUfAGGCf 249 UfCfCfAGGCfCf 250 AD-15097
    CfUfGGATsT UfAUfGAGGGUfG
    CfTsT
    833-851 GcAcCcUcAuAgGcCuGgATsT 251 P*uCfcAfgGfcC 252 AD-15107
    fuAfuGfaGfgGf
    uGfcTsT
    833-851 GcAcCcUcAuAgGcCuGgATsT 253 UfCfCfAGGCfCf 254 AD-15117
    UfAUfGAGGGUfG
    CfTsT
    833-851 GfcAfcCfcUfcAfuAfgGfcC 255 UCCAGgcCUauGA 256 AD-15127
    fuGfgAfTsT GGGugcTsT
    833-851 GCfACfCfCfUfCfAUfAGGCf 257 UCCAGgcCUauGA 258 AD-15137
    CfUfGGATsT GGGugcTsT
    833-851 GcAcCcUcAuAgGcCuGgATsT 259 UCCAGgcCUauGA 260 AD-15147
    GGGugcTsT
    836-854 UGUAUCUCCUAGACACCAGTsT 261 CUGGUGUCUAGGA 262 AD-9516
    GAUACATsT
    836-854 uGuAucuccuAGAcAccAGTsT 263 CUGGUGUCuAGGA 264 AD-9642
    GAuAcATsT
    840-858 UCUCCUAGACACCAGCAUATsT 265 UAUGCUGGUGUCU 266 AD-9562
    AGGAGATsT
    840-858 ucuccuAGAcAccAGcAuATsT 267 uAUGCUGGUGUCu 268 AD-9688
    AGGAGATsT
    840-858 UfcUfcCfuAfgAfcAfcCfaG 269 P*uAfuGfcUfgG 270 AD-14677
    fcAfuAfTsT fuGfuCfuAfgGf
    aGfaTsT
    840-858 UfCfUfCfCfUfAGACfACfCf 271 UfAUfGCfUfGGU 272 AD-14687
    AGCfAUfATsT fGUfCfUfAGGAG
    ATsT
    840-858 UcUcCuAgAcAcCaGcAuATsT 273 P*uAfuGfcUfgG 274 AD-14697
    fuGfuCfuAfgGf
    aGfaTsT
    840-858 UcUcCuAgAcAcCaGcAuATsT 275 UfAUfGCfUfGGU 276 AD-14707
    fGUfCfUfAGGAG
    ATsT
    840-858 UfcUfcCfuAafAfcAfcCfaG 277 UAUGCugGUguCU 278 AD-14717
    fcAfuAfTsT AGGagaTsT
    840-858 UfCfUfCfCfUfAGACfACfCf 279 UAUGCugGUguCU 280 AD-14727
    AGCfAUfATsT AGGagaTsT
    840-858 UcUcCuAgAcAcCaGcAuATsT 281 UAUGCugGUguCU 282 AD-14737
    AGGagaTsT
    840-858 AfgGfcCfuGfgAfgUfuUfaU 283 P*cCfgAfaUfaA 284 AD-15083
    fuCfgGfTsT faCfuCfcAfgGf
    cCfuTsT
    840-858 AGGCfCfUfGGAGUfUfUfAUf 285 CfCfGAAUfAAAC 286 AD-15093
    UfCfGGTsT fUfCfCfAGGCfC
    fUfTsT
    840-858 AgGcCuGgAgUuUaUuCgGTsT 287 P*cCfgAfaUfaA 288 AD-15103
    faCfuCfcAfgGf
    cCfuTsT
    840-858 AgGcCuGgAgUuUaUuCgGTsT 289 CfCfGAAUfAAAC 290 AD-15113
    fUfCfCfAGGCfC
    fUfTsT
    840-858 AfgGfcCfuGfgAfgUfuUfaU 291 CCGAAuaAAcuCC 292 AD-15123
    fuCfgGfTsT AGGccuTsT
    840-858 AGGCfCfUfGGAGUfUfUfAUf 293 CCGAAuaAAcuCC 294 AD-15133
    UfCfGGTsT AGGccuTsT
    840-858 AgGcCuGgAgUuUaUuCgGTsT 295 CCGAAuaAAcuCC 296 AD-15143
    AGGccuTsT
    841-859 CUCCUAGACACCAGCAUACTsT 297 GUAUGCUGGUGUC 298 AD-9521
    UAGGAGTsT
    841-859 cuccuAGAcAccAGcAuAcTsT 299 GuAUGCUGGUGUC 300 AD-9647
    uAGGAGTsT
    842-860 UCCUAGACACCAGCAUACATsT 301 UGUAUGCUGGUGU 302 AD-9611
    CUAGGATsT
    842-860 uccuAGAcAccAGcAuAcATsT 303 UGuAUGCUGGUGU 304 AD-9737
    CuAGGATsT
    843-861 CCUAGACACCAGCAUACAGTsT 305 CUGUAUGCUGGUG 306 AD-9592
    UCUAGGTsT
    843-861 ccuAGAcAccAGcAuAcAGTsT 307 CUGuAUGCUGGUG 308 AD-9718
    UCuAGGTsT
    847-865 GACACCAGCAUACAGAGUGTsT 309 CACUCUGUAUGCU 310 AD-9561
    GGUGUCTsT
    847-865 GAcAccAGcAuAcAGAGuGTsT 311 cACUCUGuAUGCU 312 AD-9687
    GGUGUCTsT
    855-873 CAUACAGAGUGACCACCGGTsT 313 CCGGUGGUCACUC 314 AD-9636
    UGUAUGTsT
    855-873 cAuAcAGAGuGAccAccGGTsT 315 CCGGUGGUcACUC 316 AD-9762
    UGuAUGTsT
    860-878 AGAGUGACCACCGGGAAAUTsT 317 AUUUCCCGGUGGU 318 AD-9540
    CACUCUTsT
    860-878 AGAGuGAccAccGGGAAAuTsT 319 AUUUCCCGGUGGU 320 AD-9666
    cACUCUTsT
    861-879 GAGUGACCACCGGGAAAUCTsT 321 GAUUUCCCGGUGG 322 AD-9535
    UCACUCTsT
    861-879 GAGuGAccAccGGGAAAucTsT 323 GAUUUCCCGGUGG 324 AD-9661
    UcACUCTsT
    863-881 GUGACCACCGGGAAAUCGATsT 325 UCGAUUUCCCGGU 326 AD-9559
    GGUCACTsT
    863-881 GuGAccAccGGGAAAucGATsT 327 UCGAUUUCCCGGU 328 AD-9685
    GGUcACTsT
    865-883 GACCACCGGGAAAUCGAGGTsT 329 CCUCGAUUUCCCG 330 AD-9533
    GUGGUCTsT
    865-883 GAccAccGGGAAAucGAGGTsT 331 CCUCGAUUUCCCG 332 AD-9659
    GUGGUCTsT
    866-884 ACCACCGGGAAAUCGAGGGTsT 333 CCCUCGAUUUCCC 334 AD-9612
    GGUGGUTsT
    866-884 AccAccGGGAAAucGAGGGTsT 335 CCCUCGAUUUCCC 336 AD-9738
    GGUGGUTsT
    867-885 CCACCGGGAAAUCGAGGGCTsT 337 GCCCUCGAUUUCC 338 AD-9557
    CGGUGGTsT
    867-885 ccAccGGGAAAucGAGGGcTsT 339 GCCCUCGAUUUCC 340 AD-9683
    CGGUGGTsT
    875-893 AAAUCGAGGGCAGGGUCAUTsT 341 AUGACCCUGCCCU 342 AD-9531
    CGAUUUTsT
    875-893 AAAucGAGGGcAGGGucAuTsT 343 AUGACCCUGCCCU 344 AD-9657
    CGAUUUTsT
    875-893 AfaAfuCfgAfgGfgCfaGfgG 345 P*aUfgAfcCfcU 346 AD-14673
    fuCfaUfTsT fgCfcCfuCfgAf
    uUfuTsT
    875-893 AAAUfCfGAGGGCfAGGGUfCf 347 AUfGACfCfCfUf 348 AD-14683
    AUfTsT GCfCfCfUfCfGA
    UfUfUfTsT
    875-893 AaAuCgAgGgCaGgGuCaUTsT 349 P*aUfgAfcCfcU 350 AD-14693
    fgCfcCfuCfgAf
    uUfuTsT
    875-893 AaAuCgAgGgCaGgGuCaUTsT 351 AUfGACfCfCfUf 352 AD-14703
    GCfCfCfUfCfGA
    UfUfUfTsT
    875-893 AfaAfuCfgAfgGfgCfaGfgG 353 AUGACccUGccCU 354 AD-14713
    fuCfaUfTsT CGAuuuTsT
    875-893 AAAUfCfGAGGGCfAGGGUfCf 355 AUGACccUGccCU 356 AD-14723
    AUfTsT CGAuuuTsT
    875-893 AaAuCgAgGgCaGgGuCaUTsT 357 AUGACccUGccCU 358 AD-14733
    CGAuuuTsT
    875-893 CfgGfcAfcCfcUfcAfuAfgG 359 P*cAfgGfcCfuA 360 AD-15079
    fcCfuGfTsT fuGfaGfgGfuGf
    cCfgTsT
    875-893 CfGGCfACfCfCfUfCfAUfAG 361 CfAGGCfCfUfAU 362 AD-15089
    GCfCfUfGTsT fGAGGGUfGCfCf
    GTsT
    875-893 CgGcAcCcUcAuAgGcCuGTsT 363 P*cAfgGfcCfuA 364 AD-15099
    fuGfaGfgGfuGf
    cCfgTsT
    875-893 CgGcAcCcUcAuAgGcCuGTsT 365 CfAGGCfCfUfAU 366 AD-15109
    fGAGGGUfGCfCf
    GTsT
    875-893 CfgGfcAfcCfcUfcAfuAfgG 367 CAGGCcuAUgaGG 368 AD-15119
    fcCfuGfTsT GUGccgTsT
    875-893 CfGGCfACfCfCfUfCfAUfAG 369 CAGGCcuAUgaGG 370 AD-15129
    GCfCfUfGTsT GUGccgTsT
    875-893 CgGcAcCcUcAuAgGcCuGTsT 371 CAGGCcuAUgaGG 372 AD-15139
    GUGccgTsT
    877-895 AUCGAGGGCAGGGUCAUGGTsT 373 CCAUGACCCUGCC 374 AD-9542
    CUCGAUTsT
    877-895 AucGAGGGcAGGGucAuGGTsT 375 CcAUGACCCUGCC 376 AD-9668
    CUCGAUTsT
    878-896 cGAGGGcAGGGucAuGGucTsT 377 GACcAUGACCCUG 378 AD-9739
    CCCUCGTsT
    880-898 GAGGGCAGGGUCAUGGUCATsT 379 UGACCAUGACCCU 380 AD-9637
    GCCCUCTsT
    880-898 GAGGGcAGGGucAuGGucATsT 381 UGACcAUGACCCU 382 AD-9763
    GCCCUCTsT
    882-900 GGGCAGGGUCAUGGUCACCTsT 383 GGUGACCAUGACC 384 AD-9630
    CUGCCCTsT
    882-900 GGGcAGGGucAuGGucAccTsT 385 GGUGACcAUGACC 386 AD-9756
    CUGCCCTsT
    885-903 CAGGGUCAUGGUCACCGACTsT 387 GUCGGUGACCAUG 388 AD-9593
    ACCCUGTsT
    885-903 cAGGGucAuGGucAccGAcTsT 389 GUCGGUGACcAUG 390 AD-9719
    ACCCUGTsT
    886-904 AGGGUCAUGGUCACCGACUTsT 391 AGUCGGUGACCAU 392 AD-9601
    GACCCUTsT
    886-904 AGGGucAuGGucAccGAcuTsT 393 AGUCGGUGACcAU 394 AD-9727
    GACCCUTsT
    892-910 AUGGUCACCGACUUCGAGATsT 395 UCUCGAAGUCGGU 396 AD-9573
    GACCAUTsT
    892-910 AuGGucAccGAcuucGAGATsT 397 UCUCGAAGUCGGU 398 AD-9699
    GACcAUTsT
    899-917 CCGACUUCGAGAAUGUGCCTT 399 GGCACAUUCUCGA 400 AD-15228
    AGUCGGTT
    921-939 GGAGGACGGGACCCGCUUCTT 401 GAAGCGGGUCCCG 402 AD-15395
    UCCUCCTT
    993- CAGCGGCCGGGAUGCCGGCTsT 403 GCCGGCAUCCCGG 404 AD-9602
    1011 CCGCUGTsT
    993- cAGcGGccGGGAuGccGGcTsT 405 GCCGGcAUCCCGG 406 AD-9728
    1011 CCGCUGTsT
    1020- GGGUGCCAGCAUGCGCAGCTT 407 GCUGCGCAUGCUG 408 AD-15386
    1038 GCACCCTT
    1038- CCUGCGCGUGCUCAACUGCTsT 409 GCAGUUGAGCACG 410 AD-9580
    1056 CGCAGGTsT
    1038- ccuGcGcGuGcucAAcuGcTsT 411 GcAGUUGAGcACG 412 AD-9706
    1056 CGcAGGTsT
    1040- UGCGCGUGCUCAACUGCCATsT 413 UGGCAGUUGAGCA 414 AD-9581
    1058 CGCGCATsT
    1040- uGcGcGuGcucAAcuGccATsT 415 UGGcAGUUGAGcA 416 AD-9707
    1058 CGCGcATsT
    1042- CGCGUGCUCAACUGCCAAGTsT 417 CUUGGCAGUUGAG 418 AD-9543
    1060 CACGCGTsT
    1042- cGcGuGcucAAcuGccAAGTsT 419 CUUGGcAGUUGAG 420 AD-9669
    1060 cACGCGTsT
    1053- CUGCCAAGGGAAGGGCACGTsT 421 CGUGCCCUUCCCU 422 AD-9574
    1071 UGGCAGTsT
    1053- cuGccAAGGGAAGGGcAcGTsT 423 CGUGCCCUUCCCU 424 AD-9700
    1071 UGGcAGTsT
    1057- CAAGGGAAGGGCACGGUUATT 425 UAACCGUGCCCUU 426 AD-15320
    1075 CCCUUGTT
    1058- AAGGGAAGGGCACGGUUAGTT 427 CUAACCGUGCCCU 428 AD-15321
    1076 UCCCUUTT
    1059- AGGGAAGGGCACGGUUAGCTT 429 GCUAACCGUGCCC 430 AD-15199
    1077 UUCCCUTT
    1060- GGGAAGGGCACGGUUAGCGTT 431 CGCUAACCGUGCC 432 AD-15167
    1078 CUUCCCTT
    1061- GGAAGGGCACGGUUAGCGGTT 433 CCGCUAACCGUGC 434 AD-15164
    1079 CCUUCCTT
    1062- GAAGGGCACGGUUAGCGGCTT 435 GCCGCUAACCGUG 436 AD-15166
    1080 CCCUUCTT
    1063- AAGGGCACGGUUAGCGGCATT 437 UGCCGCUAACCGU 438 AD-15322
    1081 GCCCUUTT
    1064- AGGGCACGGUUAGCGGCACTT 439 GUGCCGCUAACCG 440 AD-15200
    1082 UGCCCUTT
    1068- CACGGUUAGCGGCACCCUCTT 441 GAGGGUGCCGCUA 442 AD-15213
    1086 ACCGUGTT
    1069- ACGGUUAGCGGCACCCUCATT 443 UGAGGGUGCCGCU 444 AD-15229
    1087 AACCGUTT
    1072- GUUAGCGGCACCCUCAUAGTT 445 CUAUGAGGGUGCC 446 AD-15215
    1090 GCUAACTT
    1073- UUAGCGGCACCCUCAUAGGTT 447 CCUAUGAGGGUGC 448 AD-15214
    1091 CGCUAATT
    1076- GCGGCACCCUCAUAGGCCUTsT 449 AGGCCUAUGAGGG 450 AD-9315
    1094 UGCCGCTsT
    1079- GCACCCUCAUAGGCCUGGATsT 451 UCCAGGCCUAUGA 452 AD-9326
    1097 GGGUGCTsT
    1085- UCAUAGGCCUGGAGUUUAUTsT 453 AUAAACUCCAGGC 454 AD-9318
    1103 CUAUGATsT
    1090- GGCCUGGAGUUUAUUCGGATsT 455 UCCGAAUAAACUC 456 AD-9323
    1108 CAGGCCTsT
    1091- GCCUGGAGUUUAUUCGGAATsT 457 UUCCGAAUAAACU 458 AD-9314
    1109 CCAGGCTsT
    1091- GccuGGAGuuuAuucGGAATsT 459 UUCCGAAuAAACU 460 AD-10792
    1109 CcAGGCTsT
    1091- GccuGGAGuuuAuucGGAATsT 461 UUCCGAAUAACUC 462 AD-10796
    1109 CAGGCTsT
    1093- CUGGAGUUUAUUCGGAAAATsT 463 UUUUCCGAAUAAA 464 AD-9638
    1111 CUCCAGTsT
    1093- cuGGAGuuuAuucGGAAAATsT 465 UUUUCCGAAuAAA 466 AD-9764
    1111 CUCcAGTsT
    1095- GGAGUUUAUUCGGAAAAGCTsT 467 GCUUUUCCGAAUA 468 AD-9525
    1113 AACUCCTsT
    1095- GGAGuuuAuucGGAAAAGcTsT 469 GCUUUUCCGAAuA 470 AD-9651
    1113 AACUCCTsT
    1096- GAGUUUAUUCGGAAAAGCCTsT 471 GGCUUUUCCGAAU 472 AD-9560
    1114 AAACUCTsT
    1096- GAGuuuAuucGGAAAAGccTsT 473 GGCUUUUCCGAAu 474 AD-9686
    1114 AAACUCTsT
    1100- UUAUUCGGAAAAGCCAGCUTsT 475 AGCUGGCUUUUCC 476 AD-9536
    1118 GAAUAATsT
    1100- uuAuucGGAAAAGccAGcuTsT 477 AGCUGGCUUUUCC 478 AD-9662
    1118 GAAuAATsT
    1154- CCCUGGCGGGUGGGUACAGTsT 479 CUGUACCCACCCG 480 AD-9584
    1172 CCAGGGTsT
    1154- cccuGGcGGGuGGGuAcAGTsT 481 CUGuACCcACCCG 482 AD-9710
    1172 CcAGGGTsT
    1155- CCUGGCGGGUGGGUACAGCTT 483 GCUGUACCCACCC 484 AD-15323
    1173 GCCAGGTT
    1157- UGGCGGGUGGGUACAGCCGTsT 485 CGGCUGUACCCAC 486 AD-9551
    1175 CCGCCATsT
    1157- uGGcGGGuGGGuAcAGccGTsT 487 CGGCUGuACCcAC 488 AD-9677
    1175 CCGCcATsT
    1158- GGCGGGUGGGUACAGCCGCTT 489 GCGGCUGUACCCA 490 AD-15230
    1176 CCCGCCTT
    1162- GGUGGGUACAGCCGCGUCCTT 491 GGACGCGGCUGUA 492 AD-15231
    1180 CCCACCTT
    1164- UGGGUACAGCCGCGUCCUCTT 493 GAGGACGCGGCUG 494 AD-15285
    1182 UACCCATT
    1172- GCCGCGUCCUCAACGCCGCTT 495 GCGGCGUUGAGGA 496 AD-15396
    1190 CGCGGCTT
    1173- CCGCGUCCUCAACGCCGCCTT 497 GGCGGCGUUGAGG 498 AD-15397
    1191 ACGCGGTT
    1216- GUCGUGCUGGUCACCGCUGTsT 499 CAGCGGUGACCAG 500 AD-9600
    1234 CACGACTsT
    1216- GucGuGcuGGucAccGcuGTsT 501 cAGCGGUGACcAG 502 AD-9726
    1234 cACGACTsT
    1217- UCGUGCUGGUCACCGCUGCTsT 503 GCAGCGGUGACCA 504 AD-9606
    1235 GCACGATsT
    1217- ucGuGcuGGucAccGcuGcTsT 505 GcAGCGGUGACcA 506 AD-9732
    1235 GcACGATsT
    1223- UGGUCACCGCUGCCGGCAATsT 507 UUGCCGGCAGCGG 508 AD-9633
    1241 UGACCATsT
    1223- uGGucAccGcuGccGGcAATsT 509 UUGCCGGcAGCGG 510 AD-9759
    1241 UGACcATsT
    1224- GGUCACCGCUGCCGGCAACTsT 511 GUUGCCGGCAGCG 512 AD-9588
    1242 GUGACCTsT
    1224- GGucAccGcuGccGGcAAcTsT 513 GUUGCCGGcAGCG 514 AD-9714
    1242 GUGACCTsT
    1227- CACCGCUGCCGGCAACUUCTsT 515 GAAGUUGCCGGCA 516 AD-9589
    1245 GCGGUGTsT
    1227- cAccGcuGccGGcAAcuucTsT 517 GAAGUUGCCGGcA 518 AD-9715
    1245 GCGGUGTsT
    1229- CCGCUGCCGGCAACUUCCGTsT 519 CGGAAGUUGCCGG 520 AD-9575
    1247 CAGCGGTsT
    1229- ccGcuGccGGcAAcuuccGTsT 521 CGGAAGUUGCCGG 522 AD-9701
    1247 cAGCGGTsT
    1230- CGCUGCCGGCAACUUCCGGTsT 523 CCGGAAGUUGCCG 524 AD-9563
    1248 GCAGCGTsT
    1230- cGcuGccGGcAAcuuccGGTsT 525 CCGGAAGUUGCCG 526 AD-9689
    1248 GcAGCGTsT
    1231- GCUGCCGGCAACUUCCGGGTsT 527 CCCGGAAGUUGCC 528 AD-9594
    1249 GGCAGCTsT
    1231- GcuGccGGcAAcuuccGGGTsT 529 CCCGGAAGUUGCC 530 AD-9720
    1249 GGcAGCTsT
    1236- CGGCAACUUCCGGGACGAUTsT 531 AUCGUCCCGGAAG 532 AD-9585
    1254 UUGCCGTsT
    1236- cGGcAAcuuccGGGAcGAuTsT 533 AUCGUCCCGGAAG 534 AD-9711
    1254 UUGCCGTsT
    1237- GGCAACUUCCGGGACGAUGTsT 535 CAUCGUCCCGGAA 536 AD-9614
    1255 GUUGCCTsT
    1237- GGcAAcuuccGGGAcGAuGTsT 537 cAUCGUCCCGGAA 538 AD-9740
    1255 GUUGCCTsT
    1243- UUCCGGGACGAUGCCUGCCTsT 539 GGCAGGCAUCGUC 540 AD-9615
    1261 CCGGAATsT
    1243- uuccGGGAcGAuGccuGccTsT 541 GGcAGGcAUCGUC 542 AD-9741
    1261 CCGGAATsT
    1248- GGACGAUGCCUGCCUCUACTsT 543 GUAGAGGCAGGCA 544 AD-9534
    1266 UCGUCCTsT
    1248- GGACGAUGCCUGCCUCUACTsT 545 GUAGAGGCAGGCA 546 AD-9534
    1266 UCGUCCTsT
    1248- GGAcGAuGccuGccucuAcTsT 547 GuAGAGGcAGGcA 548 AD-9660
    1266 UCGUCCTsT
    1279- GCUCCCGAGGUCAUCACAGTT 549 CUGUGAUGACCUC 550 AD-15324
    1297 GGGAGCTT
    1280- CUCCCGAGGUCAUCACAGUTT 551 ACUGUGAUGACCU 552 AD-15232
    1298 CGGGAGTT
    1281- UCCCGAGGUCAUCACAGUUTT 553 AACUGUGAUGACC 554 AD-15233
    1299 UCGGGATT
    1314- CCAAGACCAGCCGGUGACCTT 555 GGUCACCGGCUGG 556 AD-15234
    1332 UCUUGGTT
    1315- CAAGACCAGCCGGUGACCCTT 557 GGGUCACCGGCUG 558 AD-15286
    1333 GUCUUGTT
    1348- ACCAACUUUGGCCGCUGUGTsT 559 CACAGCGGCCAAA 560 AD-9590
    1366 GUUGGUTsT
    1348- AccAAcuuuGGccGcuGuGTsT 561 cAcAGCGGCcAAA 562 AD-9716
    1366 GUUGGUTsT
    1350- CAACUUUGGCCGCUGUGUGTsT 563 CACACAGCGGCCA 564 AD-9632
    1368 AAGUUGTsT
    1350- cAAcuuuGGccGcuGuGuGTsT 565 cAcAcAGCGGCcA 566 AD-9758
    1368 AAGUUGTsT
    1360- CGCUGUGUGGACCUCUUUGTsT 567 CAAAGAGGUCCAC 568 AD-9567
    1378 ACAGCGTsT
    1360- cGcuGuGuGGAccucuuuGTsT 569 cAAAGAGGUCcAc 570 AD-9693
    1378 AcAGCGTsT
    1390- GACAUCAUUGGUGCCUCCATsT 571 UGGAGGCACCAAU 572 AD-9586
    1408 GAUGUCTsT
    1390- GAcAucAuuGGuGccuccATsT 573 UGGAGGcACcAAU 574 AD-9712
    1408 GAUGUCTsT
    1394- UCAUUGGUGCCUCCAGCGATsT 575 UCGCUGGAGGCAC 576 AD-9564
    1412 CAAUGATsT
    1394- ucAuuGGuGccuccAGcGATsT 577 UCGCUGGAGGcAC 578 AD-9690
    1412 cAAUGATsT
    1417- AGCACCUGCUUUGUGUCACTsT 579 GUGACACAAAGCA 580 AD-9616
    1435 GGUGCUTsT
    1417- AGcAccuGcuuuGuGucAcTsT 581 GUGAcAcAAAGcA 582 AD-9742
    1435 GGUGCUTsT
    1433- CACAGAGUGGGACAUCACATT 583 UGUGAUGUCCCAC 584 AD-15398
    1451 UCUGUGTT
    1486- AUGCUGUCUGCCGAGCCGGTsT 585 CCGGCUCGGCAGA 586 AD-9617
    1504 CAGCAUTsT
    1486- AuGcuGucuGccGAGccGGTsT 587 CCGGCUCGGcAGA 588 AD-9743
    1504 cAGcAUTsT
    1491- GUCUGCCGAGCCGGAGCUCTsT 589 GAGCUCCGGCUCG 590 AD-9635
    1509 GCAGACTsT
    1491- GucuGccGAGccGGAGcucTsT 591 GAGCUCCGGCUCG 592 AD-9761
    1509 GcAGACTsT
    1521- GUUGAGGCAGAGACUGAUCTsT 593 GAUCAGUCUCUGC 594 AD-9568
    1539 CUCAACTsT
    1521- GuuGAGGcAGAGAcuGAucTsT 595 GAUcAGUCUCUGC 596 AD-9694
    1539 CUcAACTsT
    1527- GCAGAGACUGAUCCACUUCTsT 597 GAAGUGGAUCAGU 598 AD-9576
    1545 CUCUGCTsT
    1527- GcAGAGAcuGAuccAcuucTsT 599 GAAGUGGAUcAGU 600 AD-9702
    1545 CUCUGCTsT
    1529- AGAGACUGAUCCACUUCUCTsT 601 GAGAAGUGGAUCA 602 AD-9627
    1547 GUCUCUTsT
    1529- AGAGAcuGAuccAcuucucTsT 603 GAGAAGUGGAUcA 604 AD-9753
    1547 GUCUCUTsT
    1543- UUCUCUGCCAAAGAUGUCATsT 605 UGACAUCUUUGGC 606 AD-9628
    1561 AGAGAATsT
    1543- uucucuGccAAAGAuGucATsT 607 UGAcAUCUUUGGc 608 AD-9754
    1561 AGAGAATsT
    1545- CUCUGCCAAAGAUGUCAUCTsT 609 GAUGACAUCUUUG 610 AD-9631
    1563 GCAGAGTsT
    1545- cucuGccAAAGAuGucAucTsT 611 GAUGAcAUCUUUG 612 AD-9757
    1563 GcAGAGTsT
    1580- CUGAGGACCAGCGGGUACUTsT 613 AGUACCCGCUGGU 614 AD-9595
    1598 CCUCAGTsT
    1580- cuGAGGAccAGcGGGuAcuTsT 615 AGuACCCGCUGGU 616 AD-9721
    1598 CCUcAGTsT
    1581- UGAGGACCAGCGGGUACUGTsT 617 CAGUACCCGCUGG 618 AD-9544
    1599 UCCUCATsT
    1581- uGAGGAccAGcGGGuAcuGTsT 619 cAGuACCCGCUGG 620 AD-9670
    1599 UCCUcATsT
    1666- ACUGUAUGGUCAGCACACUTT 621 AGUGUGCUGACCA 622 AD-15235
    1684 UACAGUTT
    1668- UGUAUGGUCAGCACACUCGTT 623 CGAGUGUGCUGAC 624 AD-15236
    1686 CAUACATT
    1669- GUAUGGUCAGCACACUCGGTT 625 CCGAGUGUGCUGA 626 AD-15168
    1687 CCAUACTT
    1697- GGAUGGCCACAGCCGUCGCTT 627 GCGACGGCUGUGG 628 AD-15174
    1715 CCAUCCTT
    1698- GAUGGCCACAGCCGUCGCCTT 629 GGCGACGGCUGUG 630 AD-15325
    1716 GCCAUCTT
    1806- CAAGCUGGUCUGCCGGGCCTT 631 GGCCCGGCAGACC 632 AD-15326
    1824 AGCUUGTT
    1815- CUGCCGGGCCCACAACGCUTsT 633 AGCGUUGUGGGCC 634 AD-9570
    1833 CGGCAGTsT
    1815- cuGccGGGcccAcAAcGcuTsT 635 AGCGUUGUGGGCC 636 AD-9696
    1833 CGGcAGTsT
    1816- UGCCGGGCCCACAACGCUUTsT 637 AAGCGUUGUGGGC 638 AD-9566
    1834 CCGGCATsT
    1816- uGccGGGcccAcAAcGcuuTsT 639 AAGCGUUGUGGGC 640 AD-9692
    1834 CCGGcATsT
    1818- CCGGGCCCACAACGCUUUUTsT 641 AAAAGCGUUGUGG 642 AD-9532
    1836 GCCCGGTsT
    1818- ccGGGcccAcAAcGcuuuuTsT 643 AAAAGCGUUGUGG 644 AD-9658
    1836 GCCCGGTsT
    1820- GGGCCCACAACGCUUUUGGTsT 645 CCAAAAGCGUUGU 646 AD-9549
    1838 GGGCCCTsT
    1820- GGGcccAcAAcGcuuuuGGTsT 647 CcAAAAGCGUUGU 648 AD-9675
    1838 GGGCCCTsT
    1840- GGUGAGGGUGUCUACGCCATsT 649 UGGCGUAGACACC 650 AD-9541
    1858 CUCACCTsT
    1840- GGuGAGGGuGucuAcGccATsT 651 UGGCGuAGAcACC 652 AD-9667
    1858 CUcACCTsT
    1843- GAGGGUGUCUACGCCAUUGTsT 653 CAAUGGCGUAGAC 654 AD-9550
    1861 ACCCUCTsT
    1843- GAGGGuGucuAcGccAuuGTsT 655 cAAUGGCGuAGAc 656 AD-9676
    1861 ACCCUCTsT
    1861- GCCAGGUGCUGCCUGCUACTsT 657 GUAGCAGGCAGCA 658 AD-9571
    1879 CCUGGCTsT
    1861- GccAGGuGcuGccuGcuAcTsT 659 GuAGcAGGcAGcA 660 AD-9697
    1879 CCUGGCTsT
    1862- CCAGGUGCUGCCUGCUACCTsT 661 GGUAGCAGGCAGC 662 AD-9572
    1880 ACCUGGTsT
    1862- ccAGGuGcuGccuGcuAccTsT 663 GGuAGcAGGcAGc 664 AD-9698
    1880 ACCUGGTsT
    2008- ACCCACAAGCCGCCUGUGCTT 665 GCACAGGCGGCUU 666 AD-15327
    2026 GUGGGUTT
    2023- GUGCUGAGGCCACGAGGUCTsT 667 GACCUCGUGGCCU 668 AD-9639
    2041 CAGCACTsT
    2023- GuGcuGAGGccAcGAGGucTsT 669 GACCUCGUGGCCU 670 AD-9765
    2041 cAGcACTsT
    2024- UGCUGAGGCCACGAGGUCATsT 671 UGACCUCGUGGCC 672 AD-9518
    2042 UCAGCATsT
    2024- UGCUGAGGCCACGAGGUCATsT 673 UGACCUCGUGGCC 674 AD-9518
    2042 UCAGCATsT
    2024- uGcuGAGGccAcGAGGucATsT 675 UGACCUCGUGGCC 676 AD-9644
    2042 UcAGcATsT
    2024- UfgCfuGfaGfgCfcAfcGfaG 677 P*uGfaCfcUfcG 678 AD-14672
    2042 fgUfcAfTsT fuGfgCfcUfcAf
    gCfaTsT
    2024- UfGCfUfGAGGCfCfACfGAGG 679 UfGACfCfUfCfG 680 AD-14682
    2042 UfCfATsT UfGGCfCfUfCfA
    GCfATsT
    2024- UgCuGaGgCcAcGaGgUcATsT 681 P*uGfaCfcUfcG 682 AD-14692
    2042 fuGfgCfcUfcAf
    gCfaTsT
    2024- UgCuGaGgCcAcGaGgUcATsT 683 UfGACfCfUfCfG 684 AD-14702
    2042 UfGGCfCfUfCfA
    GCfATsT
    2024- UfgCfuGfaGfgCfcAfcGfaG 685 UGACCucGUggCC 686 AD-14712
    2042 fgUfcAfTsT UCAgcaTsT
    2024- UfGCfUfGAGGCfCfACfGAGG 687 UGACCucGUggCC 688 AD-14722
    2042 UfCfATsT UCAgcaTsT
    2024- UgCuGaGgCcAcGaGgUcATsT 689 UGACCucGUggCC 690 AD-14732
    2042 UCAgcaTsT
    2024- GfuGfgUfcAfgCfgGfcCfgG 691 P*cAfuCfcCfgG 692 AD-15078
    2042 fgAfuGfTsT fcCfgCfuGfaCf
    cAfcTsT
    2024- GUfGGUfCfAGCfGGCfCfGGG 693 CfAUfCfCfCfGG 694 AD-15088
    2042 AUfGTsT CfCfGCfUfGACf
    CfACfTsT
    2024- GuGgUcAgCgGcCgGgAuGTsT 695 P*cAfuCfcCfgG 696 AD-15098
    2042 fcCfgCfuGfaCf
    cAfcTsT
    2024- GuGgUcAgCgGcCgGgAuGTsT 697 CfAUfCfCfCfGG 698 AD-15108
    2042 CfCfGCfUfGACf
    CfACfTsT
    2024- GfuGfgUfcAfgCfgGfcCfgG 699 CAUCCcgGCcgCU 700 AD-15118
    2042 fgAfuGfTsT GACcacTsT
    2024- GUfGGUfCfAGCfGGCfCfGGG 701 CAUCCcgGCcgCU 702 AD-15128
    2042 AUfGTsT GACcacTsT
    2024- GuGgUcAgCgGcCgGgAuGTsT 703 CAUCCcgGCcgCU 704 AD-15138
    2042 GACcacTsT
    2030- GGCCACGAGGUCAGCCCAATT 705 UUGGGCUGACCUC 706 AD-15237
    2048 GUGGCCTT
    2035- CGAGGUCAGCCCAACCAGUTT 707 ACUGGUUGGGCUG 708 AD-15287
    2053 ACCUCGTT
    2039- GUCAGCCCAACCAGUGCGUTT 709 ACGCACUGGUUGG 710 AD-15238
    2057 GCUGACTT
    2041- CAGCCCAACCAGUGCGUGGTT 711 CCACGCACUGGUU 712 AD-15328
    2059 GGGCUGTT
    2062- CACAGGGAGGCCAGCAUCCTT 713 GGAUGCUGGCCUC 714 AD-15399
    2080 CCUGUGTT
    2072- CCAGCAUCCACGCUUCCUGTsT 715 CAGGAAGCGUGGA 716 AD-9582
    2090 UGCUGGTsT
    2072- ccAGcAuccAcGcuuccuGTsT 717 cAGGAAGCGUGGA 718 AD-9708
    2090 UGCUGGTsT
    2118- AGUCAAGGAGCAUGGAAUCTsT 719 GAUUCCAUGCUCC 720 AD-9545
    2136 UUGACUTsT
    2118- AGucAAGGAGcAuGGAAucTsT 721 GAUUCcAUGCUCC 722 AD-9671
    2136 UUGACUTsT
    2118- AfgUfcAfaGfgAfgCfaUfgG 723 P*gAfuUfcCfaU 724 AD-14674
    2136 faAfuCfTsT fgCfuCfcUfuGf
    aCfuTsT
    2118- AGUfCfAAGGAGCfAUfGGAAU 725 GAUfUfCfCfAUf 726 AD-14684
    2136 fCfTsT GCfUfCfCfUfUf
    GACfUfTsT
    2118- AgUcAaGgAgCaUgGaAuCTsT 727 P*gAfuUfcCfaU 728 AD-14694
    2136 fgCfuCfcUfuGf
    aCfuTsT
    2118- AgUcAaGgAgCaUgGaAuCTsT 729 GAUfUfCfCfAUf 730 AD-14704
    2136 GCfUfCfCfUfUf
    GACfUfTsT
    2118- AfgUfcAfaGfgAfgCfaUfgG 731 GAUUCcaUGcuCC 732 AD-14714
    2136 faAfuCfTsT UUGacuTsT
    2118- AGUfCfAAGGAGCfAUfGGAAU 733 GAUUCcaUGcuCC 734 AD-14724
    2136 fCfTsT UUGacuTsT
    2118- AgUcAaGgAgCaUgGaAuCTsT 735 GAUUCcaUGcuCC 736 AD-14734
    2136 UUGacuTsT
    2118- GfcGfgCfaCfcCfuCfaUfaG 737 P*aGfgCfcUfaU 738 AD-15080
    2136 fgCfcUfTsT fgAfgGfgUfgCf
    cGfcTsT
    2118- GCfGGCfACfCfCfUfCfAUfA 739 AGGCfCfUfAUfG 740 AD-15090
    2136 GGCfCfUfTsT AGGGUfGCfCfGC
    fTsT
    2118- GcGgCaCcCuCaUaGgCcUTsT 741 P*aGfgCfcUfaU 742 AD-15100
    2136 fgAfgGfgUfgCf
    cGfcTsT
    2118- GcGgCaCcCuCaUaGgCcUTsT 743 AGGCfCfUfAUfG 744 AD-15110
    2136 AGGGUfGCfCfGC
    fTsT
    2118- GfcGfgCfaCfcCfuCfaUfaG 745 AGGCCuaUGagGG 746 AD-15120
    2136 fgCfcUfTsT UGCcgcTsT
    2118- GCfGGCfACfCfCfUfCfAUfA 747 AGGCCuaUGagGG 748 AD-15130
    2136 GGCfCfUfTsT UGCcgcTsT
    2118- GcGgCaCcCuCaUaGgCcUTsT 749 AGGCCuaUGagGG 750 AD-15140
    2136 UGCcgcTsT
    2122- AAGGAGCAUGGAAUCCCGGTsT 751 CCGGGAUUCCAUG 752 AD-9522
    2140 CUCCUUTsT
    2122- AAGGAGcAuGGAAucccGGTsT 753 CCGGGAUUCcAUG 754 AD-9648
    2140 CUCCUUTsT
    2123- AGGAGCAUGGAAUCCCGGCTsT 755 GCCGGGAUUCCAU 756 AD-9552
    2141 GCUCCUTsT
    2123- AGGAGcAuGGAAucccGGcTsT 757 GCCGGGAUUCcAU 758 AD-9678
    2141 GCUCCUTsT
    2125- GAGCAUGGAAUCCCGGCCCTsT 759 GGGCCGGGAUUCC 760 AD-9618
    2143 AUGCUCTsT
    2125- GAGcAuGGAAucccGGcccTsT 761 GGGCCGGGAUUCc 762 AD-9744
    2143 AUGCUCTsT
    2230- GCCUACGCCGUAGACAACATT 763 UGUUGUCUACGGC 764 AD-15239
    2248 GUAGGCTT
    2231- CCUACGCCGUAGACAACACTT 765 GUGUUGUCUACGG 766 AD-15212
    2249 CGUAGGTT
    2232- CUACGCCGUAGACAACACGTT 767 CGUGUUGUCUACG 768 AD-15240
    2250 GCGUAGTT
    2233- UACGCCGUAGACAACACGUTT 769 ACGUGUUGUCUAC 770 AD-15177
    2251 GGCGUATT
    2235- CGCCGUAGACAACACGUGUTT 771 ACACGUGUUGUCU 772 AD-15179
    2253 ACGGCGTT
    2236- GCCGUAGACAACACGUGUGTT 773 CACACGUGUUGUC 774 AD-15180
    2254 UACGGCTT
    2237- CCGUAGACAACACGUGUGUTT 775 ACACACGUGUUGU 776 AD-15241
    2255 CUACGGTT
    2238- CGUAGACAACACGUGUGUATT 777 UACACACGUGUUG 778 AD-15268
    2256 UCUACGTT
    2240- UAGACAACACGUGUGUAGUTT 779 ACUACACACGUGU 780 AD-15242
    2258 UGUCUATT
    2241- AGACAACACGUGUGUAGUCTT 781 GACUACACACGUG 782 AD-15216
    2259 UUGUCUTT
    2242- GACAACACGUGUGUAGUCATT 783 UGACUACACACGU 784 AD-15176
    2260 GUUGUCTT
    2243- ACAACACGUGUGUAGUCAGTT 785 CUGACUACACACG 786 AD-15181
    2261 UGUUGUTT
    2244- CAACACGUGUGUAGUCAGGTT 787 CCUGACUACACAC 788 AD-15243
    2262 GUGUUGTT
    2247- CACGUGUGUAGUCAGGAGCTT 789 GCUCCUGACUACA 790 AD-15182
    2265 CACGUGTT
    2248- ACGUGUGUAGUCAGGAGCCTT 791 GGCUCCUGACUAC 792 AD-15244
    2266 ACACGUTT
    2249- CGUGUGUAGUCAGGAGCCGTT 793 CGGCUCCUGACUA 794 AD-15387
    2267 CACACGTT
    2251- UGUGUAGUCAGGAGCCGGGTT 795 CCCGGCUCCUGAC 796 AD-15245
    2269 UACACATT
    2257- GUCAGGAGCCGGGACGUCATsT 797 UGACGUCCCGGCU 798 AD-9555
    2275 CCUGACTsT
    2257- GucAGGAGccGGGAcGucATsT 799 UGACGUCCCGGCU 800 AD-9681
    2275 CCUGACTsT
    2258- UCAGGAGCCGGGACGUCAGTsT 801 CUGACGUCCCGGC 802 AD-9619
    2276 UCCUGATsT
    2258- ucAGGAGccGGGAcGucAGTsT 803 CUGACGUCCCGGC 804 AD-9745
    2276 UCCUGATsT
    2259- CAGGAGCCGGGACGUCAGCTsT 805 GCUGACGUCCCGG 806 AD-9620
    2277 CUCCUGTsT
    2259- cAGGAGccGGGAcGucAGcTsT 807 GCUGACGUCCCGG 808 AD-9746
    2277 CUCCUGTsT
    2263- AGCCGGGACGUCAGCACUATT 809 UAGUGCUGACGUC 810 AD-15288
    2281 CCGGCUTT
    2265- CCGGGACGUCAGCACUACATT 811 UGUAGUGCUGACG 812 AD-15246
    2283 UCCCGGTT
    2303- CCGUGACAGCCGUUGCCAUTT 813 AUGGCAACGGCUG 814 AD-15289
    2321 UCACGGTT
    2317- GCCAUCUGCUGCCGGAGCCTsT 815 GGCUCCGGCAGCA 816 AD-9324
    2335 GAUGGCTsT
    2375- CCCAUCCCAGGAUGGGUGUTT 817 ACACCCAUCCUGG 818 AD-15329
    2393 GAUGGGTT
    2377- CAUCCCAGGAUGGGUGUCUTT 819 AGACACCCAUCCU 820 AD-15330
    2395 GGGAUGTT
    2420- AGCUUMAAAUGGUUCCGATT 821 UCGGAACCAUUUU 822 AD-15169
    2438 AAAGCUTT
    2421- GCUUUAAAAUGGUUCCGACTT 823 GUCGGAACCAUUU 824 AD-15201
    2439 UAAAGCTT
    2422- CUUMAAAUGGUUCCGACUTT 825 AGUCGGAACCAUU 826 AD-15331
    2440 UUAAAGTT
    2423- UUUAAAAUGGUUCCGACUUTT 827 AAGUCGGAACCAU 828 AD-15190
    2441 UUUAAATT
    2424- UUAAAAUGGUUCCGACUUGTT 829 CAAGUCGGAACCA 830 AD-15247
    2442 UUUUAATT
    2425- UAAAAUGGUUCCGACUUGUTT 831 ACAAGUCGGAACC 832 AD-15248
    2443 AUUUUATT
    2426- AAAAUGGUUCCGACUUGUCTT 833 GACAAGUCGGAAC 834 AD-15175
    2444 CAUUUUTT
    2427- AAAUGGUUCCGACUUGUCCTT 835 GGACAAGUCGGAA 836 AD-15249
    2445 CCAUUUTT
    2428- AAUGGUUCCGACUUGUCCCTT 837 GGGACAAGUCGGA 838 AD-15250
    2446 ACCAUUTT
    2431- GGUUCCGACUUGUCCCUCUTT 839 AGAGGGACAAGUC 840 AD-15400
    2449 GGAACCTT
    2457- CUCCAUGGCCUGGCACGAGTT 841 CUCGUGCCAGGCC 842 AD-15332
    2475 AUGGAGTT
    2459- CCAUGGCCUGGCACGAGGGTT 843 CCCUCGUGCCAGG 844 AD-15388
    2477 CCAUGGTT
    2545- GAACUCACUCACUCUGGGUTT 845 ACCCAGAGUGAGU 846 AD-15333
    2563 GAGUUCTT
    2549- UCACUCACUCUGGGUGCCUTT 847 AGGCACCCAGAGU 848 AD-15334
    2567 GAGUGATT
    2616- UUUCACCAUUCAAACAGGUTT 849 ACCUGUUUGAAUG 850 AD-15335
    2634 GUGAAATT
    2622- CAUUCAAACAGGUCGAGCUTT 851 AGCUCGACCUGUU 852 AD-15183
    2640 UGAAUGTT
    2623- AUUCAAACAGGUCGAGCUGTT 853 CAGCUCGACCUGU 854 AD-15202
    2641 UUGAAUTT
    2624- UUCAAACAGGUCGAGCUGUTT 855 ACAGCUCGACCUG 856 AD-15203
    2642 UUUGAATT
    2625- UCAAACAGGUCGAGCUGUGTT 857 CACAGCUCGACCU 858 AD-15272
    2643 GUUUGATT
    2626- CAAACAGGUCGAGCUGUGCTT 859 GCACAGCUCGACC 860 AD-15217
    2644 UGUUUGTT
    2627- AAACAGGUCGAGCUGUGCUTT 861 AGCACAGCUCGAC 862 AD-15290
    2645 CUGUUUTT
    2628- AACAGGUCGAGCUGUGCUCTT 863 GAGCACAGCUCGA 864 AD-15218
    2646 CCUGUUTT
    2630- CAGGUCGAGCUGUGCUCGGTT 865 CCGAGCACAGCUC 866 AD-15389
    2648 GACCUGTT
    2631- AGGUCGAGCUGUGCUCGGGTT 867 CCCGAGCACAGCU 868 AD-15336
    2649 CGACCUTT
    2633- GUCGAGCUGUGCUCGGGUGTT 869 CACCCGAGCACAG 870 AD-15337
    2651 CUCGACTT
    2634- UCGAGCUGUGCUCGGGUGCTT 871 GCACCCGAGCACA 872 AD-15191
    2652 GCUCGATT
    2657- AGCUGCUCCCAAUGUGCCGTT 873 CGGCACAUUGGGA 874 AD-15390
    2675 GCAGCUTT
    2658- GCUGCUCCCAAUGUGCCGATT 875 UCGGCACAUUGGG 876 AD-15338
    2676 AGCAGCTT
    2660- UGCUCCCAAUGUGCCGAUGTT 877 CAUCGGCACAUUG 878 AD-15204
    2678 GGAGCATT
    2663- UCCCAAUGUGCCGAUGUCCTT 879 GGACAUCGGCACA 880 AD-15251
    2681 UUGGGATT
    2665- CCAAUGUGCCGAUGUCCGUTT 881 ACGGACAUCGGCA 882 AD-15205
    2683 CAUUGGTT
    2666- CAAUGUGCCGAUGUCCGUGTT 883 CACGGACAUCGGC 884 AD-15171
    2684 ACAUUGTT
    2667- AAUGUGCCGAUGUCCGUGGTT 885 CCACGGACAUCGG 886 AD-15252
    2685 CACAUUTT
    2673- CCGAUGUCCGUGGGCAGAATT 887 UUCUGCCCACGGA 888 AD-15339
    2691 CAUCGGTT
    2675- GAUGUCCGUGGGCAGAAUGTT 889 CAUUCUGCCCACG 890 AD-15253
    2693 GACAUCTT
    2678- GUCCGUGGGCAGAAUGACUTT 891 AGUCAUUCUGCCC 892 AD-15340
    2696 ACGGACTT
    2679- UCCGUGGGCAGAAUGACUUTT 893 AAGUCAUUCUGCC 894 AD-15291
    2697 CACGGATT
    2683- UGGGCAGAAUGACUUDUAUTT 895 AUAAAAGUCAUUC 896 AD-15341
    2701 UGCCCATT
    2694- ACUUUUAUUGAGCUCUUGUTT 897 ACAAGAGCUCAAU 898 AD-15401
    2712 AAAAGUTT
    2700- AUUGAGCUCUUGUUCCGUGTT 899 CACGGAACAAGAG 900 AD-15342
    2718 CUCAAUTT
    2704- AGCUCUUGUUCCGUGCCAGTT 901 CUGGCACGGAACA 902 AD-15343
    2722 AGAGCUTT
    2705- GCUCUUGUUCCGUGCCAGGTT 903 CCUGGCACGGAAC 904 AD-15292
    2723 AAGAGCTT
    2710- UGUUCCGUGCCAGGCAUUCTT 905 GAAUGCCUGGCAC 906 AD-15344
    2728 GGAACATT
    2711- GUUCCGUGCCAGGCAUUCATT 907 UGAAUGCCUGGCA 908 AD-15254
    2729 CGGAACTT
    2712- UUCCGUGCCAGGCAUUCAATT 909 UUGAAUGCCUGGC 910 AD-15345
    2730 ACGGAATT
    2715- CGUGCCAGGCAUUCAAUCCTT 911 GGAUUGAAUGCCU 912 AD-15206
    2733 GGCACGTT
    2716- GUGCCAGGCAUUCAAUCCUTT 913 AGGAUUGAAUGCC 914 AD-15346
    2734 UGGCACTT
    2728- CAAUCCUCAGGUCUCCACCTT 915 GGUGGAGACCUGA 916 AD-15347
    2746 GGAUUGTT
    2743- CACCAAGGAGGCAGGAUUCTsT 917 GAAUCCUGCCUCC 918 AD-9577
    2761 UUGGUGTsT
    2743- cAccAAGGAGGcAGGAuucTsT 919 GAAUCCUGCCUCC 920 AD-9703
    2761 UUGGUGTsT
    2743- CfaCfcAfaGfgAfgGfcAfgG 921 P*gAfaUfcCfuG 922 AD-14678
    2761 faUfuCfTsT fcCfuCfcUfuGf
    gUfgTsT
    2743- CfACfCfAAGGAGGCfAGGAUf 923 GAAUfCfCfUfGC 924 AD-14688
    2761 UfCfTsT fCfUfCfCfUfUf
    GGUfGTsT
    2743- CaCcAaGgAgGcAgGaUuCTsT 925 P*gAfaUfcCfuG 926 AD-14698
    2761 fcCfuCfcUfuGf
    gUfgTsT
    2743- CaCcAaGgAgGcAgGaUuCTsT 927 GAAUfCfCfUfGC 928 AD-14708
    2761 fCfUfCfCfUfUf
    GGUfGTsT
    2743- CfaCfcAfaGfgAfgGfcAfgG 929 GAAUCcuGCcuCC 930 AD-14718
    2761 faUfuCfTsT UUGgugTsT
    2743- CfACfCfAAGGAGGCfAGGAUf 931 GAAUCcuGCcuCC 932 AD-14728
    2761 UfCfTsT UUGgugTsT
    2743- CaCcAaGgAgGcAgGaUuCTsT 933 GAAUCcuGCcuCC 934 AD-14738
    2761 UUGgugTsT
    2743- GfgCfcUfgGfaGfuUfuAfuU 935 P*uCfcGfaAfuA 936 AD-15084
    2761 fcGfgAfTsT faAfcUfcCfaGf
    gCfcTsT
    2743- GGCfCfUfGGAGUfUfUfAUfU 937 UfCfCfGAAUfAA 938 AD-15094
    2761 fCfGGATsT ACfUfCfCfAGGC
    fCfTsT
    2743- GgCcUgGaGuUuAuUcGgATsT 939 P*uCfcGfaAfuA 940 AD-15104
    2761 faAfcUfcCfaGf
    gCfcTsT
    2743- GgCcUgGaGuUuAuUcGgATsT 941 UfCfCfGAAUfAA 942 AD-15114
    2761 ACfUfCfCfAGGC
    fCfTsT
    2743- GfgCfcUfgGfaGfuUfuAfuU 943 UCCGAauAAacUC 944 AD-15124
    2761 fcGfgAfTsT CAGgccTsT
    2743- GGCfCfUfGGAGUfUfUfAUfU 945 UCCGAauAAacUC 946 AD-15134
    2761 fCfGGATsT CAGgccTsT
    2743- GgCcUgGaGuUuAuUcGgATsT 947 UCCGAauAAacUC 948 AD-15144
    2761 CAGgccTsT
    2753- GCAGGAUUCUUCCCAUGGATT 949 UCCAUGGGAAGAA 950 AD-15391
    2771 UCCUGCTT
    2794- UGCAGGGACAAACAUCGUUTT 951 AACGAUGUUUGUC 952 AD-15348
    2812 CCUGCATT
    2795- GCAGGGACAAACAUCGUUGTT 953 CAACGAUGUUUGU 954 AD-15349
    2813 CCCUGCTT
    2797- AGGGACAAACAUCGUUGGGTT 955 CCCAACGAUGUUU 956 AD-15170
    2815 GUCCCUTT
    2841- CCCUCAUCUCCAGCUAACUTT 957 AGUUAGCUGGAGA 958 AD-15350
    2859 UGAGGGTT
    2845- CAUCUCCAGCUAACUGUGGTT 959 CCACAGUUAGCUG 960 AD-15402
    2863 GAGAUGTT
    2878- GCUCCCUGAUUAAUGGAGGTT 961 CCUCCAUUAAUCA 962 AD-15293
    2896 GGGAGCTT
    2881- CCCUGAUUAAUGGAGGCUUTT 963 AAGCCUCCAUUAA 964 AD-15351
    2899 UCAGGGTT
    2882- CCUGAUUAAUGGAGGCUUATT 965 UAAGCCUCCAUUA 966 AD-15403
    2900 AUCAGGTT
    2884- UGAUUAAUGGAGGCUUAGCTT 967 GCUAAGCCUCCAU 968 AD-15404
    2902 UAAUCATT
    2885- GAUUAAUGGAGGCUUAGCUTT 969 AGCUAAGCCUCCA 970 AD-15207
    2903 UUAAUCTT
    2886- AUUAAUGGAGGCUUAGCUUTT 971 AAGCUAAGCCUCC 972 AD-15352
    2904 AUUAAUTT
    2887- UUAAUGGAGGCUUAGCUUUTT 973 AAAGCUAAGCCUC 974 AD-15255
    2905 CAUUAATT
    2903- UUUCUGGAUGGCAUCUAGCTsT 975 GCUAGAUGCCAUC 976 AD-9603
    2921 CAGAAATsT
    2903- uuucuGGAuGGcAucuAGcTsT 977 GCuAGAUGCcAUC 978 AD-9729
    2921 cAGAAATsT
    2904- UUCUGGAUGGCAUCUAGCCTsT 979 GGCUAGAUGCCAU 980 AD-9599
    2922 CCAGAATsT
    2904- uucuGGAuGGcAucuAGccTsT 981 GGCuAGAUGCcAU 982 AD-9725
    2922 CcAGAATsT
    2905- UCUGGAUGGCAUCUAGCCATsT 983 UGGCUAGAUGCCA 984 AD-9621
    2923 UCCAGATsT
    2905- ucuGGAuGGcAucuAGccATsT 985 UGGCuAGAUGCcA 986 AD-9747
    2923 UCcAGATsT
    2925- AGGCUGGAGACAGGUGCGCTT 987 GCGCACCUGUCUC 988 AD-15405
    2943 CAGCCUTT
    2926- GGCUGGAGACAGGUGCGCCTT 989 GGCGCACCUGUCU 990 AD-15353
    2944 CCAGCCTT
    2927- GCUGGAGACAGGUGCGCCCTT 991 GGGCGCACCUGUC 992 AD-15354
    2945 UCCAGCTT
    2972- UUCCUGAGCCACCUUUACUTT 993 AGUAAAGGUGGCU 994 AD-15406
    2990 CAGGAATT
    2973- UCCUGAGCCACCUUUACUCTT 995 GAGUAAAGGUGGC 996 AD-15407
    2991 UCAGGATT
    2974- CCUGAGCCACCUUUACUCUTT 997 AGAGUAAAGGUGG 998 AD-15355
    2992 CUCAGGTT
    2976- UGAGCCACCUUUACUCUGCTT 999 GCAGAGUAAAGGU 1000 AD-15356
    2994 GGCUCATT
    2978- AGCCACCUUUACUCUGCUCTT 1001 GAGCAGAGUAAAG 1002 AD-15357
    2996 GUGGCUTT
    2981- CACCUUUACUCUGCUCUAUTT 1003 AUAGAGCAGAGUA 1004 AD-15269
    2999 AAGGUGTT
    2987- UACUCUGCUCUAUGCCAGGTsT 1005 CCUGGCAUAGAGC 1006 AD-9565
    3005 AGAGUATsT
    2987- uAcucuGcucuAuGccAGGTsT 1007 CCUGGcAuAGAGc 1008 AD-9691
    3005 AGAGuATsT
    2998- AUGCCAGGCUGUGCUAGCATT 1009 UGCUAGCACAGCC 1010 AD-15358
    3016 UGGCAUTT
    3003- AGGCUGUGCUAGCAACACCTT 1011 GGUGUUGCUAGCA 1012 AD-15359
    3021 CAGCCUTT
    3006- CUGUGCUAGCAACACCCAATT 1013 UUGGGUGUUGCUA 1014 AD-15360
    3024 GCACAGTT
    3010- GCUAGCAACACCCAAAGGUTT 1015 ACCUUUGGGUGUU 1016 AD-15219
    3028 GCUAGCTT
    3038- GGAGCCAUCACCUAGGACUTT 1017 AGUCCUAGGUGAU 1018 AD-15361
    3056 GGCUCCTT
    3046- CACCUAGGACUGACUCGGCTT 1019 GCCGAGUCAGUCC 1020 AD-15273
    3064 UAGGUGTT
    3051- AGGACUGACUCGGCAGUGUTT 1021 ACACUGCCGAGUC 1022 AD-15362
    3069 AGUCCUTT
    3052- GGACUGACUCGGCAGUGUGTT 1023 CACACUGCCGAGU 1024 AD-15192
    3070 CAGUCCTT
    3074- UGGUGCAUGCACUGUCUCATT 1025 UGAGACAGUGCAU 1026 AD-15256
    3092 GCACCATT
    3080- AUGCACUGUCUCAGCCAACTT 1027 GUUGGCUGAGACA 1028 AD-15363
    3098 GUGCAUTT
    3085- CUGUCUCAGCCAACCCGCUTT 1029 AGCGGGUUGGCUG 1030 AD-15364
    3103 AGACAGTT
    3089- CUCAGCCAACCCGCUCCACTsT 1031 GUGGAGCGGGUUG 1032 AD-9604
    3107 GCUGAGTsT
    3089- cucAGccAAcccGcuccAcTsT 1033 GUGGAGCGGGUUG 1034 AD-9730
    3107 GCUGAGTsT
    3093- GCCAACCCGCUCCACUACCTsT 1035 GGUAGUGGAGCGG 1036 AD-9527
    3111 GUUGGCTsT
    3093- GccAAcccGcuccAcuAccTsT 1037 GGuAGUGGAGCGG 1038 AD-9653
    3111 GUUGGCTsT
    3096- AACCCGCUCCACUACCCGGTT 1039 CCGGGUAGUGGAG 1040 AD-15365
    3114 CGGGUUTT
    3099- CCGCUCCACUACCCGGCAGTT 1041 CUGCCGGGUAGUG 1042 AD-15294
    3117 GAGCGGTT
    3107- CUACCCGGCAGGGUACACATT 1043 UGUGUACCCUGCC 1044 AD-15173
    3125 GGGUAGTT
    3108- UACCCGGCAGGGUACACAUTT 1045 AUGUGUACCCUGC 1046 AD-15366
    3126 CGGGUATT
    3109- ACCCGGCAGGGUACACAUUTT 1047 AAUGUGUACCCUG 1048 AD-15367
    3127 CCGGGUTT
    3110- CCCGGCAGGGUACACAUUCTT 1049 GAAUGUGUACCCU 1050 AD-15257
    3128 GCCGGGTT
    3112- CGGCAGGGUACACAUUCGCTT 1051 GCGAAUGUGUACC 1052 AD-15184
    3130 CUGCCGTT
    3114- GCAGGGUACACAUUCGCACTT 1053 GUGCGAAUGUGUA 1054 AD-15185
    3132 CCCUGCTT
    3115- CAGGGUACACAUUCGCACCTT 1055 GGUGCGAAUGUGU 1056 AD-15258
    3133 ACCCUGTT
    3116- AGGGUACACAUUCGCACCCTT 1057 GGGUGCGAAUGUG 1058 AD-15186
    3134 UACCCUTT
    3196- GGAACUGAGCCAGAAACGCTT 1059 GCGUUUCUGGCUC 1060 AD-15274
    3214 AGUUCCTT
    3197- GAACUGAGCCAGAAACGCATT 1061 UGCGUUUCUGGCU 1062 AD-15368
    3215 CAGUUCTT
    3198- AACUGAGCCAGAAACGCAGTT 1063 CUGCGUUUCUGGC 1064 AD-15369
    3216 UCAGUUTT
    3201- UGAGCCAGAAACGCAGAUUTT 1065 AAUCUGCGUUUCU 1066 AD-15370
    3219 GGCUCATT
    3207- AGAAACGCAGAUUGGGCUGTT 1067 CAGCCCAAUCUGC 1068 AD-15259
    3225 GUUUCUTT
    3210- AACGCAGAUUGGGCUGGCUTT 1069 AGCCAGCCCAAUC 1070 AD-15408
    3228 UGCGUUTT
    3233- AGCCAAGCCUCUUCUUACUTsT 1071 AGUAAGAAGAGGC 1072 AD-9597
    3251 UUGGCUTsT
    3233- AGccAAGccucuucuuAcuTsT 1073 AGuAAGAAGAGGC 1074 AD-9723
    3251 UUGGCUTsT
    3233- AfgCfcAfaGfcCfuCfuUfcU 1075 P*aGfuAfaGfaA 1076 AD-14680
    3251 fuAfcUfTsT fgAfgGfcUfuGf
    gCfuTsT
    3233- AGCfCfAAGCfCfUfCfUfUfC 1077 AGUfAAGAAGAGG 1078 AD-14690
    3251 fUfUfACfUfTsT CfUfUfGGCfUfT
    sT
    3233- AgCcAaGcCuCuUcUuAcUTsT 1079 P*aGfuAfaGfaA 1080 AD-14700
    3251 fgAfgGfcUfuGf
    gCfuTsT
    3233- AgCcAaGcCuCuUcUuAcUTsT 1081 AGUfAAGAAGAGG 1082 AD-14710
    3251 CfUfUfGGCfUfT
    sT
    3233- AfgCfcAfaGfcCfuCfuUfcU 1083 AGUAAgaAGagGC 1084 AD-14720
    3251 fuAfcUfTsT UUGgcuTsT
    3233- AGCfCfAAGCfCfUfCfUfUfC 1085 AGUAAgaAGagGC 1086 AD-14730
    3251 fUfUfACfUfTsT UUGgcuTsT
    3233- AgCcAaGcCuCuUcUuAcUTsT 1087 AGUAAgaAGagGC 1088 AD-14740
    3251 UUGgcuTsT
    3233- UfgGfuUfcCfcUfgAfgGfaC 1089 P*gCfuGfgUfcC 1090 AD-15086
    3251 fcAfgCfTsT fuCfaGfgGfaAf
    cCfaTsT
    3233- UfGGUfUfCfCfCfUfGAGGAC 1091 GCfUfGGUfCfCf 1092 AD-15096
    3251 fCfAGCfTsT UfCfAGGGAACfC
    fATsT
    3233- UgGuUcCcUgAgGaCcAgCTsT 1093 P*gCfuGfgUfcC 1094 AD-15106
    3251 fuCfaGfgGfaAf
    cCfaTsT
    3233- UgGuUcCcUgAgGaCcAgCTsT 1095 GCfUfGGUfCfCf 1096 AD-15116
    3251 UfCfAGGGAACfC
    fATsT
    3233- UfgGfuUfcCfcUfgAfgGfaC 1097 GCUGGucCUcaGG 1098 AD-15126
    3251 fcAfgCfTsT GAAccaTsT
    3233- UfGGUfUfCfCfCfUfGAGGAC 1099 GCUGGucCUcaGG 1100 AD-15136
    3251 fCfAGCfTsT GAAccaTsT
    3233- UgGuUcCcUgAgGaCcAgCTsT 1101 GCUGGucCUcaGG 1102 AD-15146
    3251 GAAccaTsT
    3242- UCUUCUUACUUCACCCGGCTT 1103 GCCGGGUGAAGUA 1104 AD-15260
    3260 AGAAGATT
    3243- CUUCUUACUUCACCCGGCUTT 1105 AGCCGGGUGAAGU 1106 AD-15371
    3261 AAGAAGTT
    3244- UUCUUACUUCACCCGGCUGTT 1107 CAGCCGGGUGAAG 1108 AD-15372
    3262 UAAGAATT
    3262- GGGCUCCUCAUUUUUACGGTT 1109 CCGUAAAAAUGAG 1110 AD-15172
    3280 GAGCCCTT
    3263- GGCUCCUCAUUUUUACGGGTT 1111 CCCGUAAAAAUGA 1112 AD-15295
    3281 GGAGCCTT
    3264- GCUCCUCAUUUUUACGGGUTT 1113 ACCCGUAAAAAUG 1114 AD-15373
    3282 AGGAGCTT
    3265- CUCCUCAUUUUUACGGGUATT 1115 UACCCGUAAAAAU 1116 AD-15163
    3283 GAGGAGTT
    3266- UCCUCAUUUUUACGGGUAATT 1117 UUACCCGUAAAAA 1118 AD-15165
    3284 UGAGGATT
    3267- CCUCAUUUUUACGGGUAACTT 1119 GUUACCCGUAAAA 1120 AD-15374
    3285 AUGAGGTT
    3268- CUCAUUUUUACGGGUAACATT 1121 UGUUACCCGUAAA 1122 AD-15296
    3286 AAUGAGTT
    3270- CAUUUUUACGGGUAACAGUTT 1123 ACUGUUACCCGUA 1124 AD-15261
    3288 AAAAUGTT
    3271- AUUUUUACGGGUAACAGUGTT 1125 CACUGUUACCCGU 1126 AD-15375
    3289 AAAAAUTT
    3274- UUUACGGGUAACAGUGAGGTT 1127 CCUCACUGUUACC 1128 AD-15262
    3292 CGUAAATT
    3308- CAGACCAGGAAGCUCGGUGTT 1129 CACCGAGCUUCCU 1130 AD-15376
    3326 GGUCUGTT
    3310- GACCAGGAAGCUCGGUGAGTT 1131 CUCACCGAGCUUC 1132 AD-15377
    3328 CUGGUCTT
    3312- CCAGGAAGCUCGGUGAGUGTT 1133 CACUCACCGAGCU 1134 AD-15409
    3330 UCCUGGTT
    3315- GGAAGCUCGGUGAGUGAUGTT 1135 CAUCACUCACCGA 1136 AD-15378
    3333 GCUUCCTT
    3324- GUGAGUGAUGGCAGAACGATT 1137 UCGUUCUGCCAUC 1138 AD-15410
    3342 ACUCACTT
    3326- GAGUGAUGGCAGAACGAUGTT 1139 CAUCGUUCUGCCA 1140 AD-15379
    3344 UCACUCTT
    3330- GAUGGCAGAACGAUGCCUGTT 1141 CAGGCAUCGUUCU 1142 AD-15187
    3348 GCCAUCTT
    3336- AGAACGAUGCCUGCAGGCATT 1143 UGCCUGCAGGCAU 1144 AD-15263
    3354 CGUUCUTT
    3339- ACGAUGCCUGCAGGCAUGGTT 1145 CCAUGCCUGCAGG 1146 AD-15264
    3357 CAUCGUTT
    3348- GCAGGCAUGGAACUUUUUCTT 1147 GAAAAAGUUCCAU 1148 AD-15297
    3366 GCCUGCTT
    3356- GGAACUUUUUCCGUUAUCATT 1149 UGAUAACGGAAAA 1150 AD-15208
    3374 AGUUCCTT
    3357- GAACUUUUUCCGUUAUCACTT 1151 GUGAUAACGGAAA 1152 AD-15209
    3375 AAGUUCTT
    3358- AACUUUUUCCGUUAUCACCTT 1153 GGUGAUAACGGAA 1154 AD-15193
    3376 AAAGUUTT
    3370- UAUCACCCAGGCCUGAUUCTT 1155 GAAUCAGGCCUGG 1156 AD-15380
    3388 GUGAUATT
    3378- AGGCCUGAUUCACUGGCCUTT 1157 AGGCCAGUGAAUC 1158 AD-15298
    3396 AGGCCUTT
    3383- UGAUUCACUGGCCUGGCGGTT 1159 CCGCCAGGCCAGU 1160 AD-15299
    3401 GAAUCATT
    3385- AUUCACUGGCCUGGCGGAGTT 1161 CUCCGCCAGGCCA 1162 AD-15265
    3403 GUGAAUTT
    3406- GCUUCUAAGGCAUGGUCGGTT 1163 CCGACCAUGCCUU 1164 AD-15381
    3424 AGAAGCTT
    3407- CUUCUAAGGCAUGGUCGGGTT 1165 CCCGACCAUGCCU 1166 AD-15210
    3425 UAGAAGTT
    3429- GAGGGCCAACAACUGUCCCTT 1167 GGGACAGUUGUUG 1168 AD-15270
    3447 GCCCUCTT
    3440- ACUGUCCCUCCUUGAGCACTsT 1169 GUGCUCAAGGAGG 1170 AD-9591
    3458 GACAGUTsT
    3440- AcuGucccuccuuGAGcAcTsT 1171 GUGCUcAAGGAGG 1172 AD-9717
    3458 GAcAGUTsT
    3441- CUGUCCCUCCUUGAGCACCTsT 1173 GGUGCUCAAGGAG 1174 AD-9622
    3459 GGACAGTsT
    3441- cuGucccuccuuGAGcAccTsT 1175 GGUGCUcAAGGAG 1176 AD-9748
    3459 GGAcAGTsT
    3480- ACAUUUAUCUUUUGGGUCUTsT 1177 AGACCCAAAAGAU 1178 AD-9587
    3498 AAAUGUTsT
    3480- AcAuuuAucuuuuGGGucuTsT 1179 AGACCcAAAAGAu 1180 AD-9713
    3498 AAAUGUTsT
    3480- AfcAfuUfuAfuCfuUfuUfgG 1181 P*aGfaCfcCfaA 1182 AD-14679
    3498 fgUfcUfTsT faAfgAfuAfaAf
    uGfuTsT
    3480- ACfAUfUfUfAUfCfUfUfUfU 1183 AGACfCfCfAAAA 1184 AD-14689
    3498 fGGGUfCfUfTsT GAUfAAAUfGUfT
    sT
    3480- AcAuUuAuCuUuUgGgUcUTsT 1185 P*aGfaCfcCfaA 1186 AD-14699
    3498 faAfgAfuAfaAf
    uGfuTsT
    3480- AcAuUuAuCuUuUgGgUcUTsT 1187 AGACfCfCfAAAA 1188 AD-14709
    3498 GAUfAAAUfGUfT
    sT
    3480- AfcAfuUfuAfuCfuUfuUfgG 1189 AGACCcaAAagAU 1190 AD-14719
    3498 fgUfcUfTsT AAAuguTsT
    3480- ACfAUfUfUfAUfCfUfUfUfU 1191 AGACCcaAAagAU 1192 AD-14729
    3498 fGGGUfCfUfTsT AAAuguTsT
    3480- AcAuUuAuCuUuUgGgUcUTsT 1193 AGACCcaAAagAU 1194 AD-14739
    3498 AAAuguTsT
    3480- GfcCfaUfcUfgCfuGfcCfgG 1195 P*gGfcUfcCfgG 1196 AD-15085
    3498 faGfcCfTsT fcAfgCfaGfaUf
    gGfcTsT
    3480- GCfCfAUfCfUfGCfUfGCfCf 1197 GGCfUfCfCfGGC 1198 AD-15095
    3498 GGAGCfCfTsT fAGCfAGAUfGGC
    fTsT
    3480- GcCaUcUgCuGcCgGaGcCTsT 1199 P*gGfcUfcCfgG 1200 AD-15105
    3498 fcAfgCfaGfaUf
    gGfcTsT
    3480- GcCaUcUgCuGcCgGaGcCTsT 1201 GGCfUfCfCfGGC 1202 AD-15115
    3498 fAGCfAGAUfGGC
    fTsT
    3480- GfcCfaUfcUfgCfuGfcCfgG 1203 GGCUCauGCagCA 1204 AD-15125
    3498 faGfcCfTsT GAUggcTsT
    3480- GCfCfAUfCfUfGCfUfGCfCf 1205 GGCUCauGCagCA 1206 AD-15135
    3498 GGAGCfCfTsT GAUggcTsT
    3480- GcCaUcUgCuGcCgGaGcCTsT 1207 GGCUCauGCagCA 1208 AD-15145
    3498 GAUggcTsT
    3481- CAUUUAUCUUUUGGGUCUGTsT 1209 CAGACCCAAAAGA 1210 AD-9578
    3499 UAAAUGTsT
    3481- cAuuuAucuuuuGGGucuGTsT 1211 cAGACCcAAAAGA 1212 AD-9704
    3499 uAAAUGTsT
    3485- UAUCUUUUGGGUCUGUCCUTsT 1213 AGGACAGACCCAA 1214 AD-9558
    3503 AAGAUATsT
    3485- uAucuuuuGGGucuGuccuTsT 1215 AGGAcAGACCcAA 1216 AD-9684
    3503 AAGAuATsT
    3504- CUCUGUUGCCUUUUUACAGTsT 1217 CUGUAAAAAGGCA 1218 AD-9634
    3522 ACAGAGTsT
    3504- cucuGuuGccuuuuuAcAGTsT 1219 CUGuAAAAAGGcA 1220 AD-9760
    3522 AcAGAGTsT
    3512- CCUUUUUACAGCCAACUUUTT 1221 AAAGUUGGCUGUA 1222 AD-15411
    3530 AAAAGGTT
    3521- AGCCAACUUUUCUAGACCUTT 1223 AGGUCUAGAAAAG 1224 AD-15266
    3539 UUGGCUTT
    3526- ACUUUUCUAGACCUGUUUUTT 1225 AAAACAGGUCUAG 1226 AD-15382
    3544 AAAAGUTT
    3530- UUCUAGACCUGUUUUGCUUTsT 1227 AAGCAAAACAGGU 1228 AD-9554
    3548 CUAGAATsT
    3530- uucuAGAccuGuuuuGcuuTsT 1229 AAGcAAAAcAGGU 1230 AD-9680
    3548 CuAGAATsT
    3530- UfuCfuAfgAfcCfuGfuUfuU 1231 P*aAfgCfaAfaA 1232 AD-14676
    3548 fgCfuUfTsT fcAfgGfuCfuAf
    gAfaTsT
    3530- UfUfCfUfAGACfCfUfGUfUf 1233 AAGCfAAAACfAG 1234 AD-14686
    3548 UfUfGCfUfUfTsT GUfCfUfAGAATs
    T
    3530- UuCuAgAcCuGuUuUgCuUTsT 1235 P*aAfgCfaAfaA 1236 AD-14696
    3548 fcAfgGfuCfuAf
    gAfaTsT
    3530- UuCuAgAcCuGuUuUgCuUTsT 1237 AAGCfAAAACfAG 1238 AD-14706
    3548 GUfCfUfAGAATs
    T
    3530- UfuCfuAfgAfcCfuGfuUfuU 1239 AAGcAaaACagGU 1240 AD-14716
    3548 ffCfuUfTsT CUAgaaTsT
    3530- UfUfCfUfAGACfCfUfGUfUf 1241 AAGcAaaACagGU 1242 AD-14726
    3548 UfUfGCfUfUfTsT CUAgaaTsT
    3530- UuCuAgAcCuGuUuUgCuUTsT 1243 AAGcAaaACagGU 1244 AD-14736
    3548 CUAgaaTsT
    3530- CfaUfaGfgCfcUfgGfaGfuU 1245 P*aAfuAfaAfcU 1246 AD-15082
    3548 fuAfuUfTsT fcCfaGfgCfcUf
    aUfgTsT
    3530- CfAUfAGGCfCfUfGGAGUfUf 1247 AAUfAAACfUfCf 1248 AD-15092
    3548 UfAUfUfTsT CfAGGCfCfUfAU
    fGTsT
    3530- CaUaGgCcUgGaGuUuAuUTsT 1249 P*aAfuAfaAfcU 1250 AD-15102
    3548 fcCfaGfgCfcUf
    aUfgTsT
    3530- CaUaGgCcUgGaGuUuAuUTsT 1251 AAUfAAACfUfCf 1252 AD-15112
    3548 CfAGGCfCfUfAU
    fGTsT
    3530- CfaUfaGfgCfcUfgGfaGfuU 1253 AAUAAacUCcaGG 1254 AD-15122
    3548 fuAfuUfTsT CCUaugTsT
    3530- CfAUfAGGCfCfUfGGAGUfUf 1255 AAUAAacUCcaGG 1256 AD-15132
    3548 UfAUfUfTsT CCUaugTsT
    3530- CaUaGgCcUgGaGuUuAuUTsT 1257 AAUAAacUCcaGG 1258 AD-15142
    3548 CCUaugTsT
    3531- UCUAGACCUGUUUUGCUUUTsT 1259 AAAGCAAAACAGG 1260 AD-9553
    3549 UCUAGATsT
    3531- ucuAGAccuGuuuuGcuuuTsT 1261 AAAGcAAAAcAGG 1262 AD-9679
    3549 UCuAGATsT
    3531- UfcUfaGfaCfcUfgUfuUfuG 1263 P*aAfaGfcAfaA 1264 AD-14675
    3549 fcUfuUfTsT faCfaGfgUfcUf
    aGfaTsT
    3531- UfCfUfAGACfCfUfGUfUfUf 1265 AAAGCfAAAACfA 1266 AD-14685
    3549 UfGCfUfUfUfTsT GGUfCfUfAGATs
    T
    3531- UcUaGaCcUgUuUuGcUuUTsT 1267 P*aAfaGfcAfaA 1268 AD-14695
    3549 faCfaGfgUfcUf
    aGfaTsT
    3531- UcUaGaCcUgUuUuGcUuUTsT 1269 AAAGCfAAAACfA 1270 AD-14705
    3549 GGUfCfUfAGATs
    T
    3531- UfcUfaGfaCfcUfgUfuUfuG 1271 AAAGCaaAAcaGG 1272 AD-14715
    3549 fcUfuUfTsT UCUagaTsT
    3531- UfCfUfAGACfCfUfGUfUfUf 1273 AAAGCaaAAcaGG 1274 AD-14725
    3549 UfGCfUfUfUfTsT UCUagaTsT
    3531- UcUaGaCcUgUuUuGcUuUTsT 1275 AAAGCaaAAcaGG 1276 AD-14735
    3549 UCUagaTsT
    3531- UfcAfuAfgGfcCfuGfgAfgU 1277 P*aUfaAfaCfuC 1278 AD-15081
    3549 fuUfaUfTsT fcAfgGfcCfuAf
    uGfaTsT
    3531- UfCfAUfAGGCfCfUfGGAGUf 1297 AUfAAACfUfCfC 1280 AD-15091
    3549 UfUfAUfTsT fAGGCfCfUfAUf
    GATsT
    3531- UcAuAgGcCuGgAgUuUaUTsT 1281 P*aUfaAfaCfuC 1282 AD-15101
    3549 fcAfgGfcCfuAf
    uGfaTsT
    3531- UcAuAgGcCuGgAgUuUaUTsT 1283 AUfAAACfUfCfC 1284 AD-15111
    3549 fAGGCfCfUfAUf
    GATsT
    3531- UfcAfuAfgGfcCfuGfgAfgU 1285 AUAAAcuCCagGC 1286 AD-15121
    3549 fuUfaUfTsT CUAugaTsT
    3531- UfCfAUfAGGCfCfUfGGAGUf 1287 AUAAAcuCCagGC 1288 AD-15131
    3549 UfUfAUfTsT CUAugaTsT
    3531- UcAuAgGcCuGgAgUuUaUTsT 1289 AUAAAcuCCagGC 1290 AD-15141
    3549 CUAugaTsT
    3557- UGAAGAUAUUUAUUCUGGGTsT 1291 CCCAGAAUAAAUA 1292 AD-9626
    3575 UCUUCATsT
    3557- uGAAGAuAuuuAuucuGGGTsT 1293 CCcAGAAuAAAuA 1294 AD-9752
    3575 UCUUcATsT
    3570- UCUGGGUUUUGUAGCAUUUTsT 1295 AAAUGCUACAAAA 1296 AD-9629
    3588 CCCAGATsT
    3570- ucuGGGuuuuGuAGcAuuuTsT 1297 AAAUGCuAcAAAA 1298 AD-9755
    3588 CCcAGATsT
    3613- AUAAAAACAAACAAACGUUTT 1299 AACGUUUGUUUGU 1300 AD-15412
    3631 UUUUAUTT
    3617- AAACAAACAAACGUUGUCCTT 1301 GGACAACGUUUGU 1302 AD-15211
    3635 UUGUUUTT
    3618- AACAAACAAACGUUGUCCUTT 1303 AGGACAACGUUUG 1304 AD-15300
    3636 UUUGUUTT
    *Target: target in human PCSK9 gene, access.
    # NM_174936
    U, C, A, G: corresponding ribonucleotide;
    T: deoxythymidine;
    u, c, a, g: corresponding 2′-O-methyl ribonucleotide;
    Uf, Cf, Af, Gf: corresponding 2′-deoxy-2′-fluoro ribonucleotide;
    where nucleotides are written in sequence, they are connected by 3′-5′ phosphodiester groups;
    nucleotides with interjected “s” are connected by 3′-O-5′-O phosphorothiodiester groups;
    unless denoted by prefix “P*”, oligonucleotides are devoid of a 5′-phosphate group on the 5′-most nucleotide;
    all oligonucleotides bear 3′-OH on the 3′-most nucleotide.
  • TABLE 5
    Sequences of modified dsRNA targeted to PCSK9
    U, C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g:
    corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding 2′-deoxy-
    2′-fluoro ribonucleotide; where nucleotides are written in sequence, they are
    connected by 3′-5′ phosphodiester groups; nucleotides with interjected “s” are
    connected by 3′-O-5′-O phosphorothiodiester groups; unless denoted by prefix “P*”, 
    oligonucleotides are devoid of a 5′-phosphate group on the 5′-most nucleotide; all
    oligonucleotides bear 3′-OH on the 3′-most nucleotide.
    Sense strand sequence  SEQ ID Antisense-strand SEQ ID
    Duplex # (5′-3′)1 NO: sequence (5′-3′)1 NO:
    AD-10792 GccuGGAGuuuAuucGGAATsT 1305 UUCCGAAuAAACUCcAGGCT 1306
    sT
    AD-10793 GccuGGAGuuuAuucGGAATsT 1307 uUcCGAAuAAACUccAGGCT 1308
    sT
    AD-10796 GccuGGAGuuuAuucGGAATsT 1309 UUCCGAAUAAACUCCAGGCT 1310
    sT
    AD-12038 GccuGGAGuuuAuucGGAATsT 1311 uUCCGAAUAAACUCCAGGCT 1312
    sT
    AD-12039 GccuGGAGuuuAuucGGAATsT 1313 UuCCGAAUAAACUCCAGGCT 1314
    sT
    AD-12040 GccuGGAGuuuAuucGGAATsT 1315 UUcCGAAUAAACUCCAGGCT 1316
    sT
    AD-12041 GccuGGAGuuuAuucGGAATsT 1317 UUCcGAAUAAACUCCAGGCT 1318
    sT
    AD-12042 GCCUGGAGUUUAUUCGGAATsT 1319 uUCCGAAUAAACUCCAGGCT 1320
    sT
    AD-12043 GCCUGGAGUUUAUUCGGAATsT 1321 UuCCGAAUAAACUCCAGGCT 1322
    sT
    AD-12044 GCCUGGAGUUUAUUCGGAATsT 1323 UUcCGAAUAAACUCCAGGCT 1324
    sT
    AD-12045 GCCUGGAGUUUAUUCGGAATsT 1325 UUCcGAAUAAACUCCAGGCT 1326
    sT
    AD-12046 GccuGGAGuuuAuucGGAA 1327 UUCCGAAUAAACUCCAGGCs 1328
    csu
    AD-12047 GccuGGAGuuuAuucGGAAA 1329 UUUCCGAAUAAACUCCAGGC 1330
    scsu
    AD-12048 GccuGGAGuuuAuucGGAAAA 1331 UUUUCCGAAUAAACUCCAGG 1332
    Cscsu
    AD-12049 GccuGGAGuuuAuucGGAAAAG 1333 CUUUUCCGAAUAAACUCCAG 1334
    GCscsu
    AD-12050 GccuGGAGuuuAuucGGAATTab 1335 UUCCGAAUAAACUCCAGGCT 1336
    Tab
    AD-12051 GccuGGAGuuuAuucGGAAATTab 1337 UUUCCGAAuAAACUCCAGGC 1338
    TTab
    AD-12052 GccuGGAGuuuAuucGGAAAATTab 1339 UUUUCCGAAUAAACUCCAGG 1340
    CTTab
    AD-12053 GccuGGAGuuuAuucGGAAAAGTTab 1341 CUUUUCCGAAUAAACUCCAG 1342
    GCTTab
    AD-12054 GCCUGGAGUUUAUUCGGAATsT 1343 UUCCGAAUAAACUCCAGGCs 1344
    csu
    AD-12055 GccuGGAGuuuAuucGGAATsT 1345 UUCCGAAUAAACUCCAGGCs 1346
    csu
    AD-12056 GcCuGgAgUuUaUuCgGaA 1347 UUCCGAAUAAACUCCAGGCT 1348
    Tab
    AD-12057 GcCuGgAgUuUaUuCgGaA 1349 UUCCGAAUAAACUCCAGGCT 1350
    sT
    AD-12058 GcCuGgAgUuUaUuCgGaA 1351 UUCCGAAuAAACUCcAGGCT 1352
    sT
    AD-12059 GcCuGgAgUuUaUuCgGaA 1353 uUcCGAAuAAACUccAGGCT 1354
    sT
    AD-12060 GcCuGgAgUuUaUuCgGaA 1355 UUCCGaaUAaaCUCCAggc 1356
    AD-12061 GcCuGgnAgUuUaUuCgGaATsT 1357 UUCCGaaUAaaCUCCAggcT 1358
    sT
    AD-12062 GcCuGgAgUuUaUuCgGaATTab 1359 UUCCGaaUAaaCUCCAggcT 1360
    Tab
    AD-12063 GcCuGgAgUuUaUuCgGaA 1361 UUCCGaaUAaaCUCCAggcs 1362
    csu
    AD-12064 GcCuGgnAgUuUaUuCgGaATsT 1363 UUCCGAAuAAACUCcAGGCT 1364
    sT
    AD-12065 GcCuGgAgUuUaUuCgGaATTab 1365 UUCCGAAuAAACUCcAGGCT 1366
    Tab
    AD-12066 GcCuGgAgUuUaUuCgGaA 1367 UUCCGAAuAAACUCcAGGCs 1368
    csu
    AD-12067 GcCuGgnAgUuUaUuCgGaATsT 1369 UUCCGAAUAAACUCCAGGCT 1370
    sT
    AD-12068 GcCuGgAgUuUaUuCgGaATTab 1371 UUCCGAAUAAACUCCAGGCT 1372
    Tab
    AD-12069 GcCuGgAgUuUaUuCgGaA 1373 UUCCGAAUAAACUCCAGGCs 1374
    csu
    AD-12338 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1375 P*uUfcCfgAfaUfaAfaCf 1376
    f uCfcAfgGfc
    AD-12339 GcCuGgAgUuUaUuCgGaA 1377 P*uUfcCfgAfaUfaAfaCf 1378
    uCfcAfgGfc
    AD-12340 GccuGGAGuuuAuucGGAA 1379 P*uUfcCfgAfaUfaAfaCf 1380
    uCfcAfgGfc
    AD-12341 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1381 P*uUfcCfgAfaUfaAfaCf 1382
    fTsT uCfcAfgGfcTsT
    AD-12342 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1383 UUCCGAAuAAACUCcAGGCT 1384
    fTsT sT
    AD-12343 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1385 uUcCGAAuAAACUccAGGCT 1386
    fTsT sT
    AD-12344 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1387 UUCCGAAUAAACUCCAGGCT 1388
    fTsT sT
    AD-12345 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1389 UUCCGAAUAAACUCCAGGCs 1390
    fTsT csu
    AD-12346 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1391 UUCCGaaUAaaCUCCAggcs 1392
    fTsT csu
    AD-12347 GCCUGGAGUUUAUUCGGAATsT 1393 P*uUfcCfgAfaUfaAfaCf 1394
    uCfcAfgGfcTsT
    AD-12348 GccuGGAGuuuAuucGGAATsT 1395 P*uUfcCfgAfaUfaAfaCf 1396
    uCfcAfgGfcTsT
    AD-12349 GcCuGgnAgUuUaUuCgGaATsT 1397 P*uUfcCfgAfaUfaAfaCf 1398
    uCfcAfgGfcTsT
    AD-12350 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1399 P*uUfcCfgAfaUfaAfaCf 1400
    fTTab uCfcAfgGfcTTab
    AD-12351 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1401 P*uUfcCfgAfaUfaAfaCf 1402
    f uCfcAfgGfcsCfsu
    AD-12352 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1403 UUCCGaaUAaaCUCCAggcs 1404
    f csu
    AD-12354 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1405 UUCCGAAUAAACUCCAGGCs 1406
    f csu
    AD-12355 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1407 UUCCGAAuAAACUCcAGGCT 1408
    f sT
    AD-12356 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1409 uUcCGAAuAAACUccAGGCT 1410
    f sT
    AD-12357 GmocCmouGmogAm02gUmouUmoaUmo 1411 UUCCGaaUAaaCUCCAggc 1412
    uCmogGmoaA
    AD-12358 GmocCmouGmogAm02gUmouUmoaUmo 1413 P*uUfcCfgAfaUfaAfaCf 1414
    uCmogGmoaA uCfcAfgGfc
    AD-12359 GmocCmouGmogAm02gUmouUmoaUmo 1415 P*uUfcCfgAfaUfaAfaCf 1416
    uCmogGmoaA uCfcAfgGfcsCfsu
    AD-12360 GmocCmouGmogAm02gUmouUmoaUmo 1417 UUCCGAAUAAACUCCAGGCs 1418
    uCmogGmoaA csu
    AD-12361 GmocCmouGmogAm02gUmouUmoaUmo 1419 UUCCGAAuAAACUCcAGGCT 1420
    uCmogGmoaA sT
    AD-12362 GmocCmouGmogAm02gUmouUmoaUmo 1421 uUcCGAAuAAACUccAGGCT 1422
    uCmogGmoaA sT
    AD-12363 GmocCmouGmogAm02gUmouUmoaUmo 1423 UUCCGaaUAaaCUCCAggcs 1424
    uCmogGmoaA csu
    AD-12364 GmocCmouGmogAmogUmouUmoaUmou 1425 UUCCGaaUAaaCUCCAggcT 1426
    CmogGmoaATsT sT
    AD-12365 GmocCmouGmogAmogUmouUmoaUmou 1427 UUCCGAAuAAACUCcAGGCT 1428
    CmogGmoaATsT sT
    AD-12366 GmocCmouGmogAmogUmouUmoaUmou 1429 UUCCGAAUAAACUCCAGGCT 1430
    CmogGmoaATsT sT
    AD-12367 GmocmocmouGGAGmoumoumouAmoum 1431 UUCCGaaUAaaCUCCAggcT 1432
    oumocGGAATsT sT
    AD-12368 GmocmocmouGGAGmoumoumouAmoum 1433 UUCCGAAuAAACUCcAGGCT 1434
    oumocGGAATsT sT
    AD-12369 GmocmocmouGGAGmoumoumouAmoum 1435 UUCCGAAUAAACUCCAGGCT 1436
    oumocGGAATsT sT
    AD-12370 GmocmocmouGGAGmoumoumouAmoum 1437 P*UfUfCfCfGAAUfAAACf 1438
    oumocGGAATsT UfCfCfAGGCfTsT
    AD-12371 GmocmocmouGGAGmoumoumouAmoum 1439 P*UfUfCfCfGAAUfAAACf 1440
    oumocGGAATsT UfCfCfAGGCfsCfsUf
    AD-12372 GmocmocmouGGAGmoumoumouAmoum 1441 P*uUfcCfgAfaUfaAfaCf 1442
    oumocGGAATsT uCfcAfgGfcsCfsu
    AD-12373 GmocmocmouGGAGmoumoumouAmoum 1443 UUCCGAAUAAACUCCAGGCT 1444
    oumocGGAATsT sT
    AD-12374 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1445 UfUfCfCfGAAUfAAACfUf 1446
    TsT CfCfAGGCfTsT
    AD-12375 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1447 UUCCGAAUAAACUCCAGGCT 1448
    TsT sT
    AD-12377 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1449 uUcCGAAuAAACUccAGGCT 1450
    TsT sT
    AD-12378 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1451 UUCCGaaUAaaCUCCAggcs 1452
    TsT csu
    AD-12379 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1453 UUCCGAAUAAACUCCAGGCs 1454
    TsT csu
    AD-12380 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1455 P*uUfcCfgAfaUfaAfaCf 1456
    TsT uCfcAfgGfcsCfsu
    AD-12381 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1457 P*uUfcCfgAfaUfaAfaCf 1458
    TsT uCfcAfgGfcTsT
    AD-12382 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1459 P*UfUfCfCfGAAUfAAACf 1460
    TsT UfCfCfAGGCfTsT
    AD-12383 GCCUGGAGUUUAUUCGGAATsT 1461 P*UfUfCfCfGAAUfAAACf 1462
    UfCfCfAGGCfTsT
    AD-12384 GccuGGAGuuuAuucGGAATsT 1463 P*UfUfCfCfGAAUfAAACf 1464
    UfCfCfAGGCfTsT
    AD-12385 GcCuGgnAgUuUaUuCgGaATsT 1465 P*UfUfCfCfGAAUfAAACf 1466
    UfCfCfAGGCfTsT
    AD-12386 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1467 P*UfUfCfCfGAAUfAAACf 1468
    f UfCfCfAGGCfTsT
    AD-12387 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1469 UfUfCfCfGAAUfAAACfUf 1470
    A CfCfAGGCfsCfsUf
    AD-12388 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1471 P*uUfcCfgAfaUfaAfaCf 1472
    A uCfcAfgGfc
    AD-12389 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1473 P*uUfcCfgAfaUfaAfaCf 1474
    A uCfcAfgGfcsCfsu
    AD-12390 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1475 UUCCGAAUAAACUCCAGGCs 1476
    A csu
    AD-12391 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1477 UUCCGaaUAaaCUCCAggc 1478
    A
    AD-12392 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1479 UUCCGAAUAAACUCCAGGCT 1480
    A sT
    AD-12393 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1481 UUCCGAAuAAACUCcAGGCT 1482
    A sT
    AD-12394 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1483 uUcCGAAuAAACUccAGGCT 1484
    A sT
    AD-12395 GmocCmouGmogAmogUmouUmoaUmou 1485 P*UfUfCfCfGAAUfAAACf 1486
    CmogGmoaATsT UfCfCfAGGCfsCfsUf
    AD-12396 GmocCmouGmogAm02gUmouUmoaUmo 1487 P*UfUfCfCfGAAUfAAACf 1488
    uCmogGmoaA UfCfCfAGGCfsCfsUf
    AD-12397 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1489 P*UfUfCfCfGAAUfAAACf 1490
    f UfCfCfAGGCfsCfsUf
    AD-12398 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1491 P*UfUfCfCfGAAUfAAACf 1492
    fTsT UfCfCfAGGCfsCfsUf
    AD-12399 GcCuGgnAgUuUaUuCgGaATsT 1493 P*UfUfCfCfGAAUfAAACf 1494
    UfCfCfAGGCfsCfsUf
    AD-12400 GCCUGGAGUUUAUUCGGAATsT 1495 P*UfUfCfCfGAAUfAAACf 1496
    UfCfCfAGGCfsCfsUf
    AD-12401 GccuGGAGuuuAuucGGAATsT 1497 P*UfUfCfCfGAAUfAAACf 1498
    UfCfCfAGGCfsCfsUf
    AD-12402 GccuGGAGuuuAuucGGAA 1499 P*UfUfCfCfGAAUfAAACf 1500
    UfCfCfAGGCfsCfsUf
    AD-12403 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1501 P*UfUfCfCfGAAUfAAACf 1502
    A UfCfCfAGGCfsCfsUf
    AD-9314 GCCUGGAGUUUAUUCGGAATsT 1503 UUCCGAAUAAACUCCAGGCT 1504
    sT
    AD-10794 ucAuAGGccuGGAGuuuAudTsdT 1525 AuAAACUCcAGGCCuAUGAd 1526
    TsdT
    AD-10795 ucAuAGGccuGGAGuuuAudTsdT 1527 AuAAACUccAGGcCuAuGAd 1528
    TsdT
    AD-10797 ucAuAGGccuGGAGuuuAudTsdT 1529 AUAAACUCCAGGCCUAUGAd 1530
    TsdT
  • TABLE 6
    dsRNA targeted to PCSK9: mismatches and modifications
    Strand: S/Sense; AS/Antisense; U, C, A, G: corresponding ribonucleotide;
    T: deoxythymidine; u, c, a, g: corresponding 2′-O-methyl ribonucleotide;
    Uf, Cf, Af, Gf: corresponding 2′-deoxy-2′-fluoro ribonucleotide; Y1
    corresponds to DFT difluorotoluyl ribo(or deoxyribo)nucleotide; where
    nucleotides are written in sequence, they are connected by 3′-5′ phospho-
    diester groups; nucleotides with interjected “s” are connected by 3′- 
    O-5′-O phosphorothiodiester groups; unless denoted by prefix “P*”,
    oligonucleotides are devoid of a 5′-phosphate group on the 5′-most
    nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide
    Duplex # Strand SEQ ID NO: Sequence (5′ to 3′)
    AD-9680 S 1531 uucuAGAccuGuuuuGcuudTsdT
    AS 1532 AAGcAAAAcAGGUCuAGAAdTsdT
    AD-3267 S 1535 uucuAGAcCuGuuuuGcuuTsT
    AS 1536 AAGcAAAAcAGGUCuAGAATsT
    AD-3268 S 1537 uucuAGAccUGuuuuGcuuTsT
    AS 1538 AAGcAAAAcAGGUCuAGAATsT
    AD-3269 S 1539 uucuAGAcCUGuuuuGcuuTsT
    AS 1540 AAGcAAAAcAGGUCuAGAATsT
    AD-3270 S 1541 uucuAGAcY1uGuuuuGcuuTsT
    AS 1542 AAGcAAAAcAGGUCuAGAATsT
    AD-3271 S 1543 uucuAGAcY1UGuuuuGcuuTsT
    AS 1544 AAGcAAAAcAGGUCuAGAATsT
    AD-3272 S 1545 uucuAGAccY1GuuuuGcuuTsT
    AS 1546 AAGcAAAAcAGGUCuAGAATsT
    AD-3273 S 1547 uucuAGAcCY1GuuuuGcuuTsT
    AS 1548 AAGcAAAAcAGGUCuAGAATsT
    AD-3274 S 1549 uucuAGAccuY1uuuuGcuuTsT
    AS 1550 AAGcAAAAcAGGUCuAGAATsT
    AD-3275 S 1551 uucuAGAcCUY1uuuuGcuuTsT
    AS 1552 AAGcAAAAcAGGUCuAGAATsT
    AD-14676 S 1553 UfuCfuAfgAfcCfuGfuUfuUfgCfuUfTsT
    AS 1554 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3276 S 1555 UfuCfuAfgAfcCuGfuUfuUfgCfuUfTsT
    AS 1556 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3277 S 1557 UfuCfuAfgAfcCfUGfuUfuUfgCfuUfTsT
    AS 1558 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3278 S 1559 UfuCfuAfgAfcCUGfuUfuUfgCfuUfTsT
    AS 1560 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3279 S 1561 UfuCfuAfgAfcY1uGfuUfuUfgCfuUfTsT
    AS 1562 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3280 S 1563 UfuCfuAfgAfcY1UGfuUfuUfgCfuUfTsT
    AS 1564 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3281 S 1565 UfuCfuAfgAfcCfY1GfuUfuUfgCfuUfTsT
    AS 1566 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3282 S 1567 UfuCfuAfgAfcCY1GfuUfuUfgCfuUfTsT
    AS 1568 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3283 S 1569 UfuCfuAfgAfcCfuY1uUfuUfgCfuUfTsT
    AS 1570 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3284 S 1571 UfuCfuAfgAfcCUY1uUfuUfgCfuUfTsT
    AS 1572 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-10792 S 459 GccuGGAGuuuAuucGGAATsT
    AS 460 UUCCGAAuAAACUCcAGGCTsT
    AD-3254 S 1573 GccuGGAGuY1uAuucGGAATsT
    AS 1574 UUCCGAAuAAACUCcAGGCTsT
    AD-3255 S 1575 GccuGGAGUY1uAuucGGAATsT
    AS 1576 UUCCGAAuAAACUCcAGGCTsT
  • TABLE 7
    Sequences of unmodified siRNA flanking AD-9680
    *Target: target in human PCSK9 gene, access. # NM_174936 U, C, A, G:
    corresponding ribonucleotide; dT: deoxythymidine; where nucleotides
    are written in sequence, they are connected by 3′-5′ phosphodiester
    groups; nucleotides with interjected “s” are connected by 3′-O-5′-O
    phosphorothiodiester groups.
    Duplex # Strand Sequence (5′ to 3′) *Target SEQ ID NO:
    AD-22169-b1 sense CAGCCAACUUUUCUAGACCdTsdT 3520 1577
    antis GGUCUAGAAAAGUUGGCUGdTsdT 3520 1578
    AD-22170-b1 sense AGCCAACUUUUCUAGACCUdTsdT 3521 1579
    antis AGGUCUAGAAAAGUUGGCUdTsdT 3521 1580
    AD-22171-b1 sense GCCAACUUUUCUAGACCUGdTsdT 3522 1581
    antis CAGGUCUAGAAAAGUUGGCdTsdT 3522 1582
    AD-22172-b1 sense CCAACUUUUCUAGACCUGUdTsdT 3523 1583
    antis ACAGGUCUAGAAAAGUUGGdTsdT 3523 1584
    AD-22173-b1 sense CAACUUUUCUAGACCUGUUdTsdT 3524 1585
    antis AACAGGUCUAGAAAAGUUGdTsdT 3524 1586
    AD-22174-b1 sense AACUUUUCUAGACCUGUUUdTsdT 3525 1587
    antis AAACAGGUCUAGAAAAGUUdTsdT 3525 1588
    AD-22175-b1 sense ACUUUUCUAGACCUGUUUUdTsdT 3526 1589
    antis AAAACAGGUCUAGAAAAGUdTsdT 3526 1590
    AD-22176-b1 sense CUUUUCUAGACCUGUUUUGdTsdT 3527 1591
    antis CAAAACAGGUCUAGAAAAGdTsdT 3527 1592
    AD-22177-b1 sense UUUUCUAGACCUGUUUUGCdTsdT 3528 1593
    antis GCAAAACAGGUCUAGAAAAdTsdT 3528 1594
    AD-22178-b1 sense UUUCUAGACCUGUUUUGCUdTsdT 3529 1595
    antis AGCAAAACAGGUCUAGAAAdTsdT 3529 1596
    AD-22179-b1 sense UCUAGACCUGUUUUGCUUUdTsdT 3531 1597
    antis AAAGCAAAACAGGUCUAGAdTsdT 3531 1598
    AD-22180-b1 sense CUAGACCUGUUUUGCUUUUdTsdT 3532 1599
    antis AAAAGCAAAACAGGUCUAGdTsdT 3532 1600
    AD-22181-b1 sense UAGACCUGUUUUGCUUUUGdTsdT 3533 1601
    antis CAAAAGCAAAACAGGUCUAdTsdT 3533 1602
    AD-22182-b1 sense AGACCUGUUUUGCUUUUGUdTsdT 3534 1603
    antis ACAAAAGCAAAACAGGUCUdTsdT 3534 1604
    AD-22183-b1 sense GACCUGUUUUGCUUUUGUAdTsdT 3535 1605
    antis UACAAAAGCAAAACAGGUCdTsdT 3535 1606
    AD-22184-b1 sense ACCUGUUUUGCUUUUGUAAdTsdT 3536 1607
    antis UUACAAAAGCAAAACAGGUdTsdT 3536 1608
    AD-22185-b1 sense CCUGUUUUGCUUUUGUAACdTsdT 3537 1609
    antis GUUACAAAAGCAAAACAGGdTsdT 3537 1610
    AD-22186-b1 sense CUGUUUUGCUUUUGUAACUdTsdT 3538 1611
    antis AGUUACAAAAGCAAAACAGdTsdT 3538 1612
    AD-22187-b1 sense UGUUUUGCUUUUGUAACUUdTsdT 3539 1613
    antis AAGUUACAAAAGCAAAACAdTsdT 3539 1614
    AD-22188-b1 sense GUUUUGCUUUUGUAACUUGdTsdT 3540 1615
    antis CAAGUUACAAAAGCAAAACdTsdT 3540 1616
    AD-22189-b1 sense UUUUGCUUUUGUAACUUGAdTsdT 3541 1617
    antis UCAAGUUACAAAAGCAAAAdTsdT 3541 1618
    AD-22190-b1 sense UUUGCUUUUGUAACUUGAAdTsdT 3542 1619
    antis UUCAAGUUACAAAAGCAAAdTsdT 3542 1620
    AD-22191-b1 sense UUGCUUUUGUAACUUGAAGdTsdT 3543 1621
    antis CUUCAAGUUACAAAAGCAAdTsdT 3543 1622
    AD-22192-b1 sense UGCUUUUGUAACUUGAAGAdTsdT 3544 1623
    antis UCUUCAAGUUACAAAAGCAdTsdT 3544 1624
    AD-22193-b1 sense GCUUUUGUAACUUGAAGAUdTsdT 3545 1625
    antis AUCUUCAAGUUACAAAAGCdTsdT 3545 1626
    AD-22194-b1 sense CUUUUGUAACUUGAAGAUAdTsdT 3546 1627
    antis UAUCUUCAAGUUACAAAAGdTsdT 3546 1628
    AD-22195-b1 sense UUUUGUAACUUGAAGAUAUdTsdT 3547 1629
    antis AUAUCUUCAAGUUACAAAAdTsdT 3547 1630
    AD-22196-b1 sense UUUGUAACUUGAAGAUAUUdTsdT 3548 1631
    antis AAUAUCUUCAAGUUACAAAdTsdT 3548 1632
    AD-22197-b1 sense UUGUAACUUGAAGAUAUUUdTsdT 3549 1633
    antis AAAUAUCUUCAAGUUACAAdTsdT 3549 1634
    AD-22198-b1 sense UGUAACUUGAAGAUAUUUAdTsdT 3550 1635
    antis UAAABAUCUUCAAGUUACAdTsdT 3550 1636
    AD-22199-b1 sense GUAACUUGAAGAUAUUUAUdTsdT 3551 1637
    antis AUAAAUAUCUUCAAGUUACdTsdT 3551 1638
    AD-22200-b1 sense UAACUUGAAGAUAUUUAUUdTsdT 3552 1639
    antis AAUAAAUAUCUUCAAGUUAdTsdT 3552 1640
    AD-22201-b1 sense AACUUGAAGAUAUUUAUUCdTsdT 3553 1641
    antis GAAUAAAUAUCUUCAAGUUdTsdT 3553 1642
    AD-22202-b1 sense ACUUGAAGAUAUUUAUUCUdTsdT 3554 1643
    antis AGAAUAAAUAUCUUCAAGUdTsdT 3554 1644
    AD-22203-b1 sense CUUGAAGAUAUUUAUUCUGdTsdT 3555 1645
    antis CAGAAUAAAUAUCUUCAAGdTsdT 3555 1646
    AD-22204-b1 sense UUGAAGAUAUUUAUUCUGGdTsdT 3556 1647
    antis CCAGAAUAAAUAUCUUCAAdTsdT 3556 1648
    AD-22205-b1 sense UGAAGAUAUUUAUUCUGGGdTsdT 3557 1649
    antis CCCAGAAUAAAUAUCUUCAdTsdT 3557 1650
    AD-22206-b1 sense GAAGAUAUUUAUUCUGGGUdTsdT 3558 1651
    antis ACCCAGAAUAAAUAUCUUCdTsdT 3558 1652
  • TABLE 8
    Sequences of modified siRNA flanking AD-9680
    *Target: 5′ nutlcoetide of target sequence in human PCSK9 gene, access.
    # NM_174936 U, C, A, G: corresponding ribonucleotide; dT: deoxythymidine;
    u, c, a, g: corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf:
    corresponding 2′-deoxy-2′-fluoro ribonucleotide; Y1 corresponds to DFT
    difluorotoluyl ribo(or deoxyribo)nucleotide; where nucleotides are 
    written in sequence, they are connected by 3′-5′ phosphodiester groups;
    nucleotides with interjected “s” are connected by 3′-O-5′-O phosphoro-
    thiodiester groups; unless denoted by prefix “P*”, oligonucleotides are
    devoid of a 5′-phosphate group on the 5′-most nucleotide; all oligo-
    nucleotides bear 3′-OH on the 3′-most nucleotide
    Duplex # Strand Sequence (5′ to 3′) *Target SEQ ID NO:
    AD-22098-b1 sense cAGccAAcuuuucuAGAccdTsdT 3520 1653
    antis GGUCuAGAAAAGUUGGCUGdTsdT 3520 1654
    AD-22099-b1 sense AGccAAcuuuucuAGAccudTsdT 3521 1655
    antis AGGUCuAGAAAAGUUGGCUdTsdT 3521 1656
    AD-22100-b1 sense GccAAcuuuucuAGAccuGdTsdT 3522 1657
    antis cAGGUCuAGAAAAGUUGGCdTsdT 3522 1658
    AD-22101-b1 sense ccAAcuuuucuAGAccuGudTsdT 3523 1659
    antis AcAGGUCuAGAAAAGUUGGdTsdT 3523 1660
    AD-22102-b1 sense cAAcuuuucuAGAccuGuudTsdT 3524 1661
    antis AAcAGGUCuAGAAAAGUUGdTsdT 3524 1662
    AD-22103-b1 sense AAcuuuucuAGAccuGuuudTsdT 3525 1663
    antis AAAcAGGUCuAGAAAAGUUdTsdT 3525 1664
    AD-22104-b1 sense AcuuuucuAGAccuGuuuudTsdT 3526 1665
    antis AAAAcAGGUCuAGAAAAGUdTsdT 3526 1666
    AD-22105-b1 sense cuuuucuAGAccuGuuuuGdTsdT 3527 1667
    antis cAAAAcAGGUCuAGAAAAGdTsdT 3527 1668
    AD-22106-b1 sense uuuucuAGAccuGuuuuGcdTsdT 3528 1669
    antis GcAAAAcAGGUCuAGAAAAdTsdT 3528 1670
    AD-22107-b1 sense uuucuAGAccuGuuuuGcudTsdT 3529 1671
    antis AGcAAAAcAGGUCuAGAAAdTsdT 3529 1672
    AD-22108-b1 sense ucuAGAccuGuuuuGcuuudTsdT 3531 1673
    antis AAAGcAAAAcAGGUCuAGAdTsdT 3531 1674
    AD-22109-b1 sense cuAGAccuGuuuuGcuuuudTsdT 3532 1675
    antis AAAAGcAAAAcAGGUCuAGdTsdT 3532 1676
    AD-22110-b1 sense uAGAccuGuuuuGcuuuuGdTsdT 3533 1677
    antis cAAAAGcAAAAcAGGUCuAdTsdT 3533 1678
    AD-22111-b1 sense AGAccuGuuuuGcuuuuGudTsdT 3534 1679
    antis AcAAAAGcAAAAcAGGUCUdTsdT 3534 1680
    AD-22112-b1 sense GAccuGuuuuGcuuuuGuAdTsdT 3535 1681
    antis uAcAAAAGcAAAAcAGGUCdTsdT 3535 1682
    AD-22113-b1 sense AccuGuuuuGcuuuuGuAAdTsdT 3536 1683
    antis UuAcAAAAGcAAAAcAGGUdTsdT 3536 1684
    AD-22114-b1 sense ccuGuuuuGcuuuuGuAAcdTsdT 3537 1685
    antis GUuAcAAAAGcAAAAcAGGdTsdT 3537 1686
    AD-22115-b1 sense cuGuuuuGcuuuuGuAAcudTsdT 3538 1687
    antis AGUuAcAAAAGcAAAAcAGdTsdT 3538 1688
    sense uGuuuuGcuuuuGuAAcuudTsdT 3539 1689
    antis AAGUuAcAAAAGcAAAAcAdTsdT 3539 1690
    AD-22116-b1 sense GuuuuGcuuuuGuAAcuuGdTsdT 3540 1691
    antis cAAGUuAcAAAAGcAAAACdTsdT 3540 1692
    AD-22117-b1 sense uuuuGcuuuuGuAAcuuGAdTsdT 3541 1693
    antis UcAAGUuAcAAAAGcAAAAdTsdT 3541 1694
    AD-22118-b1 sense uuuGcuuuuGuAAcuuGAAdTsdT 3542 1695
    antis UUcAAGUuAcAAAAGcAAAdTsdT 3542 1696
    AD-22119-b1 sense uuGcuuuuGuAAcuuGAAGdTsdT 3543 1697
    antis CUUcAAGUuAcAAAAGcAAdTsdT 3543 1698
    AD-22120-b1 sense uGcuuuuGuAAcuuGAAGAdTsdT 3544 1699
    antis UCUUcAAGUuAcAAAAGcAdTsdT 3544 1700
    AD-22121-b1 sense GcuuuuGuAAcuuGAAGAudTsdT 3545 1701
    antis AUCUUcAAGUuAcAAAAGCdTsdT 3545 1702
    AD-22122-b1 sense cuuuuGuAAcuuGAAGAuAdTsdT 3546 1703
    antis uAUCUUcAAGUuAcAAAAGdTsdT 3546 1704
    AD-22123-b1 sense uuuuGuAAcuuGAAGAuAudTsdT 3547 1705
    antis AuAUCUUcAAGUuAcAAAAdTsdT 3547 1706
    AD-22124-b1 sense uuuGuAAcuuGAAGAuAuudTsdT 3548 1707
    antis AAuAUCUUcAAGUuAcAAAdTsdT 3548 1708
    AD-22125-b1 sense uuGuAAcuuGAAGAuAuuudTsdT 3549 1709
    antis AAAuAUCUUcAAGUuAcAAdTsdT 3549 1710
    AD-22126-b1 sense uGuAAcuuGAAGAuAuuuAdTsdT 3550 1711
    antis uAAAuAUCUUcAAGUuAcAdTsdT 3550 1712
    AD-22127-b1 sense GuAAcuuGAAGAuAuuuAudTsdT 3551 1713
    antis AuAAAuAUCUUcAAGUuACdTsdT 3551 1714
    AD-22128-b1 sense uAAcuuGAAGAuAuuuAuudTsdT 3552 1715
    antis AAuAAAuAUCUUcAAGUuAdTsdT 3552 1716
    AD-22129-b1 sense AAcuuGAAGAuAuuuAuucdTsdT 3553 1717
    antis GAAuAAAuAUCUUcAAGUUdTsdT 3553 1718
    AD-22130-b1 sense AcuuGAAGAuAuuuAuucudTsdT 3554 1719
    antis AGAAuAAAuAUCUUcAAGUdTsdT 3554 1720
    AD-22131-b1 sense cuuGAAGAuAuuuAuucuGdTsdT 3555 1721
    antis cAGAAuAAAuAUCUUcAAGdTsdT 3555 1722
    AD-22132-b1 sense uuGAAGAuAuuuAuucuGGdTsdT 3556 1723
    antis CcAGAAuAAAuAUCUUcAAdTsdT 3556 1724
    AD-22133-b1 sense uGAAGAuAuuuAuucuGGGdTsdT 3557 1725
    antis CCcAGAAuAAAuAUCUUcAdTsdT 3557 1726
    AD-22134-b1 sense GAAGAuAuuuAuucuGGGudTsdT 3558 1727
    antis ACCcAGAAuAAAuAUCUUCdTsdT 3558 1728
  • TABLE 9
    Sequences of XBP-1 dsRNAs
    *Target refers to target gene and location of target sequence.
    NM_001004210 is the gene for rat XBP-1. XM_001103095 is the sequence
    for Macaca mulatta (rhesus monkey) XBP-1.
    SEQ ID SEQ ID
    Target* NO sense (5′-3′) NO antisense (5′-3′)
    NM_001004210 1729 CCCAGCUGAUUAGUGUCUA 1753 UAGACACUAAUCAGCUGGG
    1128-1146
    NM_001004210 1730 CCAGCUGAUUAGUGUCUAA 1754 UUAGACACUAAUCAGCUGG
    1129-1147
    NM_001004210 1731 CUCCCAGAGGUCUACCCAG 1755 CUGGGUAGACCUCUGGGAG
     677-695
    NM_001004210 1732 GAUCACCCUGAAUUCAUUG 1756 CAAUGAAUUCAGGGUGAUC
     893-911
    NM_001004210 1733 UCACCCUGAAUUCAUUGUC 1757 GACAAUGAAUUCAGGGUGA
     895-913
    NM_001004210 1734 CCCCAGCUGAUUAGUGUCU 1758 AGACACUAAUCAGCUGGGG
    1127-1145
    NM_001004210 1735 AUCACCCUGAAUUCAUUGU 1759 ACAAUGAAUUCAGGGUGAU
     894-912
    NM_001004210 1736 CAUUUAUUUAAAACUACCC 1760 GGGUAGUUUUAAAUAAAUG
    1760-1778
    NM_001004210 1737 ACUGAAAAACAGAGUAGCA 1761 UGCUACUCUGUUUUUCAGU
     215-233
    NM_001004210 1738 CCAUUUAUUUAAAACUACC 1762 GGUAGUUUUAAAUAAAUGG
    1759-1777
    NM_001004210 1739 UUGAGAACCAGGAGUUAAG 1763 CUUAACUCCUGGUUCUCAA
     367-385
    NM_001004210 1740 CACCCUGAAUUCAUUGUCU 1764 AGACAAUGAAUUCAGGGUG
     896-914
    NM_001004210 1741 AACUGAAAAACAGAGUAGC 1765 GCUACUCUGUUUUUCAGUU
     214-232
    NM_001004210 1742 CUGAAAAACAGAGUAGCAG 1766 CUGCUACUCUGUUUUUCAG
     216-234
    XM_001103095 1743 AGAAAAUCAGCUUUUACGA 1767 UCGUAAAAGCUGAUUUUCU
     387-405
    XM_001103095 1744 UCCCCAGCUGAUUAGUGUC 1768 GACACUAAUCAGCUGGGGA
    1151-1169
    XM_001103095 1745 UACUUAUUAUGUAAGGGUC 1769 GACCCUUACAUAAUAAGUA
    1466-1484
    XM_001103095 1746 UAUCUUAAAAGGGUGGUAG 1770 CUACCACCCUUUUAAGAUA
    1435-1453
    XM_001103095 1747 CCAUGGAUUCUGGCGGUAU 1771 AUACCGCCAGAAUCCAUGG
     577-595
    XM_001103095 1748 UUAAUGAACUAAUUCGUUU 1772 AAACGAAUUAGUUCAUUAA
     790-808
    XM_001103095 1749 AGGGUCAUUAGACAAAUGU 1773 ACAUUUGUCUAAUGACCCU
    1479-1497
    XM_001103095 1750 UGAACUAAUUCGUUUUGAC 1774 GUCAAAACGAAUUAGUUCA
     794-812
    XM_001103095 1751 UUCCCCAGCUGAUUAGUGU 1775 ACACUAAUCAGCUGGGGAA
    1150-1168
    XM_001103095 1752 UAUGUAAGGGUCAUUAGAC 1776 GUCUAAUGACCCUUACAUA
    1473-1491
  • TABLE 10
    Target gene name and target sequence
    location for dsRNA targeting XBP-1
    Duplex # Target gene and location of target sequence
    AD18027 NM_001004210_1128-1146
    AD18028 NM_001004210_1129-1147
    AD18029 NM_001004210_677-695
    AD18030 NM_001004210_893-911
    AD18031 NM_001004210_895-913
    AD18032 NM_001004210_1127-1145
    AD18033 NM_001004210_894-912
    AD18034 NM_001004210_1760-1778
    AD18035 NM_001004210_215-233
    AD18036 NM_001004210_1759-1777
    AD18037 NM_001004210_367-385
    AD18038 NM_001004210_896-914
    AD18039 NM_001004210_214-232
    AD18040 NM_001004210_216-234
    AD18041 XM_001103095_387-405
    AD18042 XM_001103095_1151-1169
    AD18043 XM_001103095_1466-1484
    AD18044 XM_001103095_1435-1453
    AD18045 XM_001103095_577-595
    AD18046 XM_001103095_790-808
    AD18047 XM_001103095_1479-1497
    AD18048 XM_001103095_794-812
    AD18049 XM_001103095_1150-1168
    AD18050 XM_001103095_1473-1491
    *Target refers to target gene and location of target sequence.
    NM_001004210 is the gene for rat XBP-1.
    XM_001103095 is the sequence for Macaca mulatta (rhesus monkey) XBP-1.
  • TABLE 11
    Sequences of dsRNA targeting XBP-1, with Endolight chemistry modifications
    U, C, A, G: corresponding ribonucleotide; dT: deoxythymidine; u, c, a, g:
    corresponding 2′-O-methyl ribonucleotide; where nucleotides are written in
    sequence, they are connected by 3′-5′ phosphodiester groups; nucleotides
    with interjected “s” are connected by 3′-O-5′-O phosphorothiodiester
    groups.
    SEQ SEQ
    ID ID
    Duplex # NO Sense (5′-3′) NO Antisense (5′-3′)
    AD18027 4166 cccAGcuGAuuAGuGucuAdTsdT 1800 uAGAcACuAAUcAGCUGGGdTsdT
    AD18028 1777 ccAGcuGAuuAGuGucuAAdTsdT 1801 UuAGAcACuAAUcAGCUGGdTsdT
    AD18029 1778 cucccAGAGGucuAcccAGdTsdT 1802 CUGGGuAGACCUCUGGGAGdTsdT
    AD18030 1779 GAucAcccuGAAuucAuuGdTsdT 1803 cAAUGAAUUcAGGGUGAUCdTsdT
    AD18031 1780 ucAcccuGAAuucAuuGucdTsdT 1804 GAcAAUGAAUUcAGGGUGAdTsdT
    AD18032 1781 ccccAGcuGAuuAGuGucudTsdT 1805 AGAcACuAAUcAGCUGGGGdTsdT
    AD18033 1782 AucAcccuGAAuucAuuGudTsdT 1806 AcAAUGAAUUcAGGGUGAUdTsdT
    AD18034 1783 cAuuuAuuuAAAAcuAcccdTsdT 1807 GGGuAGUUUuAAAuAAAUGdTsdT
    AD18035 1784 AcuGAAAAAcAGAGuAGcAdTsdT 1808 UGCuACUCUGUUUUUcAGUdTsdT
    AD18036 1785 ccAuuuAuuuAAAAcuAccdTsdT 1809 GGuAGUUUuAAAuAAAUGGdTsdT
    AD18037 1786 uuGAGAAccAGGAGuuAAGdTsdT 1810 CUuAACUCCUGGUUCUcAAdTsdT
    AD18038 1787 cAcccuGAAuucAuuGucudTsdT 1811 AGAcAAUGAAUUcAGGGUGdTsdT
    AD18039 1788 AAcuGAAAAAcAGAGuAGcdTsdT 1812 GCuACUCUGUUUUUcAGUUdTsdT
    AD18040 1789 cuGAAAAAcAGAGuAGcAGdTsdT 1813 CUGCuACUCUGUUUUUcAGdTsdT
    AD18041 1790 AGAAAAucAGcuuuuAcGAdTsdT 1814 UCGuAAAAGCUGAUUUUCUdTsdT
    AD18042 1791 uccccAGcuGAuuAGuGucdTsdT 1815 GAcACuAAUcAGCUGGGGAdTsdT
    AD18043 1792 uAcuuAuuAuGuAAGGGucdTsdT 1816 GACCCUuAcAuAAuAAGuAdTsdT
    AD18044 1793 uAucuuAAAAGGGuGGuAGdTsdT 1817 CuACcACCCUUUuAAGAuAdTsdT
    AD18045 1794 ccAuGGAuucuGGcGGuAudTsdT 1818 AuACCGCcAGAAUCcAUGGdTsdT
    AD18046 1795 uuAAuGAAcuAAuucGuuudTsdT 1819 AAACGAAUuAGUUcAUuAAdTsdT
    AD18047 1796 AGGGucAuuAGAcAAAuGudTsdT 1820 AcAUUUGUCuAAUGACCCUdTsdT
    AD18048 1797 uGAAcuAAuucGuuuuGAcdTsdT 1821 GUcAAAACGAAUuAGUUcAdTsdT
    AD18049 1798 uuccccAGcuGAuuAGuGudTsdT 1822 AcACuAAUcAGCUGGGGAAdTsdT
    AD18050 1799 uAuGuAAGGGucAuuAGAcdTsdT 1823 GUCuAAUGACCCUuAcAuAdTsdT
  • TABLE 12
    Sequences of dsRNA targeting both human and rhesus monkey XBP-1.
    *Target refers location of target sequence in NM_005080 (human XBP-1 mRNA).
     Sense and antisense sequences are described with optional dinucleotide
    (NN) overhangs.
    SEQ ID SEQ ID
    Target* sense (5′-3′) NO antisense (5′-3′) NO
     100-118 CUGCUUCUGUCGGGGCAGCNN 1824 GCUGCCCCGACAGAAGCAGNN 28
    1011-1029 GAGCUGGGUAUCUCAAAUCNN 1825 GAUUUGAGAUACCCAGCUCNN 28
     101-119 UGCUUCUGUCGGGGCAGCCNN 1826 GGCUGCCCCGACAGAAGCANN 28
    1012-1030 AGCUGGGUAUCUCAAAUCUNN 1827 AGAUUUGAGAUACCCAGCUNN 28
    1013-1031 GCUGGGUAUCUCAAAUCUGNN 1828 CAGAUUUGAGAUACCCAGCNN 28
    1014-1032 CUGGGUAUCUCAAAUCUGCNN 1829 GCAGAUUUGAGAUACCCAGNN 28
    1015-1033 UGGGUAUCUCAAAUCUGCUNN 1830 AGCAGAUUUGAGAUACCCANN 28
    1016-1034 GGGUAUCUCAAAUCUGCUUNN 1831 AAGCAGAUUUGAGAUACCCNN 28
    1017-1035 GGUAUCUCAAAUCUGCUUUNN 1832 AAAGCAGAUUUGAGAUACCNN 28
    1018-1036 GUAUCUCAAAUCUGCUUUCNN 1833 GAAAGCAGAUUUGAGAUACNN 28
    1019-1037 UAUCUCAAAUCUGCUUUCANN 1834 UGAAAGCAGAUUUGAGAUANN 28
    1020-1038 AUCUCAAAUCUGCUUUCAUNN 1835 AUGAAAGCAGAUUUGAGAUNN 28
    1021-1039 UCUCAAAUCUGCUUUCAUCNN 1836 GAUGAAAGCAGAUUUGAGANN 28
     102-120 GCUUCUGUCGGGGCAGCCCNN 1837 GGGCUGCCCCGACAGAAGCNN 28
    1022-1040 CUCAAAUCUGCUUUCAUCCNN 1838 GGAUGAAAGCAGAUUUGAGNN 28
    1023-1041 UCAAAUCUGCUUUCAUCCANN 1839 UGGAUGAAAGCAGAUUUGANN 28
    1024-1042 CAAAUCUGCUUUCAUCCAGNN 1840 CUGGAUGAAAGCAGAUUUGNN 28
    1025-1043 AAAUCUGCUUUCAUCCAGCNN 1841 GCUGGAUGAAAGCAGAUUUNN 29
    1026-1044 AAUCUGCUUUCAUCCAGCCNN 1842 GGCUGGAUGAAAGCAGAUUNN 29
    1027-1045 AUCUGCUUUCAUCCAGCCANN 1843 UGGCUGGAUGAAAGCAGAUNN 29
    1028-1046 UCUGCUUUCAUCCAGCCACNN 1844 GUGGCUGGAUGAAAGCAGANN 29
    1029-1047 CUGCUUUCAUCCAGCCACUNN 1845 AGUGGCUGGAUGAAAGCAGNN 29
    1030-1048 UGCUUUCAUCCAGCCACUGNN 1846 CAGUGGCUGGAUGAAAGCANN 29
    1031-1049 GCUUUCAUCCAGCCACUGCNN 1847 GCAGUGGCUGGAUGAAAGCNN 29
     103-121 CUUCUGUCGGGGCAGCCCGNN 1848 CGGGCUGCCCCGACAGAAGNN 29
    1032-1050 CUUUCAUCCAGCCACUGCCNN 1849 GGCAGUGGCUGGAUGAAAGNN 29
    1033-1051 UUUCAUCCAGCCACUGCCCNN 1850 GGGCAGUGGCUGGAUGAAANN 29
     104-122 UUCUGUCGGGGCAGCCCGCNN 1851 GCGGGCUGCCCCGACAGAANN 29
     105-123 UCUGUCGGGGCAGCCCGCCNN 1852 GGCGGGCUGCCCCGACAGANN 29
    1056-1074 CCAUCUUCCUGCCUACUGGNN 1853 CCAGUAGGCAGGAAGAUGGNN 29
    1057-1075 CAUCUUCCUGCCUACUGGANN 1854 UCCAGUAGGCAGGAAGAUGNN 29
    1058-1076 AUCUUCCUGCCUACUGGAUNN 1855 AUCCAGUAGGCAGGAAGAUNN 29
    1059-1077 UCUUCCUGCCUACUGGAUGNN 1856 CAUCCAGUAGGCAGGAAGANN 29
    1060-1078 CUUCCUGCCUACUGGAUGCNN 1857 GCAUCCAGUAGGCAGGAAGNN 29
    1061-1079 UUCCUGCCUACUGGAUGCUNN 1858 AGCAUCCAGUAGGCAGGAANN 29
     106-124 CUGUCGGGGCAGCCCGCCUNN 1859 AGGCGGGCUGCCCCGACAGNN 29
    1062-1080 UCCUGCCUACUGGAUGCUUNN 1860 AAGCAUCCAGUAGGCAGGANN 29
    1063-1081 CCUGCCUACUGGAUGCUUANN 1861 UAAGCAUCCAGUAGGCAGGNN 29
    1064-1082 CUGCCUACUGGAUGCUUACNN 1862 GUAAGCAUCCAGUAGGCAGNN 29
    1065-1083 UGCCUACUGGAUGCUUACANN 1863 UGUAAGCAUCCAGUAGGCANN 29
    1066-1084 GCCUACUGGAUGCUUACAGNN 1864 CUGUAAGCAUCCAGUAGGCNN 29
    1067-1085 CCUACUGGAUGCUUACAGUNN 1865 ACUGUAAGCAUCCAGUAGGNN 29
    1068-1086 CUACUGGAUGCUUACAGUGNN 1866 CACUGUAAGCAUCCAGUAGNN 29
    1069-1087 UACUGGAUGCUUACAGUGANN 1867 UCACUGUAAGCAUCCAGUANN 29
    1070-1088 ACUGGAUGCUUACAGUGACNN 1868 GUCACUGUAAGCAUCCAGUNN 29
    1071-1089 CUGGAUGCUUACAGUGACUNN 1869 AGUCACUGUAAGCAUCCAGNN 29
     107-125 UGUCGGGGCAGCCCGCCUCNN 1870 GAGGCGGGCUGCCCCGACANN 29
    1072-1090 UGGAUGCUUACAGUGACUGNN 1871 CAGUCACUGUAAGCAUCCANN 29
    1073-1091 GGAUGCUUACAGUGACUGUNN 1872 ACAGUCACUGUAAGCAUCCNN 29
    1074-1092 GAUGCUUACAGUGACUGUGNN 1873 CACAGUCACUGUAAGCAUCNN 29
    1075-1093 AUGCUUACAGUGACUGUGGNN 1874 CCACAGUCACUGUAAGCAUNN 29
    1076-1094 UGCUUACAGUGACUGUGGANN 1875 UCCACAGUCACUGUAAGCANN 29
    1077-1095 GCUUACAGUGACUGUGGAUNN 1876 AUCCACAGUCACUGUAAGCNN 29
    1078-1096 CUUACAGUGACUGUGGAUANN 1877 UAUCCACAGUCACUGUAAGNN 29
     108-126 GUCGGGGCAGCCCGCCUCCNN 1878 GGAGGCGGGCUGCCCCGACNN 29
     109-127 UCGGGGCAGCCCGCCUCCGNN 1879 CGGAGGCGGGCUGCCCCGANN 29
     110-128 CGGGGCAGCCCGCCUCCGCNN 1880 GCGGAGGCGGGCUGCCCCGNN 29
     111-129 GGGGCAGCCCGCCUCCGCCNN 1881 GGCGGAGGCGGGCUGCCCCNN 29
    1116-1134 UUCAGUGACAUGUCCUCUCNN 1882 GAGAGGACAUGUCACUGAANN 29
     112-130 GGGCAGCCCGCCUCCGCCGNN 1883 CGGCGGAGGCGGGCUGCCCNN 29
     113-131 GGCAGCCCGCCUCCGCCGCNN 1884 GCGGCGGAGGCGGGCUGCCNN 29
    1136-1154 GCUUGGUGUAAACCAUUCUNN 1885 AGAAUGGUUUACACCAAGCNN 29
    1137-1155 CUUGGUGUAAACCAUUCUUNN 1886 AAGAAUGGUUUACACCAAGNN 29
    1138-1156 UUGGUGUAAACCAUUCUUGNN 1887 CAAGAAUGGUUUACACCAANN 29
    1139-1157 UGGUGUAAACCAUUCUUGGNN 1888 CCAAGAAUGGUUUACACCANN 29
    1140-1158 GGUGUAAACCAUUCUUGGGNN 1889 CCCAAGAAUGGUUUACACCNN 29
    1141-1159 GUGUAAACCAUUCUUGGGANN 1890 UCCCAAGAAUGGUUUACACNN 29
     114-132 GCAGCCCGCCUCCGCCGCCNN 1891 GGCGGCGGAGGCGGGCUGCNN 29
    1142-1160 UGUAAACCAUUCUUGGGAGNN 1892 CUCCCAAGAAUGGUUUACANN 29
    1143-1161 GUAAACCAUUCUUGGGAGGNN 1893 CCUCCCAAGAAUGGUUUACNN 29
    1144-1162 UAAACCAUUCUUGGGAGGANN 1894 UCCUCCCAAGAAUGGUUUANN 29
    1145-1163 AAACCAUUCUUGGGAGGACNN 1895 GUCCUCCCAAGAAUGGUUUNN 29
    1146-1164 AACCAUUCUUGGGAGGACANN 1896 UGUCCUCCCAAGAAUGGUUNN 29
    1147-1165 ACCAUUCUUGGGAGGACACNN 1897 GUGUCCUCCCAAGAAUGGUNN 29
    1148-1166 CCAUUCUUGGGAGGACACUNN 1898 AGUGUCCUCCCAAGAAUGGNN 29
    1149-1167 CAUUCUUGGGAGGACACUUNN 1899 AAGUGUCCUCCCAAGAAUGNN 29
    1150-1168 AUUCUUGGGAGGACACUUUNN 1900 AAAGUGUCCUCCCAAGAAUNN 29
    1151-1169 UUCUUGGGAGGACACUUUUNN 1901 AAAAGUGUCCUCCCAAGAANN 29
     115-133 CAGCCCGCCUCCGCCGCCGNN 1902 CGGCGGCGGAGGCGGGCUGNN 29
    1152-1170 UCUUGGGAGGACACUUUUGNN 1903 CAAAAGUGUCCUCCCAAGANN 29
    1153-1171 CUUGGGAGGACACUUUUGCNN 1904 GCAAAAGUGUCCUCCCAAGNN 29
    1154-1172 UUGGGAGGACACUUUUGCCNN 1905 GGCAAAAGUGUCCUCCCAANN 29
    1155-1173 UGGGAGGACACUUUUGCCANN 1906 UGGCAAAAGUGUCCUCCCANN 29
    1156-1174 GGGAGGACACUUUUGCCAANN 1907 UUGGCAAAAGUGUCCUCCCNN 29
    1157-1175 GGAGGACACUUUUGCCAAUNN 1908 AUUGGCAAAAGUGUCCUCCNN 29
    1158-1176 GAGGACACUUUUGCCAAUGNN 1909 CAUUGGCAAAAGUGUCCUCNN 29
    1159-1177 AGGACACUUUUGCCAAUGANN 1910 UCAUUGGCAAAAGUGUCCUNN 29
    1160-1178 GGACACUUUUGCCAAUGAANN 1911 UUCAUUGGCAAAAGUGUCCNN 29
    1161-1179 GACACUUUUGCCAAUGAACNN 1912 GUUCAUUGGCAAAAGUGUCNN 29
     116-134 AGCCCGCCUCCGCCGCCGGNN 1913 CCGGCGGCGGAGGCGGGCUNN 29
    1162-1180 ACACUUUUGCCAAUGAACUNN 1914 AGUUCAUUGGCAAAAGUGUNN 29
     117-135 GCCCGCCUCCGCCGCCGGANN 1915 UCCGGCGGCGGAGGCGGGCNN 29
     118-136 CCCGCCUCCGCCGCCGGAGNN 1916 CUCCGGCGGCGGAGGCGGGNN 29
    1182-1200 UUUCCCCAGCUGAUUAGUGNN 1917 CACUAAUCAGCUGGGGAAANN 29
    1183-1201 UUCCCCAGCUGAUUAGUGUNN 1918 ACACUAAUCAGCUGGGGAANN 29
    1184-1202 UCCCCAGCUGAUUAGUGUCNN 1919 GACACUAAUCAGCUGGGGANN 29
    1185-1203 CCCCAGCUGAUUAGUGUCUNN 1920 AGACACUAAUCAGCUGGGGNN 29
    1186-1204 CCCAGCUGAUUAGUGUCUANN 1921 UAGACACUAAUCAGCUGGGNN 29
    1187-1205 CCAGCUGAUUAGUGUCUAANN 1922 UUAGACACUAAUCAGCUGGNN 29
    1188-1206 CAGCUGAUUAGUGUCUAAGNN 1923 CUUAGACACUAAUCAGCUGNN 29
    1189-1207 AGCUGAUUAGUGUCUAAGGNN 1924 CCUUAGACACUAAUCAGCUNN 29
    1190-1208 GCUGAUUAGUGUCUAAGGANN 1925 UCCUUAGACACUAAUCAGCNN 29
    1191-1209 CUGAUUAGUGUCUAAGGAANN 1926 UUCCUUAGACACUAAUCAGNN 29
     119-137 CCGCCUCCGCCGCCGGAGCNN 1927 GCUCCGGCGGCGGAGGCGGNN 29
    1192-1210 UGAUUAGUGUCUAAGGAAUNN 1928 AUUCCUUAGACACUAAUCANN 29
    1193-1211 GAUUAGUGUCUAAGGAAUGNN 1929 CAUUCCUUAGACACUAAUCNN 29
    1194-1212 AUUAGUGUCUAAGGAAUGANN 1930 UCAUUCCUUAGACACUAAUNN 29
    1195-1213 UUAGUGUCUAAGGAAUGAUNN 1931 AUCAUUCCUUAGACACUAANN 29
    1196-1214 UAGUGUCUAAGGAAUGAUCNN 1932 GAUCAUUCCUUAGACACUANN 29
    1197-1215 AGUGUCUAAGGAAUGAUCCNN 1933 GGAUCAUUCCUUAGACACUNN 29
    1198-1216 GUGUCUAAGGAAUGAUCCANN 1934 UGGAUCAUUCCUUAGACACNN 29
     120-138 CGCCUCCGCCGCCGGAGCCNN 1935 GGCUCCGGCGGCGGAGGCGNN 29
     121-139 GCCUCCGCCGCCGGAGCCCNN 1936 GGGCUCCGGCGGCGGAGGCNN 29
    1218-1236 UACUGUUGCCCUUUUCCUUNN 1937 AAGGAAAAGGGCAACAGUANN 29
    1219-1237 ACUGUUGCCCUUUUCCUUGNN 1938 CAAGGAAAAGGGCAACAGUNN 29
    1220-1238 CUGUUGCCCUUUUCCUUGANN 1939 UCAAGGAAAAGGGCAACAGNN 29
    1221-1239 UGUUGCCCUUUUCCUUGACNN 1940 GUCAAGGAAAAGGGCAACANN 29
     122-140 CCUCCGCCGCCGGAGCCCCNN 1941 GGGGCUCCGGCGGCGGAGGNN 30
    1222-1240 GUUGCCCUUUUCCUUGACUNN 1942 AGUCAAGGAAAAGGGCAACNN 30
    1223-1241 UUGCCCUUUUCCUUGACUANN 1943 UAGUCAAGGAAAAGGGCAANN 30
    1224-1242 UGCCCUUUUCCUUGACUAUNN 1944 AUAGUCAAGGAAAAGGGCANN 30
    1225-1243 GCCCUUUUCCUUGACUAUUNN 1945 AAUAGUCAAGGAAAAGGGCNN 30
    1226-1244 CCCUUUUCCUUGACUAUUANN 1946 UAAUAGUCAAGGAAAAGGGNN 30
    1227-1245 CCUUUUCCUUGACUAUUACNN 1947 GUAAUAGUCAAGGAAAAGGNN 30
    1228-1246 CUUUUCCUUGACUAUUACANN 1948 UGUAAUAGUCAAGGAAAAGNN 30
    1229-1247 UUUUCCUUGACUAUUACACNN 1949 GUGUAAUAGUCAAGGAAAANN 30
    1230-1248 UUUCCUUGACUAUUACACUNN 1950 AGUGUAAUAGUCAAGGAAANN 30
    1231-1249 UUCCUUGACUAUUACACUGNN 1951 CAGUGUAAUAGUCAAGGAANN 30
     123-141 CUCCGCCGCCGGAGCCCCGNN 1952 CGGGGCUCCGGCGGCGGAGNN 30
    1232-1250 UCCUUGACUAUUACACUGCNN 1953 GCAGUGUAAUAGUCAAGGANN 30
    1233-1251 CCUUGACUAUUACACUGCCNN 1954 GGCAGUGUAAUAGUCAAGGNN 30
    1234-1252 CUUGACUAUUACACUGCCUNN 1955 AGGCAGUGUAAUAGUCAAGNN 30
    1235-1253 UUGACUAUUACACUGCCUGNN 1956 CAGGCAGUGUAAUAGUCAANN 30
    1236-1254 UGACUAUUACACUGCCUGGNN 1957 CCAGGCAGUGUAAUAGUCANN 30
    1237-1255 GACUAUUACACUGCCUGGANN 1958 UCCAGGCAGUGUAAUAGUCNN 30
    1238-1256 ACUAUUACACUGCCUGGAGNN 1959 CUCCAGGCAGUGUAAUAGUNN 30
    1239-1257 CUAUUACACUGCCUGGAGGNN 1960 CCUCCAGGCAGUGUAAUAGNN 30
    1240-1258 UAUUACACUGCCUGGAGGANN 1961 UCCUCCAGGCAGUGUAAUANN 30
    1241-1259 AUUACACUGCCUGGAGGAUNN 1962 AUCCUCCAGGCAGUGUAAUNN 30
     124-142 UCCGCCGCCGGAGCCCCGGNN 1963 CCGGGGCUCCGGCGGCGGANN 30
    1242-1260 UUACACUGCCUGGAGGAUANN 1964 UAUCCUCCAGGCAGUGUAANN 30
    1243-1261 UACACUGCCUGGAGGAUAGNN 1965 CUAUCCUCCAGGCAGUGUANN 30
    1244-1262 ACACUGCCUGGAGGAUAGCNN 1966 GCUAUCCUCCAGGCAGUGUNN 30
    1245-1263 CACUGCCUGGAGGAUAGCANN 1967 UGCUAUCCUCCAGGCAGUGNN 30
    1246-1264 ACUGCCUGGAGGAUAGCAGNN 1968 CUGCUAUCCUCCAGGCAGUNN 30
     125-143 CCGCCGCCGGAGCCCCGGCNN 1969 GCCGGGGCTCCGGCGGCGGNN 30
     126-144 CGCCGCCGGAGCCCCGGCCNN 1970 GGCCGGGGCTCCGGCGGCGNN 30
     127-145 GCCGCCGGAGCCCCGGCCGNN 1971 CGGCCGGGGCTCCGGCGGCNN 30
    1280-1298 CUUCAUUCAAAAAGCCAAANN 1972 UUUGGCUUUUUGAAUGAAGNN 30
    1281-1299 UUCAUUCAAAAAGCCAAAANN 1973 UUUUGGCUUUUUGAAUGAANN 30
     128-146 CCGCCGGAGCCCCGGCCGGNN 1974 CCGGCCGGGGCTCCGGCGGNN 30
    1282-1300 UCAUUCAAAAAGCCAAAAUNN 1975 AUUUUGGCUUUUUGAAUGANN 30
    1283-1301 CAUUCAAAAAGCCAAAAUANN 1976 UAUUUUGGCUUUUUGAAUGNN 30
    1284-1302 AUUCAAAAAGCCAAAAUAGNN 1977 CUAUUUUGGCUUUUUGAAUNN 30
    1285-1303 UUCAAAAAGCCAAAAUAGANN 1978 UCUAUUUUGGCUUUUUGAANN 30
    1286-1304 UCAAAAAGCCAAAAUAGAGNN 1979 CUCUAUUUUGGCUUUUUGANN 30
    1287-1305 CAAAAAGCCAAAAUAGAGANN 1980 UCUCUAUUUUGGCUUUUUGNN 30
    1288-1306 AAAAAGCCAAAAUAGAGAGNN 1981 CUCUCUAUUUUGGCUUUUUNN 30
    1289-1307 AAAAGCCAAAAUAGAGAGUNN 1982 ACUCUCUAUUUUGGCUUUUNN 30
    1290-1308 AAAGCCAAAAUAGAGAGUANN 1983 UACUCUCUAUUUUGGCUUUNN 30
     129-147 CGCCGGAGCCCCGGCCGGCNN 1984 GCCGGCCGGGGCTCCGGCGNN 30
     130-148 GCCGGAGCCCCGGCCGGCCNN 1985 GGCCGGCCGGGGCTCCGGCNN 30
    1310-1328 ACAGUCCUAGAGAAUUCCUNN 1986 AGGAAUUCUCUAGGACUGUNN 30
     131-149 CCGGAGCCCCGGCCGGCCANN 1987 TGGCCGGCCGGGGCTCCGGNN 30
     132-150 CGGAGCCCCGGCCGGCCAGNN 1988 CTGGCCGGCCGGGGCTCCGNN 30
    1330-1348 UAUUUGUUCAGAUCUCAUANN 1989 UAUGAGAUCUGAACAAAUANN 30
    1331-1349 AUUUGUUCAGAUCUCAUAGNN 1990 CUAUGAGAUCUGAACAAAUNN 30
     133-151 GGAGCCCCGGCCGGCCAGGNN 1991 CCTGGCCGGCCGGGGCTCCNN 30
    1332-1350 UUUGUUCAGAUCUCAUAGANN 1992 UCUAUGAGAUCUGAACAAANN 30
    1333-1351 UUGUUCAGAUCUCAUAGAUNN 1993 AUCUAUGAGAUCUGAACAANN 30
    1334-1352 UGUUCAGAUCUCAUAGAUGNN 1994 CAUCUAUGAGAUCUGAACANN 30
    1335-1353 GUUCAGAUCUCAUAGAUGANN 1995 UCAUCUAUGAGAUCUGAACNN 30
     134-152 GAGCCCCGGCCGGCCAGGCNN 1996 GCCTGGCCGGCCGGGGCTCNN 30
     135-153 AGCCCCGGCCGGCCAGGCCNN 1997 GGCCTGGCCGGCCGGGGCTNN 30
     136-154 GCCCCGGCCGGCCAGGCCCNN 1998 GGGCCTGGCCGGCCGGGGCNN 30
    1365-1383 UGUCUUUUGACAUCCAGCANN 1999 UGCUGGAUGUCAAAAGACANN 30
    1366-1384 GUCUUUUGACAUCCAGCAGNN 2000 CUGCUGGAUGUCAAAAGACNN 30
    1367-1385 UCUUUUGACAUCCAGCAGUNN 2001 ACUGCUGGAUGUCAAAAGANN 30
    1368-1386 CUUUUGACAUCCAGCAGUCNN 2002 GACUGCUGGAUGUCAAAAGNN 30
    1369-1387 UUUUGACAUCCAGCAGUCCNN 2003 GGACUGCUGGAUGUCAAAANN 30
    1370-1388 UUUGACAUCCAGCAGUCCANN 2004 UGGACUGCUGGAUGUCAAANN 30
    1371-1389 UUGACAUCCAGCAGUCCAANN 2005 UUGGACUGCUGGAUGUCAANN 30
     137-155 CCCCGGCCGGCCAGGCCCUNN 2006 AGGGCCUGGCCGGCCGGGGNN 30
     138-156 CCCGGCCGGCCAGGCCCUGNN 2007 CAGGGCCUGGCCGGCCGGGNN 30
    1391-1409 GUAUUGAGACAUAUUACUGNN 2008 CAGUAAUAUGUCUCAAUACNN 30
     139-157 CCGGCCGGCCAGGCCCUGCNN 2009 GCAGGGCCUGGCCGGCCGGNN 30
     140-158 CGGCCGGCCAGGCCCUGCCNN 2010 GGCAGGGCCUGGCCGGCCGNN 30
     141-159 GGCCGGCCAGGCCCUGCCGNN 2011 CGGCAGGGCCUGGCCGGCCNN 30
    1414-1432 UAAGAAAUAUUACUAUAAUNN 2012 AUUAUAGUAAUAUUUCUUANN 30
    1415-1433 AAGAAAUAUUACUAUAAUUNN 2013 AAUUAUAGUAAUAUUUCUUNN 30
    1416-1434 AGAAAUAUUACUAUAAUUGNN 2014 CAAUUAUAGUAAUAUUUCUNN 30
    1417-1435 GAAAUAUUACUAUAAUUGANN 2015 UCAAUUAUAGUAAUAUUUCNN 30
    1418-1436 AAAUAUUACUAUAAUUGAGNN 2016 CUCAAUUAUAGUAAUAUUUNN 30
    1419-1437 AAUAUUACUAUAAUUGAGANN 2017 UCUCAAUUAUAGUAAUAUUNN 30
    1420-1438 AUAUUACUAUAAUUGAGAANN 2018 UUCUCAAUUAUAGUAAUAUNN 30
    1421-1439 UAUUACUAUAAUUGAGAACNN 2019 GUUCUCAAUUAUAGUAAUANN 30
     142-160 GCCGGCCAGGCCCUGCCGCNN 2020 GCGGCAGGGCCUGGCCGGCNN 30
    1422-1440 AUUACUAUAAUUGAGAACUNN 2021 AGUUCUCAAUUAUAGUAAUNN 30
    1423-1441 UUACUAUAAUUGAGAACUANN 2022 UAGUUCUCAAUUAUAGUAANN 30
    1424-1442 UACUAUAAUUGAGAACUACNN 2023 GUAGUUCUCAAUUAUAGUANN 30
    1425-1443 ACUAUAAUUGAGAACUACANN 2024 UGUAGUUCUCAAUUAUAGUNN 30
    1426-1444 CUAUAAUUGAGAACUACAGNN 2025 CUGUAGUUCUCAAUUAUAGNN 30
    1427-1445 UAUAAUUGAGAACUACAGCNN 2026 GCUGUAGUUCUCAAUUAUANN 30
    1428-1446 AUAAUUGAGAACUACAGCUNN 2027 AGCUGUAGUUCUCAAUUAUNN 30
    1429-1447 UAAUUGAGAACUACAGCUUNN 2028 AAGCUGUAGUUCUCAAUUANN 30
    1430-1448 AAUUGAGAACUACAGCUUUNN 2029 AAAGCUGUAGUUCUCAAUUNN 30
    1431-1449 AUUGAGAACUACAGCUUUUNN 2030 AAAAGCUGUAGUUCUCAAUNN 30
     143-161 CCGGCCAGGCCCUGCCGCUNN 2031 AGCGGCAGGGCCUGGCCGGNN 30
    1432-1450 UUGAGAACUACAGCUUUUANN 2032 UAAAAGCUGUAGUUCUCAANN 30
    1433-1451 UGAGAACUACAGCUUUUAANN 2033 UUAAAAGCUGUAGUUCUCANN 30
    1434-1452 GAGAACUACAGCUUUUAAGNN 2034 CUUAAAAGCUGUAGUUCUCNN 30
    1435-1453 AGAACUACAGCUUUUAAGANN 2035 UCUUAAAAGCUGUAGUUCUNN 30
    1436-1454 GAACUACAGCUUUUAAGAUNN 2036 AUCUUAAAAGCUGUAGUUCNN 30
    1437-1455 AACUACAGCUUUUAAGAUUNN 2037 AAUCUUAAAAGCUGUAGUUNN 30
    1438-1456 ACUACAGCUUUUAAGAUUGNN 2038 CAAUCUUAAAAGCUGUAGUNN 30
    1439-1457 CUACAGCUUUUAAGAUUGUNN 2039 ACAAUCUUAAAAGCUGUAGNN 30
    1440-1458 UACAGCUUUUAAGAUUGUANN 2040 UACAAUCUUAAAAGCUGUANN 30
    1441-1459 ACAGCUUUUAAGAUUGUACNN 2041 GUACAAUCUUAAAAGCUGUNN 31
     144-162 CGGCCAGGCCCUGCCGCUCNN 2042 GAGCGGCAGGGCCUGGCCGNN 31
    1442-1460 CAGCUUUUAAGAUUGUACUNN 2043 AGUACAAUCUUAAAAGCUGNN 31
    1443-1461 AGCUUUUAAGAUUGUACUUNN 2044 AAGUACAAUCUUAAAAGCUNN 31
    1444-1462 GCUUUUAAGAUUGUACUUUNN 2045 AAAGUACAAUCUUAAAAGCNN 31
    1445-1463 CUUUUAAGAUUGUACUUUUNN 2046 AAAAGUACAAUCUUAAAAGNN 31
    1446-1464 UUUUAAGAUUGUACUUUUANN 2047 UAAAAGUACAAUCUUAAAANN 31
    1447-1465 UUUAAGAUUGUACUUUUAUNN 2048 AUAAAAGUACAAUCUUAAANN 31
    1448-1466 UUAAGAUUGUACUUUUAUCNN 2049 GAUAAAAGUACAAUCUUAANN 31
    1449-1467 UAAGAUUGUACUUUUAUCUNN 2050 AGAUAAAAGUACAAUCUUANN 31
    1450-1468 AAGAUUGUACUUUUAUCUUNN 2051 AAGAUAAAAGUACAAUCUUNN 31
    1451-1469 AGAUUGUACUUUUAUCUUANN 2052 UAAGAUAAAAGUACAAUCUNN 31
     145-163 GGCCAGGCCCUGCCGCUCANN 2053 UGAGCGGCAGGGCCUGGCCNN 31
    1452-1470 GAUUGUACUUUUAUCUUAANN 2054 UUAAGAUAAAAGUACAAUCNN 31
    1453-1471 AUUGUACUUUUAUCUUAAANN 2055 UUUAAGAUAAAAGUACAAUNN 31
    1454-1472 UUGUACUUUUAUCUUAAAANN 2056 UUUUAAGAUAAAAGUACAANN 31
    1455-1473 UGUACUUUUAUCUUAAAAGNN 2057 CUUUUAAGAUAAAAGUACANN 31
    1456-1474 GUACUUUUAUCUUAAAAGGNN 2058 CCUUUUAAGAUAAAAGUACNN 31
    1457-1475 UACUUUUAUCUUAAAAGGGNN 2059 CCCUUUUAAGAUAAAAGUANN 31
    1458-1476 ACUUUUAUCUUAAAAGGGUNN 2060 ACCCUUUUAAGAUAAAAGUNN 31
    1459-1477 CUUUUAUCUUAAAAGGGUGNN 2061 CACCCUUUUAAGAUAAAAGNN 31
    1460-1478 UUUUAUCUUAAAAGGGUGGNN 2062 CCACCCUUUUAAGAUAAAANN 31
    1461-1479 UUUAUCUUAAAAGGGUGGUNN 2063 ACCACCCUUUUAAGAUAAANN 31
     146-164 GCCAGGCCCUGCCGCUCAUNN 2064 AUGAGCGGCAGGGCCUGGCNN 31
    1462-1480 UUAUCUUAAAAGGGUGGUANN 2065 UACCACCCUUUUAAGAUAANN 31
    1463-1481 UAUCUUAAAAGGGUGGUAGNN 2066 CUACCACCCUUUUAAGAUANN 31
    1464-1482 AUCUUAAAAGGGUGGUAGUNN 2067 ACUACCACCCUUUUAAGAUNN 31
    1465-1483 UCUUAAAAGGGUGGUAGUUNN 2068 AACUACCACCCUUUUAAGANN 31
    1466-1484 CUUAAAAGGGUGGUAGUUUNN 2069 AAACUACCACCCUUUUAAGNN 31
     147-165 CCAGGCCCUGCCGCUCAUGNN 2070 CAUGAGCGGCAGGGCCUGGNN 31
     148-166 CAGGCCCUGCCGCUCAUGGNN 2071 CCAUGAGCGGCAGGGCCUGNN 31
    1486-1504 CCCUAAAAUACUUAUUAUGNN 2072 CAUAAUAAGUAUUUUAGGGNN 31
    1487-1505 CCUAAAAUACUUAUUAUGUNN 2073 ACAUAAUAAGUAUUUUAGGNN 31
    1488-1506 CUAAAAUACUUAUUAUGUANN 2074 UACAUAAUAAGUAUUUUAGNN 31
    1489-1507 UAAAAUACUUAUUAUGUAANN 2075 UUACAUAAUAAGUAUUUUANN 31
    1490-1508 AAAAUACUUAUUAUGUAAGNN 2076 CUUACAUAAUAAGUAUUUUNN 31
    1491-1509 AAAUACUUAUUAUGUAAGGNN 2077 CCUUACAUAAUAAGUAUUUNN 31
     149-167 AGGCCCUGCCGCUCAUGGUNN 2078 ACCAUGAGCGGCAGGGCCUNN 31
    1492-1510 AAUACUUAUUAUGUAAGGGNN 2079 CCCUUACAUAAUAAGUAUUNN 31
    1493-1511 AUACUUAUUAUGUAAGGGUNN 2080 ACCCUUACAUAAUAAGUAUNN 31
    1494-1512 UACUUAUUAUGUAAGGGUCNN 2081 GACCCUUACAUAAUAAGUANN 31
    1495-1513 ACUUAUUAUGUAAGGGUCANN 2082 UGACCCUUACAUAAUAAGUNN 31
    1496-1514 CUUAUUAUGUAAGGGUCAUNN 2083 AUGACCCUUACAUAAUAAGNN 31
    1497-1515 UUAUUAUGUAAGGGUCAUUNN 2084 AAUGACCCUUACAUAAUAANN 31
    1498-1516 UAUUAUGUAAGGGUCAUUANN 2085 UAAUGACCCUUACAUAAUANN 31
    1499-1517 AUUAUGUAAGGGUCAUUAGNN 2086 CUAAUGACCCUUACAUAAUNN 31
    1500-1518 UUAUGUAAGGGUCAUUAGANN 2087 UCUAAUGACCCUUACAUAANN 31
    1501-1519 UAUGUAAGGGUCAUUAGACNN 2088 GUCUAAUGACCCUUACAUANN 31
     150-168 GGCCCUGCCGCUCAUGGUGNN 2089 CACCAUGAGCGGCAGGGCCNN 31
    1502-1520 AUGUAAGGGUCAUUAGACANN 2090 UGUCUAAUGACCCUUACAUNN 31
    1503-1521 UGUAAGGGUCAUUAGACAANN 2091 UUGUCUAAUGACCCUUACANN 31
    1504-1522 GUAAGGGUCAUUAGACAAANN 2092 UUUGUCUAAUGACCCUUACNN 31
    1505-1523 UAAGGGUCAUUAGACAAAUNN 2093 AUUUGUCUAAUGACCCUUANN 31
    1506-1524 AAGGGUCAUUAGACAAAUGNN 2094 CAUUUGUCUAAUGACCCUUNN 31
    1507-1525 AGGGUCAUUAGACAAAUGUNN 2095 ACAUUUGUCUAAUGACCCUNN 31
    1508-1526 GGGUCAUUAGACAAAUGUCNN 2096 GACAUUUGUCUAAUGACCCNN 31
    1509-1527 GGUCAUUAGACAAAUGUCUNN 2097 AGACAUUUGUCUAAUGACCNN 31
    1510-1528 GUCAUUAGACAAAUGUCUUNN 2098 AAGACAUUUGUCUAAUGACNN 31
    1511-1529 UCAUUAGACAAAUGUCUUGNN 2099 CAAGACAUUUGUCUAAUGANN 31
     151-169 GCCCUGCCGCUCAUGGUGCNN 2100 GCACCAUGAGCGGCAGGGCNN 31
    1512-1530 CAUUAGACAAAUGUCUUGANN 2101 UCAAGACAUUUGUCUAAUGNN 31
    1513-1531 AUUAGACAAAUGUCUUGAANN 2102 UUCAAGACAUUUGUCUAAUNN 31
    1514-1532 UUAGACAAAUGUCUUGAAGNN 2103 CUUCAAGACAUUUGUCUAANN 31
    1515-1533 UAGACAAAUGUCUUGAAGUNN 2104 ACUUCAAGACAUUUGUCUANN 31
    1516-1534 AGACAAAUGUCUUGAAGUANN 2105 UACUUCAAGACAUUUGUCUNN 31
    1517-1535 GACAAAUGUCUUGAAGUAGNN 2106 CUACUUCAAGACAUUUGUCNN 31
    1518-1536 ACAAAUGUCUUGAAGUAGANN 2107 UCUACUUCAAGACAUUUGUNN 31
     152-170 CCCUGCCGCUCAUGGUGCCNN 2108 GGCACCAUGAGCGGCAGGGNN 31
     153-171 CCUGCCGCUCAUGGUGCCANN 2109 UGGCACCAUGAGCGGCAGGNN 31
    1541-1559 GAAUUUAUGAAUGGUUCUUNN 2110 AAGAACCAUUCAUAAAUUCNN 31
     154-172 CUGCCGCUCAUGGUGCCAGNN 2111 CUGGCACCAUGAGCGGCAGNN 31
    1542-1560 AAUUUAUGAAUGGUUCUUUNN 2112 AAAGAACCAUUCAUAAAUUNN 31
    1543-1561 AUUUAUGAAUGGUUCUUUANN 2113 UAAAGAACCAUUCAUAAAUNN 31
    1544-1562 UUUAUGAAUGGUUCUUUAUNN 2114 AUAAAGAACCAUUCAUAAANN 31
    1545-1563 UUAUGAAUGGUUCUUUAUCNN 2115 GAUAAAGAACCAUUCAUAANN 31
    1546-1564 UAUGAAUGGUUCUUUAUCANN 2116 UGAUAAAGAACCAUUCAUANN 31
    1547-1565 AUGAAUGGUUCUUUAUCAUNN 2117 AUGAUAAAGAACCAUUCAUNN 31
    1548-1566 UGAAUGGUUCUUUAUCAUUNN 2118 AAUGAUAAAGAACCAUUCANN 31
    1549-1567 GAAUGGUUCUUUAUCAUUUNN 2119 AAAUGAUAAAGAACCAUUCNN 31
    1550-1568 AAUGGUUCUUUAUCAUUUCNN 2120 GAAAUGAUAAAGAACCAUUNN 31
    1551-1569 AUGGUUCUUUAUCAUUUCUNN 2121 AGAAAUGAUAAAGAACCAUNN 31
     155-173 UGCCGCUCAUGGUGCCAGCNN 2122 GCUGGCACCAUGAGCGGCANN 31
    1552-1570 UGGUUCUUUAUCAUUUCUCNN 2123 GAGAAAUGAUAAAGAACCANN 31
    1553-1571 GGUUCUUUAUCAUUUCUCUNN 2124 AGAGAAAUGAUAAAGAACCNN 31
    1554-1572 GUUCUUUAUCAUUUCUCUUNN 2125 AAGAGAAAUGAUAAAGAACNN 31
    1555-1573 UUCUUUAUCAUUUCUCUUCNN 2126 GAAGAGAAAUGAUAAAGAANN 31
    1556-1574 UCUUUAUCAUUUCUCUUCCNN 2127 GGAAGAGAAAUGAUAAAGANN 31
    1557-1575 CUUUAUCAUUUCUCUUCCCNN 2128 GGGAAGAGAAAUGAUAAAGNN 31
    1558-1576 UUUAUCAUUUCUCUUCCCCNN 2129 GGGGAAGAGAAAUGAUAAANN 31
    1559-1577 UUAUCAUUUCUCUUCCCCCNN 2130 GGGGGAAGAGAAAUGAUAANN 31
    1560-1578 UAUCAUUUCUCUUCCCCCUNN 2131 AGGGGGAAGAGAAAUGAUANN 31
    1561-1579 AUCAUUUCUCUUCCCCCUUNN 2132 AAGGGGGAAGAGAAAUGAUNN 31
     156-174 GCCGCUCAUGGUGCCAGCCNN 2133 GGCUGGCACCAUGAGCGGCNN 31
    1562-1580 UCAUUUCUCUUCCCCCUUUNN 2134 AAAGGGGGAAGAGAAAUGANN 31
    1563-1581 CAUUUCUCUUCCCCCUUUUNN 2135 AAAAGGGGGAAGAGAAAUGNN 31
    1564-1582 AUUUCUCUUCCCCCUUUUUNN 2136 AAAAAGGGGGAAGAGAAAUNN 31
    1565-1583 UUUCUCUUCCCCCUUUUUGNN 2137 CAAAAAGGGGGAAGAGAAANN 31
    1566-1584 UUCUCUUCCCCCUUUUUGGNN 2138 CCAAAAAGGGGGAAGAGAANN 31
    1567-1585 UCUCUUCCCCCUUUUUGGCNN 2139 GCCAAAAAGGGGGAAGAGANN 31
    1568-1586 CUCUUCCCCCUUUUUGGCANN 2140 UGCCAAAAAGGGGGAAGAGNN 31
    1569-1587 UCUUCCCCCUUUUUGGCAUNN 2141 AUGCCAAAAAGGGGGAAGANN 32
    1570-1588 CUUCCCCCUUUUUGGCAUCNN 2142 GAUGCCAAAAAGGGGGAAGNN 32
    1571-1589 UUCCCCCUUUUUGGCAUCCNN 2143 GGAUGCCAAAAAGGGGGAANN 32
     157-175 CCGCUCAUGGUGCCAGCCCNN 2144 GGGCUGGCACCAUGAGCGGNN 32
    1572-1590 UCCCCCUUUUUGGCAUCCUNN 2145 AGGAUGCCAAAAAGGGGGANN 32
    1573-1591 CCCCCUUUUUGGCAUCCUGNN 2146 CAGGAUGCCAAAAAGGGGGNN 32
    1574-1592 CCCCUUUUUGGCAUCCUGGNN 2147 CCAGGAUGCCAAAAAGGGGNN 32
    1575-1593 CCCUUUUUGGCAUCCUGGCNN 2148 GCCAGGAUGCCAAAAAGGGNN 32
    1576-1594 CCUUUUUGGCAUCCUGGCUNN 2149 AGCCAGGAUGCCAAAAAGGNN 32
    1577-1595 CUUUUUGGCAUCCUGGCUUNN 2150 AAGCCAGGAUGCCAAAAAGNN 32
    1578-1596 UUUUUGGCAUCCUGGCUUGNN 2151 CAAGCCAGGAUGCCAAAAANN 32
    1579-1597 UUUUGGCAUCCUGGCUUGCNN 2152 GCAAGCCAGGAUGCCAAAANN 32
    1580-1598 UUUGGCAUCCUGGCUUGCCNN 2153 GGCAAGCCAGGAUGCCAAANN 32
    1581-1599 UUGGCAUCCUGGCUUGCCUNN 2154 AGGCAAGCCAGGAUGCCAANN 32
     158-176 CGCUCAUGGUGCCAGCCCANN 2155 UGGGCUGGCACCAUGAGCGNN 32
    1582-1600 UGGCAUCCUGGCUUGCCUCNN 2156 GAGGCAAGCCAGGAUGCCANN 32
    1583-1601 GGCAUCCUGGCUUGCCUCCNN 2157 GGAGGCAAGCCAGGAUGCCNN 32
    1584-1602 GCAUCCUGGCUUGCCUCCANN 2158 UGGAGGCAAGCCAGGAUGCNN 32
    1585-1603 CAUCCUGGCUUGCCUCCAGNN 2159 CUGGAGGCAAGCCAGGAUGNN 32
    1586-1604 AUCCUGGCUUGCCUCCAGUNN 2160 ACUGGAGGCAAGCCAGGAUNN 32
    1587-1605 UCCUGGCUUGCCUCCAGUUNN 2161 AACUGGAGGCAAGCCAGGANN 32
    1588-1606 CCUGGCUUGCCUCCAGUUUNN 2162 AAACUGGAGGCAAGCCAGGNN 32
    1589-1607 CUGGCUUGCCUCCAGUUUUNN 2163 AAAACUGGAGGCAAGCCAGNN 32
    1590-1608 UGGCUUGCCUCCAGUUUUANN 2164 UAAAACUGGAGGCAAGCCANN 32
    1591-1609 GGCUUGCCUCCAGUUUUAGNN 2165 CUAAAACUGGAGGCAAGCCNN 32
     159-177 GCUCAUGGUGCCAGCCCAGNN 2166 CUGGGCUGGCACCAUGAGCNN 32
    1592-1610 GCUUGCCUCCAGUUUUAGGNN 2167 CCUAAAACUGGAGGCAAGCNN 32
    1593-1611 CUUGCCUCCAGUUUUAGGUNN 2168 ACCUAAAACUGGAGGCAAGNN 32
    1594-1612 UUGCCUCCAGUUUUAGGUCNN 2169 GACCUAAAACUGGAGGCAANN 32
    1595-1613 UGCCUCCAGUUUUAGGUCCNN 2170 GGACCUAAAACUGGAGGCANN 32
     160-178 CUCAUGGUGCCAGCCCAGANN 2171 UCUGGGCUGGCACCAUGAGNN 32
     161-179 UCAUGGUGCCAGCCCAGAGNN 2172 CUCUGGGCUGGCACCAUGANN 32
    1615-1633 UUAGUUUGCUUCUGUAAGCNN 2173 GCUUACAGAAGCAAACUAANN 32
    1616-1634 UAGUUUGCUUCUGUAAGCANN 2174 UGCUUACAGAAGCAAACUANN 32
    1617-1635 AGUUUGCUUCUGUAAGCAANN 2175 UUGCUUACAGAAGCAAACUNN 32
     162-180 CAUGGUGCCAGCCCAGAGANN 2176 UCUCUGGGCUGGCACCAUGNN 32
     163-181 AUGGUGCCAGCCCAGAGAGNN 2177 CUCUCUGGGCUGGCACCAUNN 32
    1639-1657 GAACACCUGCUGAGGGGGCNN 2178 GCCCCCUCAGCAGGUGUUCNN 32
    1640-1658 AACACCUGCUGAGGGGGCUNN 2179 AGCCCCCUCAGCAGGUGUUNN 32
    1641-1659 ACACCUGCUGAGGGGGCUCNN 2180 GAGCCCCCUCAGCAGGUGUNN 32
     164-182 UGGUGCCAGCCCAGAGAGGNN 2181 CCUCUCUGGGCUGGCACCANN 32
    1642-1660 CACCUGCUGAGGGGGCUCUNN 2182 AGAGCCCCCUCAGCAGGUGNN 32
    1643-1661 ACCUGCUGAGGGGGCUCUUNN 2183 AAGAGCCCCCUCAGCAGGUNN 32
    1644-1662 CCUGCUGAGGGGGCUCUUUNN 2184 AAAGAGCCCCCUCAGCAGGNN 32
    1645-1663 CUGCUGAGGGGGCUCUUUCNN 2185 GAAAGAGCCCCCUCAGCAGNN 32
    1646-1664 UGCUGAGGGGGCUCUUUCCNN 2186 GGAAAGAGCCCCCUCAGCANN 32
    1647-1665 GCUGAGGGGGCUCUUUCCCNN 2187 GGGAAAGAGCCCCCUCAGCNN 32
    1648-1666 CUGAGGGGGCUCUUUCCCUNN 2188 AGGGAAAGAGCCCCCUCAGNN 32
    1649-1667 UGAGGGGGCUCUUUCCCUCNN 2189 GAGGGAAAGAGCCCCCUCANN 32
    1650-1668 GAGGGGGCUCUUUCCCUCANN 2190 UGAGGGAAAGAGCCCCCUCNN 32
     165-183 GGUGCCAGCCCAGAGAGGGNN 2191 CCCUCUCUGGGCUGGCACCNN 32
     166-184 GUGCCAGCCCAGAGAGGGGNN 2192 CCCCUCUCUGGGCUGGCACNN 32
    1670-1688 GUAUACUUCAAGUAAGAUCNN 2193 GAUCUUACUUGAAGUAUACNN 32
    1671-1689 UAUACUUCAAGUAAGAUCANN 2194 UGAUCUUACUUGAAGUAUANN 32
     167-185 UGCCAGCCCAGAGAGGGGCNN 2195 GCCCCUCUCUGGGCUGGCANN 32
    1672-1690 AUACUUCAAGUAAGAUCAANN 2196 UUGAUCUUACUUGAAGUAUNN 32
    1673-1691 UACUUCAAGUAAGAUCAAGNN 2197 CUUGAUCUUACUUGAAGUANN 32
    1674-1692 ACUUCAAGUAAGAUCAAGANN 2198 UCUUGAUCUUACUUGAAGUNN 32
    1675-1693 CUUCAAGUAAGAUCAAGAANN 2199 UUCUUGAUCUUACUUGAAGNN 32
    1676-1694 UUCAAGUAAGAUCAAGAAUNN 2200 AUUCUUGAUCUUACUUGAANN 32
    1677-1695 UCAAGUAAGAUCAAGAAUCNN 2201 GAUUCUUGAUCUUACUUGANN 32
    1678-1696 CAAGUAAGAUCAAGAAUCUNN 2202 AGAUUCUUGAUCUUACUUGNN 32
    1679-1697 AAGUAAGAUCAAGAAUCUUNN 2203 AAGAUUCUUGAUCUUACUUNN 32
    1680-1698 AGUAAGAUCAAGAAUCUUUNN 2204 AAAGAUUCUUGAUCUUACUNN 32
    1681-1699 GUAAGAUCAAGAAUCUUUUNN 2205 AAAAGAUUCUUGAUCUUACNN 32
    1682-1700 UAAGAUCAAGAAUCUUUUGNN 2206 CAAAAGAUUCUUGAUCUUANN 32
    1683-1701 AAGAUCAAGAAUCUUUUGUNN 2207 ACAAAAGAUUCUUGAUCUUNN 32
    1684-1702 AGAUCAAGAAUCUUUUGUGNN 2208 CACAAAAGAUUCUUGAUCUNN 32
    1685-1703 GAUCAAGAAUCUUUUGUGANN 2209 UCACAAAAGAUUCUUGAUCNN 32
    1686-1704 AUCAAGAAUCUUUUGUGAANN 2210 UUCACAAAAGAUUCUUGAUNN 32
    1687-1705 UCAAGAAUCUUUUGUGAAANN 2211 UUUCACAAAAGAUUCUUGANN 32
    1707-1725 UAUAGAAAUUUACUAUGUANN 2212 UACAUAGUAAAUUUCUAUANN 32
    1708-1726 AUAGAAAUUUACUAUGUAANN 2213 UUACAUAGUAAAUUUCUAUNN 32
    1709-1727 UAGAAAUUUACUAUGUAAANN 2214 UUUACAUAGUAAAUUUCUANN 32
    1710-1728 AGAAAUUUACUAUGUAAAUNN 2215 AUUUACAUAGUAAAUUUCUNN 32
    1711-1729 GAAAUUUACUAUGUAAAUGNN 2216 CAUUUACAUAGUAAAUUUCNN 32
    1712-1730 AAAUUUACUAUGUAAAUGCNN 2217 GCAUUUACAUAGUAAAUUUNN 32
    1713-1731 AAUUUACUAUGUAAAUGCUNN 2218 AGCAUUUACAUAGUAAAUUNN 32
    1714-1732 AUUUACUAUGUAAAUGCUUNN 2219 AAGCAUUUACAUAGUAAAUNN 32
    1715-1733 UUUACUAUGUAAAUGCUUGNN 2220 CAAGCAUUUACAUAGUAAANN 32
    1716-1734 UUACUAUGUAAAUGCUUGANN 2221 UCAAGCAUUUACAUAGUAANN 32
    1717-1735 UACUAUGUAAAUGCUUGAUNN 2222 AUCAAGCAUUUACAUAGUANN 32
    1718-1736 ACUAUGUAAAUGCUUGAUGNN 2223 CAUCAAGCAUUUACAUAGUNN 32
    1719-1737 CUAUGUAAAUGCUUGAUGGNN 2224 CCAUCAAGCAUUUACAUAGNN 32
    1720-1738 UAUGUAAAUGCUUGAUGGANN 2225 UCCAUCAAGCAUUUACAUANN 32
    1721-1739 AUGUAAAUGCUUGAUGGAANN 2226 UUCCAUCAAGCAUUUACAUNN 32
    1722-1740 UGUAAAUGCUUGAUGGAAUNN 2227 AUUCCAUCAAGCAUUUACANN 32
    1723-1741 GUAAAUGCUUGAUGGAAUUNN 2228 AAUUCCAUCAAGCAUUUACNN 32
    1724-1742 UAAAUGCUUGAUGGAAUUUNN 2229 AAAUUCCAUCAAGCAUUUANN 32
    1725-1743 AAAUGCUUGAUGGAAUUUUNN 2230 AAAAUUCCAUCAAGCAUUUNN 32
    1726-1744 AAUGCUUGAUGGAAUUUUUNN 2231 AAAAAUUCCAUCAAGCAUUNN 32
    1727-1745 AUGCUUGAUGGAAUUUUUUNN 2232 AAAAAAUUCCAUCAAGCAUNN 32
    1728-1746 UGCUUGAUGGAAUUUUUUCNN 2233 GAAAAAAUUCCAUCAAGCANN 32
    1729-1747 GCUUGAUGGAAUUUUUUCCNN 2234 GGAAAAAAUUCCAUCAAGCNN 32
    1730-1748 CUUGAUGGAAUUUUUUCCUNN 2235 AGGAAAAAAUUCCAUCAAGNN 32
    1731-1749 UUGAUGGAAUUUUUUCCUGNN 2236 CAGGAAAAAAUUCCAUCAANN 32
    1732-1750 UGAUGGAAUUUUUUCCUGCNN 2237 GCAGGAAAAAAUUCCAUCANN 32
    1733-1751 GAUGGAAUUUUUUCCUGCUNN 2238 AGCAGGAAAAAAUUCCAUCNN 32
    1734-1752 AUGGAAUUUUUUCCUGCUANN 2239 UAGCAGGAAAAAAUUCCAUNN 32
    1735-1753 UGGAAUUUUUUCCUGCUAGNN 2240 CUAGCAGGAAAAAAUUCCANN 32
    1736-1754 GGAAUUUUUUCCUGCUAGUNN 2241 ACUAGCAGGAAAAAAUUCCNN 33
    1737-1755 GAAUUUUUUCCUGCUAGUGNN 2242 CACUAGCAGGAAAAAAUUCNN 33
    1738-1756 AAUUUUUUCCUGCUAGUGUNN 2243 ACACUAGCAGGAAAAAAUUNN 33
    1739-1757 AUUUUUUCCUGCUAGUGUANN 2244 UACACUAGCAGGAAAAAAUNN 33
    1740-1758 UUUUUUCCUGCUAGUGUAGNN 2245 CUACACUAGCAGGAAAAAANN 33
    1741-1759 UUUUUCCUGCUAGUGUAGCNN 2246 GCUACACUAGCAGGAAAAANN 33
    1742-1760 UUUUCCUGCUAGUGUAGCUNN 2247 AGCUACACUAGCAGGAAAANN 33
    1743-1761 UUUCCUGCUAGUGUAGCUUNN 2248 AAGCUACACUAGCAGGAAANN 33
    1744-1762 UUCCUGCUAGUGUAGCUUCNN 2249 GAAGCUACACUAGCAGGAANN 33
    1745-1763 UCCUGCUAGUGUAGCUUCUNN 2250 AGAAGCUACACUAGCAGGANN 33
    1746-1764 CCUGCUAGUGUAGCUUCUGNN 2251 CAGAAGCUACACUAGCAGGNN 33
    1747-1765 CUGCUAGUGUAGCUUCUGANN 2252 UCAGAAGCUACACUAGCAGNN 33
    1748-1766 UGCUAGUGUAGCUUCUGAANN 2253 UUCAGAAGCUACACUAGCANN 33
    1749-1767 GCUAGUGUAGCUUCUGAAANN 2254 UUUCAGAAGCUACACUAGCNN 33
    1750-1768 CUAGUGUAGCUUCUGAAAGNN 2255 CUUUCAGAAGCUACACUAGNN 33
    1751-1769 UAGUGUAGCUUCUGAAAGGNN 2256 CCUUUCAGAAGCUACACUANN 33
    1752-1770 AGUGUAGCUUCUGAAAGGUNN 2257 ACCUUUCAGAAGCUACACUNN 33
    1753-1771 GUGUAGCUUCUGAAAGGUGNN 2258 CACCUUUCAGAAGCUACACNN 33
    1754-1772 UGUAGCUUCUGAAAGGUGCNN 2259 GCACCUUUCAGAAGCUACANN 33
    1755-1773 GUAGCUUCUGAAAGGUGCUNN 2260 AGCACCUUUCAGAAGCUACNN 33
    1756-1774 UAGCUUCUGAAAGGUGCUUNN 2261 AAGCACCUUUCAGAAGCUANN 33
    1757-1775 AGCUUCUGAAAGGUGCUUUNN 2262 AAAGCACCUUUCAGAAGCUNN 33
    1758-1776 GCUUCUGAAAGGUGCUUUCNN 2263 GAAAGCACCUUUCAGAAGCNN 33
    1777-1795 UCCAUUUAUUUAAAACUACNN 2264 GUAGUUUUAAAUAAAUGGANN 33
    1778-1796 CCAUUUAUUUAAAACUACCNN 2265 GGUAGUUUUAAAUAAAUGGNN 33
    1779-1797 CAUUUAUUUAAAACUACCCNN 2266 GGGUAGUUUUAAAUAAAUGNN 33
    1780-1798 AUUUAUUUAAAACUACCCANN 2267 UGGGUAGUUUUAAAUAAAUNN 33
    1781-1799 UUUAUUUAAAACUACCCAUNN 2268 AUGGGUAGUUUUAAAUAAANN 33
    1782-1800 UUAUUUAAAACUACCCAUGNN 2269 CAUGGGUAGUUUUAAAUAANN 33
    1783-1801 UAUUUAAAACUACCCAUGCNN 2270 GCAUGGGUAGUUUUAAAUANN 33
    1784-1802 AUUUAAAACUACCCAUGCANN 2271 UGCAUGGGUAGUUUUAAAUNN 33
    1785-1803 UUUAAAACUACCCAUGCAANN 2272 UUGCAUGGGUAGUUUUAAANN 33
    1786-1804 UUAAAACUACCCAUGCAAUNN 2273 AUUGCAUGGGUAGUUUUAANN 33
    1787-1805 UAAAACUACCCAUGCAAUUNN 2274 AAUUGCAUGGGUAGUUUUANN 33
    1788-1806 AAAACUACCCAUGCAAUUANN 2275 UAAUUGCAUGGGUAGUUUUNN 33
    1789-1807 AAACUACCCAUGCAAUUAANN 2276 UUAAUUGCAUGGGUAGUUUNN 33
    1790-1808 AACUACCCAUGCAAUUAAANN 2277 UUUAAUUGCAUGGGUAGUUNN 33
    1791-1809 ACUACCCAUGCAAUUAAAANN 2278 UUUUAAUUGCAUGGGUAGUNN 33
    1792-1810 CUACCCAUGCAAUUAAAAGNN 2279 CUUUUAAUUGCAUGGGUAGNN 33
    1793-1811 UACCCAUGCAAUUAAAAGGNN 2280 CCUUUUAAUUGCAUGGGUANN 33
    1794-1812 ACCCAUGCAAUUAAAAGGUNN 2281 ACCUUUUAAUUGCAUGGGUNN 33
    1795-1813 CCCAUGCAAUUAAAAGGUANN 2282 UACCUUUUAAUUGCAUGGGNN 33
    1796-1814 CCAUGCAAUUAAAAGGUACNN 2283 GUACCUUUUAAUUGCAUGGNN 33
    1797-1815 CAUGCAAUUAAAAGGUACANN 2284 UGUACCUUUUAAUUGCAUGNN 33
    1798-1816 AUGCAAUUAAAAGGUACAANN 2285 UUGUACCUUUUAAUUGCAUNN 33
    1799-1817 UGCAAUUAAAAGGUACAAUNN 2286 AUUGUACCUUUUAAUUGCANN 33
    1800-1818 GCAAUUAAAAGGUACAAUGNN 2287 CAUUGUACCUUUUAAUUGCNN 33
    1801-1819 CAAUUAAAAGGUACAAUGCNN 2288 GCAUUGUACCUUUUAAUUGNN 33
    1802-1820 AAUUAAAAGGUACAAUGCANN 2289 UGCAUUGUACCUUUUAAUUNN 33
     187-205 AGCCCGGAGGCAGCGAGCGNN 2290 CGCTCGCTGCCTCCGGGCTNN 33
     188-206 GCCCGGAGGCAGCGAGCGGNN 2291 CCGCTCGCTGCCTCCGGGCNN 33
     189-207 CCCGGAGGCAGCGAGCGGGNN 2292 CCCGCTCGCTGCCTCCGGGNN 33
     190-208 CCGGAGGCAGCGAGCGGGGNN 2293 CCCCGCTCGCTGCCTCCGGNN 33
     191-209 CGGAGGCAGCGAGCGGGGGNN 2294 CCCCCGCTCGCTGCCTCCGNN 33
     192-210 GGAGGCAGCGAGCGGGGGGNN 2295 CCCCCCGCTCGCTGCCTCCNN 33
     193-211 GAGGCAGCGAGCGGGGGGCNN 2296 GCCCCCCGCTCGCTGCCTCNN 33
     194-212 AGGCAGCGAGCGGGGGGCUNN 2297 AGCCCCCCGCUCGCUGCCUNN 33
     195-213 GGCAGCGAGCGGGGGGCUGNN 2298 CAGCCCCCCGCUCGCUGCCNN 33
     196-214 GCAGCGAGCGGGGGGCUGCNN 2299 GCAGCCCCCCGCUCGCUGCNN 33
     197-215 CAGCGAGCGGGGGGCUGCCNN 2300 GGCAGCCCCCCGCUCGCUGNN 33
     198-216 AGCGAGCGGGGGGCUGCCCNN 2301 GGGCAGCCCCCCGCUCGCUNN 33
     199-217 GCGAGCGGGGGGCUGCCCCNN 2302 GGGGCAGCCCCCCGCUCGCNN 33
     200-218 CGAGCGGGGGGCUGCCCCANN 2303 UGGGGCAGCCCCCCGCUCGNN 33
     201-219 GAGCGGGGGGCUGCCCCAGNN 2304 CUGGGGCAGCCCCCCGCUCNN 33
     202-220 AGCGGGGGGCUGCCCCAGGNN 2305 CCUGGGGCAGCCCCCCGCUNN 33
     203-221 GCGGGGGGCUGCCCCAGGCNN 2306 GCCUGGGGCAGCCCCCCGCNN 33
     204-222 CGGGGGGCUGCCCCAGGCGNN 2307 CGCCUGGGGCAGCCCCCCGNN 33
     205-223 GGGGGGCUGCCCCAGGCGCNN 2308 GCGCCUGGGGCAGCCCCCCNN 33
     206-224 GGGGGCUGCCCCAGGCGCGNN 2309 CGCGCCUGGGGCAGCCCCCNN 33
     207-225 GGGGCUGCCCCAGGCGCGCNN 2310 GCGCGCCUGGGGCAGCCCCNN 33
     208-226 GGGCUGCCCCAGGCGCGCANN 2311 UGCGCGCCUGGGGCAGCCCNN 33
     209-227 GGCUGCCCCAGGCGCGCAANN 2312 UUGCGCGCCUGGGGCAGCCNN 33
     210-228 GCUGCCCCAGGCGCGCAAGNN 2313 CUUGCGCGCCUGGGGCAGCNN 33
     211-229 CUGCCCCAGGCGCGCAAGCNN 2314 GCUUGCGCGCCUGGGGCAGNN 33
     212-230 UGCCCCAGGCGCGCAAGCGNN 2315 CGCUUGCGCGCCUGGGGCANN 33
     247-265 CUGAGCCCCGAGGAGAAGGNN 2316 CCUUCUCCUCGGGGCUCAGNN 33
     248-266 UGAGCCCCGAGGAGAAGGCNN 2317 GCCUUCUCCUCGGGGCUCANN 33
     249-267 GAGCCCCGAGGAGAAGGCGNN 2318 CGCCTTCTCCTCGGGGCTCNN 33
     250-268 AGCCCCGAGGAGAAGGCGCNN 2319 GCGCCTTCTCCTCGGGGCTNN 33
     251-269 GCCCCGAGGAGAAGGCGCUNN 2320 AGCGCCUUCUCCUCGGGGCNN 33
     252-270 CCCCGAGGAGAAGGCGCUGNN 2321 CAGCGCCUUCUCCUCGGGGNN 33
     253-271 CCCGAGGAGAAGGCGCUGANN 2322 UCAGCGCCUUCUCCUCGGGNN 33
     254-272 CCGAGGAGAAGGCGCUGAGNN 2323 CUCAGCGCCUUCUCCUCGGNN 33
     255-273 CGAGGAGAAGGCGCUGAGGNN 2324 CCUCAGCGCCUUCUCCUCGNN 33
     256-274 GAGGAGAAGGCGCUGAGGANN 2325 UCCUCAGCGCCUUCUCCUCNN 33
     257-275 AGGAGAAGGCGCUGAGGAGNN 2326 CUCCUCAGCGCCUUCUCCUNN 33
     258-276 GGAGAAGGCGCUGAGGAGGNN 2327 CCUCCUCAGCGCCUUCUCCNN 33
     259-277 GAGAAGGCGCUGAGGAGGANN 2328 UCCUCCUCAGCGCCUUCUCNN 33
     260-278 AGAAGGCGCUGAGGAGGAANN 2329 UUCCUCCUCAGCGCCUUCUNN 33
     261-279 GAAGGCGCUGAGGAGGAAANN 2330 UUUCCUCCUCAGCGCCUUCNN 33
     262-280 AAGGCGCUGAGGAGGAAACNN 2331 GUUUCCUCCUCAGCGCCUUNN 33
     263-281 AGGCGCUGAGGAGGAAACUNN 2332 AGUUUCCUCCUCAGCGCCUNN 33
     264-282 GGCGCUGAGGAGGAAACUGNN 2333 CAGUUUCCUCCUCAGCGCCNN 33
     265-283 GCGCUGAGGAGGAAACUGANN 2334 UCAGUUUCCUCCUCAGCGCNN 33
     266-284 CGCUGAGGAGGAAACUGAANN 2335 UUCAGUUUCCUCCUCAGCGNN 33
     267-285 GCUGAGGAGGAAACUGAAANN 2336 UUUCAGUUUCCUCCUCAGCNN 33
     268-286 CUGAGGAGGAAACUGAAAANN 2337 UUUUCAGUUUCCUCCUCAGNN 33
     269-287 UGAGGAGGAAACUGAAAAANN 2338 UUUUUCAGUUUCCUCCUCANN 33
     270-288 GAGGAGGAAACUGAAAAACNN 2339 GUUUUUCAGUUUCCUCCUCNN 33
     271-289 AGGAGGAAACUGAAAAACANN 2340 UGUUUUUCAGUUUCCUCCUNN 33
     272-290 GGAGGAAACUGAAAAACAGNN 2341 CUGUUUUUCAGUUUCCUCCNN 34
     273-291 GAGGAAACUGAAAAACAGANN 2342 UCUGUUUUUCAGUUUCCUCNN 34
     274-292 AGGAAACUGAAAAACAGAGNN 2343 CUCUGUUUUUCAGUUUCCUNN 34
     275-293 GGAAACUGAAAAACAGAGUNN 2344 ACUCUGUUUUUCAGUUUCCNN 34
     276-294 GAAACUGAAAAACAGAGUANN 2345 UACUCUGUUUUUCAGUUUCNN 34
     277-295 AAACUGAAAAACAGAGUAGNN 2346 CUACUCUGUUUUUCAGUUUNN 34
     278-296 AACUGAAAAACAGAGUAGCNN 2347 GCUACUCUGUUUUUCAGUUNN 34
     279-297 ACUGAAAAACAGAGUAGCANN 2348 UGCUACUCUGUUUUUCAGUNN 34
     280-298 CUGAAAAACAGAGUAGCAGNN 2349 CUGCUACUCUGUUUUUCAGNN 34
     281-299 UGAAAAACAGAGUAGCAGCNN 2350 GCUGCUACUCUGUUUUUCANN 34
     282-300 GAAAAACAGAGUAGCAGCUNN 2351 AGCUGCUACUCUGUUUUUCNN 34
     283-301 AAAAACAGAGUAGCAGCUCNN 2352 GAGCUGCUACUCUGUUUUUNN 34
     284-302 AAAACAGAGUAGCAGCUCANN 2353 UGAGCUGCUACUCUGUUUUNN 34
     285-303 AAACAGAGUAGCAGCUCAGNN 2354 CUGAGCUGCUACUCUGUUUNN 34
     286-304 AACAGAGUAGCAGCUCAGANN 2355 UCUGAGCUGCUACUCUGUUNN 34
     287-305 ACAGAGUAGCAGCUCAGACNN 2356 GUCUGAGCUGCUACUCUGUNN 34
     288-306 CAGAGUAGCAGCUCAGACUNN 2357 AGUCUGAGCUGCUACUCUGNN 34
     289-307 AGAGUAGCAGCUCAGACUGNN 2358 CAGUCUGAGCUGCUACUCUNN 34
     290-308 GAGUAGCAGCUCAGACUGCNN 2359 GCAGUCUGAGCUGCUACUCNN 34
     291-309 AGUAGCAGCUCAGACUGCCNN 2360 GGCAGUCUGAGCUGCUACUNN 34
     292-310 GUAGCAGCUCAGACUGCCANN 2361 UGGCAGUCUGAGCUGCUACNN 34
     293-311 UAGCAGCUCAGACUGCCAGNN 2362 CUGGCAGUCUGAGCUGCUANN 34
     294-312 AGCAGCUCAGACUGCCAGANN 2363 UCUGGCAGUCUGAGCUGCUNN 34
     295-313 GCAGCUCAGACUGCCAGAGNN 2364 CUCUGGCAGUCUGAGCUGCNN 34
     296-314 CAGCUCAGACUGCCAGAGANN 2365 UCUCUGGCAGUCUGAGCUGNN 34
     297-315 AGCUCAGACUGCCAGAGAUNN 2366 AUCUCUGGCAGUCUGAGCUNN 34
     298-316 GCUCAGACUGCCAGAGAUCNN 2367 GAUCUCUGGCAGUCUGAGCNN 34
     299-317 CUCAGACUGCCAGAGAUCGNN 2368 CGAUCUCUGGCAGUCUGAGNN 34
     300-318 UCAGACUGCCAGAGAUCGANN 2369 UCGAUCUCUGGCAGUCUGANN 34
     301-319 CAGACUGCCAGAGAUCGAANN 2370 UUCGAUCUCUGGCAGUCUGNN 34
     302-320 AGACUGCCAGAGAUCGAAANN 2371 UUUCGAUCUCUGGCAGUCUNN 34
     303-321 GACUGCCAGAGAUCGAAAGNN 2372 CUUUCGAUCUCUGGCAGUCNN 34
     304-322 ACUGCCAGAGAUCGAAAGANN 2373 UCUUUCGAUCUCUGGCAGUNN 34
     305-323 CUGCCAGAGAUCGAAAGAANN 2374 UUCUUUCGAUCUCUGGCAGNN 34
     325-343 GCUCGAAUGAGUGAGCUGGNN 2375 CCAGCUCACUCAUUCGAGCNN 34
     326-344 CUCGAAUGAGUGAGCUGGANN 2376 UCCAGCUCACUCAUUCGAGNN 34
     327-345 UCGAAUGAGUGAGCUGGAANN 2377 UUCCAGCUCACUCAUUCGANN 34
     328-346 CGAAUGAGUGAGCUGGAACNN 2378 GUUCCAGCUCACUCAUUCGNN 34
     329-347 GAAUGAGUGAGCUGGAACANN 2379 UGUUCCAGCUCACUCAUUCNN 34
     330-348 AAUGAGUGAGCUGGAACAGNN 2380 CUGUUCCAGCUCACUCAUUNN 34
     331-349 AUGAGUGAGCUGGAACAGCNN 2381 GCUGUUCCAGCUCACUCAUNN 34
     332-350 UGAGUGAGCUGGAACAGCANN 2382 UGCUGUUCCAGCUCACUCANN 34
     333-351 GAGUGAGCUGGAACAGCAANN 2383 UUGCUGUUCCAGCUCACUCNN 34
     334-352 AGUGAGCUGGAACAGCAAGNN 2384 CUUGCUGUUCCAGCUCACUNN 34
     335-353 GUGAGCUGGAACAGCAAGUNN 2385 ACUUGCUGUUCCAGCUCACNN 34
     336-354 UGAGCUGGAACAGCAAGUGNN 2386 CACUUGCUGUUCCAGCUCANN 34
     337-355 GAGCUGGAACAGCAAGUGGNN 2387 CCACUUGCUGUUCCAGCUCNN 34
     338-356 AGCUGGAACAGCAAGUGGUNN 2388 ACCACUUGCUGUUCCAGCUNN 34
     339-357 GCUGGAACAGCAAGUGGUANN 2389 UACCACUUGCUGUUCCAGCNN 34
     340-358 CUGGAACAGCAAGUGGUAGNN 2390 CUACCACUUGCUGUUCCAGNN 34
     341-359 UGGAACAGCAAGUGGUAGANN 2391 UCUACCACUUGCUGUUCCANN 34
     342-360 GGAACAGCAAGUGGUAGAUNN 2392 AUCUACCACUUGCUGUUCCNN 34
     343-361 GAACAGCAAGUGGUAGAUUNN 2393 AAUCUACCACUUGCUGUUCNN 34
     344-362 AACAGCAAGUGGUAGAUUUNN 2394 AAAUCUACCACUUGCUGUUNN 34
     345-363 ACAGCAAGUGGUAGAUUUANN 2395 UAAAUCUACCACUUGCUGUNN 34
     346-364 CAGCAAGUGGUAGAUUUAGNN 2396 CUAAAUCUACCACUUGCUGNN 34
     347-365 AGCAAGUGGUAGAUUUAGANN 2397 UCUAAAUCUACCACUUGCUNN 34
     348-366 GCAAGUGGUAGAUUUAGAANN 2398 UUCUAAAUCUACCACUUGCNN 34
     349-367 CAAGUGGUAGAUUUAGAAGNN 2399 CUUCUAAAUCUACCACUUGNN 34
     350-368 AAGUGGUAGAUUUAGAAGANN 2400 UCUUCUAAAUCUACCACUUNN 34
     351-369 AGUGGUAGAUUUAGAAGAANN 2401 UUCUUCUAAAUCUACCACUNN 34
     352-370 GUGGUAGAUUUAGAAGAAGNN 2402 CUUCUUCUAAAUCUACCACNN 34
     353-371 UGGUAGAUUUAGAAGAAGANN 2403 UCUUCUUCUAAAUCUACCANN 34
     354-372 GGUAGAUUUAGAAGAAGAGNN 2404 CUCUUCUUCUAAAUCUACCNN 34
     355-373 GUAGAUUUAGAAGAAGAGANN 2405 UCUCUUCUUCUAAAUCUACNN 34
     356-374 UAGAUUUAGAAGAAGAGAANN 2406 UUCUCUUCUUCUAAAUCUANN 34
     357-375 AGAUUUAGAAGAAGAGAACNN 2407 GUUCUCUUCUUCUAAAUCUNN 34
     358-376 GAUUUAGAAGAAGAGAACCNN 2408 GGUUCUCUUCUUCUAAAUCNN 34
     359-377 AUUUAGAAGAAGAGAACCANN 2409 UGGUUCUCUUCUUCUAAAUNN 34
     360-378 UUUAGAAGAAGAGAACCAANN 2410 UUGGUUCUCUUCUUCUAAANN 34
     361-379 UUAGAAGAAGAGAACCAAANN 2411 UUUGGUUCUCUUCUUCUAANN 34
     362-380 UAGAAGAAGAGAACCAAAANN 2412 UUUUGGUUCUCUUCUUCUANN 34
     363-381 AGAAGAAGAGAACCAAAAANN 2413 TTTTTGGTTCTCTTCTTCTNN 34
     364-382 GAAGAAGAGAACCAAAAACNN 2414 GTTTTTGGTTCTCTTCTTCNN 34
     365-383 AAGAAGAGAACCAAAAACUNN 2415 AGUUUUUGGUUCUCUUCUUNN 34
     366-384 AGAAGAGAACCAAAAACUUNN 2416 AAGUUUUUGGUUCUCUUCUNN 34
     367-385 GAAGAGAACCAAAAACUUUNN 2417 AAAGUUUUUGGUUCUCUUCNN 34
     368-386 AAGAGAACCAAAAACUUUUNN 2418 AAAAGUUUUUGGUUCUCUUNN 34
     369-387 AGAGAACCAAAAACUUUUGNN 2419 CAAAAGUUUUUGGUUCUCUNN 34
     370-388 GAGAACCAAAAACUUUUGCNN 2420 GCAAAAGUUUUUGGUUCUCNN 34
     371-389 AGAACCAAAAACUUUUGCUNN 2421 AGCAAAAGUUUUUGGUUCUNN 34
     372-390 GAACCAAAAACUUUUGCUANN 2422 UAGCAAAAGUUUUUGGUUCNN 34
     373-391 AACCAAAAACUUUUGCUAGNN 2423 CUAGCAAAAGUUUUUGGUUNN 34
     374-392 ACCAAAAACUUUUGCUAGANN 2424 UCUAGCAAAAGUUUUUGGUNN 34
     375-393 CCAAAAACUUUUGCUAGAANN 2425 UUCUAGCAAAAGUUUUUGGNN 34
     376-394 CAAAAACUUUUGCUAGAAANN 2426 UUUCUAGCAAAAGUUUUUGNN 34
     377-395 AAAAACUUUUGCUAGAAAANN 2427 UUUUCUAGCAAAAGUUUUUNN 34
     378-396 AAAACUUUUGCUAGAAAAUNN 2428 AUUUUCUAGCAAAAGUUUUNN 34
     379-397 AAACUUUUGCUAGAAAAUCNN 2429 GAUUUUCUAGCAAAAGUUUNN 34
     380-398 AACUUUUGCUAGAAAAUCANN 2430 UGAUUUUCUAGCAAAAGUUNN 34
     381-399 ACUUUUGCUAGAAAAUCAGNN 2431 CUGAUUUUCUAGCAAAAGUNN 34
     382-400 CUUUUGCUAGAAAAUCAGCNN 2432 GCUGAUUUUCUAGCAAAAGNN 34
     383-401 UUUUGCUAGAAAAUCAGCUNN 2433 AGCUGAUUUUCUAGCAAAANN 34
     384-402 UUUGCUAGAAAAUCAGCUUNN 2434 AAGCUGAUUUUCUAGCAAANN 34
     385-403 UUGCUAGAAAAUCAGCUUUNN 2435 AAAGCUGAUUUUCUAGCAANN 34
     386-404 UGCUAGAAAAUCAGCUUUUNN 2436 AAAAGCUGAUUUUCUAGCANN 34
     387-405 GCUAGAAAAUCAGCUUUUANN 2437 UAAAAGCUGAUUUUCUAGCNN 34
     388-406 CUAGAAAAUCAGCUUUUACNN 2438 GUAAAAGCUGAUUUUCUAGNN 34
     389-407 UAGAAAAUCAGCUUUUACGNN 2439 CGUAAAAGCUGAUUUUCUANN 34
     390-408 AGAAAAUCAGCUUUUACGANN 2440 UCGUAAAAGCUGAUUUUCUNN 34
     391-409 GAAAAUCAGCUUUUACGAGNN 2441 CUCGUAAAAGCUGAUUUUCNN 35
     392-410 AAAAUCAGCUUUUACGAGANN 2442 UCUCGUAAAAGCUGAUUUUNN 35
     393-411 AAAUCAGCUUUUACGAGAGNN 2443 CUCUCGUAAAAGCUGAUUUNN 35
     394-412 AAUCAGCUUUUACGAGAGANN 2444 UCUCUCGUAAAAGCUGAUUNN 35
     395-413 AUCAGCUUUUACGAGAGAANN 2445 UUCUCUCGUAAAAGCUGAUNN 35
     396-414 UCAGCUUUUACGAGAGAAANN 2446 UUUCUCUCGUAAAAGCUGANN 35
     397-415 CAGCUUUUACGAGAGAAAANN 2447 UUUUCUCUCGUAAAAGCUGNN 35
     398-416 AGCUUUUACGAGAGAAAACNN 2448 GUUUUCUCUCGUAAAAGCUNN 35
     399-417 GCUUUUACGAGAGAAAACUNN 2449 AGUUUUCUCUCGUAAAAGCNN 35
     400-418 CUUUUACGAGAGAAAACUCNN 2450 GAGUUUUCUCUCGUAAAAGNN 35
     401-419 UUUUACGAGAGAAAACUCANN 2451 UGAGUUUUCUCUCGUAAAANN 35
     421-439 GGCCUUGUAGUUGAGAACCNN 2452 GGUUCUCAACUACAAGGCCNN 35
     422-440 GCCUUGUAGUUGAGAACCANN 2453 UGGUUCUCAACUACAAGGCNN 35
     423-441 CCUUGUAGUUGAGAACCAGNN 2454 CUGGUUCUCAACUACAAGGNN 35
     424-442 CUUGUAGUUGAGAACCAGGNN 2455 CCUGGUUCUCAACUACAAGNN 35
     425-443 UUGUAGUUGAGAACCAGGANN 2456 UCCUGGUUCUCAACUACAANN 35
     426-444 UGUAGUUGAGAACCAGGAGNN 2457 CUCCUGGUUCUCAACUACANN 35
     427-445 GUAGUUGAGAACCAGGAGUNN 2458 ACUCCUGGUUCUCAACUACNN 35
     428-446 UAGUUGAGAACCAGGAGUUNN 2459 AACUCCUGGUUCUCAACUANN 35
     429-447 AGUUGAGAACCAGGAGUUANN 2460 UAACUCCUGGUUCUCAACUNN 35
     430-448 GUUGAGAACCAGGAGUUAANN 2461 UUAACUCCUGGUUCUCAACNN 35
     431-449 UUGAGAACCAGGAGUUAAGNN 2462 CUUAACUCCUGGUUCUCAANN 35
     432-450 UGAGAACCAGGAGUUAAGANN 2463 UCUUAACUCCUGGUUCUCANN 35
     433-451 GAGAACCAGGAGUUAAGACNN 2464 GUCUUAACUCCUGGUUCUCNN 35
     434-452 AGAACCAGGAGUUAAGACANN 2465 UGUCUUAACUCCUGGUUCUNN 35
     435-453 GAACCAGGAGUUAAGACAGNN 2466 CUGUCUUAACUCCUGGUUCNN 35
     436-454 AACCAGGAGUUAAGACAGCNN 2467 GCUGUCUUAACUCCUGGUUNN 35
     437-455 ACCAGGAGUUAAGACAGCGNN 2468 CGCUGUCUUAACUCCUGGUNN 35
     438-456 CCAGGAGUUAAGACAGCGCNN 2469 GCGCUGUCUUAACUCCUGGNN 35
      44-62 GAGCUAUGGUGGUGGUGGCNN 2470 GCCACCACCACCAUAGCUCNN 35
      45-63 AGCUAUGGUGGUGGUGGCANN 2471 UGCCACCACCACCAUAGCUNN 35
     458-476 UGGGGAUGGAUGCCCUGGUNN 2472 ACCAGGGCAUCCAUCCCCANN 35
     459-477 GGGGAUGGAUGCCCUGGUUNN 2473 AACCAGGGCAUCCAUCCCCNN 35
     460-478 GGGAUGGAUGCCCUGGUUGNN 2474 CAACCAGGGCAUCCAUCCCNN 35
     461-479 GGAUGGAUGCCCUGGUUGCNN 2475 GCAACCAGGGCAUCCAUCCNN 35
     462-480 GAUGGAUGCCCUGGUUGCUNN 2476 AGCAACCAGGGCAUCCAUCNN 35
      46-64 GCUAUGGUGGUGGUGGCAGNN 2477 CUGCCACCACCACCAUAGCNN 35
      47-65 CUAUGGUGGUGGUGGCAGCNN 2478 GCUGCCACCACCACCAUAGNN 35
     482-500 AAGAGGAGGCGGAAGCCAANN 2479 TTGGCTTCCGCCTCCTCTTNN 35
     483-501 AGAGGAGGCGGAAGCCAAGNN 2480 CTTGGCTTCCGCCTCCTCTNN 35
     484-502 GAGGAGGCGGAAGCCAAGGNN 2481 CCTTGGCTTCCGCCTCCTCNN 35
     485-503 AGGAGGCGGAAGCCAAGGGNN 2482 CCCTTGGCTTCCGCCTCCTNN 35
     486-504 GGAGGCGGAAGCCAAGGGGNN 2483 CCCCTTGGCTTCCGCCTCCNN 35
      48-66 UAUGGUGGUGGUGGCAGCCNN 2484 GGCUGCCACCACCACCAUANN 35
     487-505 GAGGCGGAAGCCAAGGGGANN 2485 TCCCCTTGGCTTCCGCCTCNN 35
     488-506 AGGCGGAAGCCAAGGGGAANN 2486 TTCCCCTTGGCTTCCGCCTNN 35
     489-507 GGCGGAAGCCAAGGGGAAUNN 2487 AUUCCCCUUGGCUUCCGCCNN 35
     490-508 GCGGAAGCCAAGGGGAAUGNN 2488 CAUUCCCCUUGGCUUCCGCNN 35
      49-67 AUGGUGGUGGUGGCAGCCGNN 2489 CGGCUGCCACCACCACCAUNN 35
      50-68 UGGUGGUGGUGGCAGCCGCNN 2490 GCGGCUGCCACCACCACCANN 35
     510-528 AGUGAGGCCAGUGGCCGGGNN 2491 CCCGGCCACUGGCCUCACUNN 35
     511-529 GUGAGGCCAGUGGCCGGGUNN 2492 ACCCGGCCACUGGCCUCACNN 35
     512-530 UGAGGCCAGUGGCCGGGUCNN 2493 GACCCGGCCACUGGCCUCANN 35
     513-531 GAGGCCAGUGGCCGGGUCUNN 2494 AGACCCGGCCACUGGCCUCNN 35
     514-532 AGGCCAGUGGCCGGGUCUGNN 2495 CAGACCCGGCCACUGGCCUNN 35
     515-533 GGCCAGUGGCCGGGUCUGCNN 2496 GCAGACCCGGCCACUGGCCNN 35
     516-534 GCCAGUGGCCGGGUCUGCUNN 2497 AGCAGACCCGGCCACUGGCNN 35
     517-535 CCAGUGGCCGGGUCUGCUGNN 2498 CAGCAGACCCGGCCACUGGNN 35
     518-536 CAGUGGCCGGGUCUGCUGANN 2499 UCAGCAGACCCGGCCACUGNN 35
     519-537 AGUGGCCGGGUCUGCUGAGNN 2500 CUCAGCAGACCCGGCCACUNN 35
     520-538 GUGGCCGGGUCUGCUGAGUNN 2501 ACUCAGCAGACCCGGCCACNN 35
     521-539 UGGCCGGGUCUGCUGAGUCNN 2502 GACUCAGCAGACCCGGCCANN 35
     522-540 GGCCGGGUCUGCUGAGUCCNN 2503 GGACUCAGCAGACCCGGCCNN 35
     523-541 GCCGGGUCUGCUGAGUCCGNN 2504 CGGACUCAGCAGACCCGGCNN 35
     524-542 CCGGGUCUGCUGAGUCCGCNN 2505 GCGGACUCAGCAGACCCGGNN 35
     525-543 CGGGUCUGCUGAGUCCGCANN 2506 UGCGGACUCAGCAGACCCGNN 35
     526-544 GGGUCUGCUGAGUCCGCAGNN 2507 CUGCGGACUCAGCAGACCCNN 35
     574-592 GUGCAGGCCCAGUUGUCACNN 2508 GUGACAACUGGGCCUGCACNN 35
     575-593 UGCAGGCCCAGUUGUCACCNN 2509 GGUGACAACUGGGCCUGCANN 35
     576-594 GCAGGCCCAGUUGUCACCCNN 2510 GGGUGACAACUGGGCCUGCNN 35
     577-595 CAGGCCCAGUUGUCACCCCNN 2511 GGGGUGACAACUGGGCCUGNN 35
     578-596 AGGCCCAGUUGUCACCCCUNN 2512 AGGGGUGACAACUGGGCCUNN 35
     579-597 GGCCCAGUUGUCACCCCUCNN 2513 GAGGGGUGACAACUGGGCCNN 35
     580-598 GCCCAGUUGUCACCCCUCCNN 2514 GGAGGGGUGACAACUGGGCNN 35
     581-599 CCCAGUUGUCACCCCUCCANN 2515 UGGAGGGGUGACAACUGGGNN 35
     582-600 CCAGUUGUCACCCCUCCAGNN 2516 CUGGAGGGGUGACAACUGGNN 35
     583-601 CAGUUGUCACCCCUCCAGANN 2517 UCUGGAGGGGUGACAACUGNN 35
     584-602 AGUUGUCACCCCUCCAGAANN 2518 UUCUGGAGGGGUGACAACUNN 35
     585-603 GUUGUCACCCCUCCAGAACNN 2519 GUUCUGGAGGGGUGACAACNN 35
     586-604 UUGUCACCCCUCCAGAACANN 2520 UGUUCUGGAGGGGUGACAANN 35
     587-605 UGUCACCCCUCCAGAACAUNN 2521 AUGUUCUGGAGGGGUGACANN 35
     588-606 GUCACCCCUCCAGAACAUCNN 2522 GAUGUUCUGGAGGGGUGACNN 35
     589-607 UCACCCCUCCAGAACAUCUNN 2523 AGAUGUUCUGGAGGGGUGANN 35
     590-608 CACCCCUCCAGAACAUCUCNN 2524 GAGAUGUUCUGGAGGGGUGNN 35
     591-609 ACCCCUCCAGAACAUCUCCNN 2525 GGAGAUGUUCUGGAGGGGUNN 35
     592-610 CCCCUCCAGAACAUCUCCCNN 2526 GGGAGAUGUUCUGGAGGGGNN 35
     593-611 CCCUCCAGAACAUCUCCCCNN 2527 GGGGAGAUGUUCUGGAGGGNN 35
     594-612 CCUCCAGAACAUCUCCCCANN 2528 UGGGGAGAUGUUCUGGAGGNN 35
     595-613 CUCCAGAACAUCUCCCCAUNN 2529 AUGGGGAGAUGUUCUGGAGNN 35
     596-614 UCCAGAACAUCUCCCCAUGNN 2530 CAUGGGGAGAUGUUCUGGANN 35
     597-615 CCAGAACAUCUCCCCAUGGNN 2531 CCAUGGGGAGAUGUUCUGGNN 35
     598-616 CAGAACAUCUCCCCAUGGANN 2532 UCCAUGGGGAGAUGUUCUGNN 35
     599-617 AGAACAUCUCCCCAUGGAUNN 2533 AUCCAUGGGGAGAUGUUCUNN 35
     600-618 GAACAUCUCCCCAUGGAUUNN 2534 AAUCCAUGGGGAGAUGUUCNN 35
     601-619 AACAUCUCCCCAUGGAUUCNN 2535 GAAUCCAUGGGGAGAUGUUNN 35
     602-620 ACAUCUCCCCAUGGAUUCUNN 2536 AGAAUCCAUGGGGAGAUGUNN 35
     603-621 CAUCUCCCCAUGGAUUCUGNN 2537 CAGAAUCCAUGGGGAGAUGNN 35
     604-622 AUCUCCCCAUGGAUUCUGGNN 2538 CCAGAAUCCAUGGGGAGAUNN 35
     605-623 UCUCCCCAUGGAUUCUGGCNN 2539 GCCAGAAUCCAUGGGGAGANN 35
     606-624 CUCCCCAUGGAUUCUGGCGNN 2540 CGCCAGAAUCCAUGGGGAGNN 35
     607-625 UCCCCAUGGAUUCUGGCGGNN 2541 CCGCCAGAAUCCAUGGGGANN 36
     608-626 CCCCAUGGAUUCUGGCGGUNN 2542 ACCGCCAGAAUCCAUGGGGNN 36
     609-627 CCCAUGGAUUCUGGCGGUANN 2543 UACCGCCAGAAUCCAUGGGNN 36
     610-628 CCAUGGAUUCUGGCGGUAUNN 2544 AUACCGCCAGAAUCCAUGGNN 36
     611-629 CAUGGAUUCUGGCGGUAUUNN 2545 AAUACCGCCAGAAUCCAUGNN 36
     612-630 AUGGAUUCUGGCGGUAUUGNN 2546 CAAUACCGCCAGAAUCCAUNN 36
     613-631 UGGAUUCUGGCGGUAUUGANN 2547 UCAAUACCGCCAGAAUCCANN 36
     614-632 GGAUUCUGGCGGUAUUGACNN 2548 GUCAAUACCGCCAGAAUCCNN 36
     615-633 GAUUCUGGCGGUAUUGACUNN 2549 AGUCAAUACCGCCAGAAUCNN 36
     616-634 AUUCUGGCGGUAUUGACUCNN 2550 GAGUCAAUACCGCCAGAAUNN 36
     617-635 UUCUGGCGGUAUUGACUCUNN 2551 AGAGUCAAUACCGCCAGAANN 36
     618-636 UCUGGCGGUAUUGACUCUUNN 2552 AAGAGUCAAUACCGCCAGANN 36
     619-637 CUGGCGGUAUUGACUCUUCNN 2553 GAAGAGUCAAUACCGCCAGNN 36
     620-638 UGGCGGUAUUGACUCUUCANN 2554 UGAAGAGUCAAUACCGCCANN 36
     621-639 GGCGGUAUUGACUCUUCAGNN 2555 CUGAAGAGUCAAUACCGCCNN 36
     622-640 GCGGUAUUGACUCUUCAGANN 2556 UCUGAAGAGUCAAUACCGCNN 36
     623-641 CGGUAUUGACUCUUCAGAUNN 2557 AUCUGAAGAGUCAAUACCGNN 36
     624-642 GGUAUUGACUCUUCAGAUUNN 2558 AAUCUGAAGAGUCAAUACCNN 36
     625-643 GUAUUGACUCUUCAGAUUCNN 2559 GAAUCUGAAGAGUCAAUACNN 36
     626-644 UAUUGACUCUUCAGAUUCANN 2560 UGAAUCUGAAGAGUCAAUANN 36
     627-645 AUUGACUCUUCAGAUUCAGNN 2561 CUGAAUCUGAAGAGUCAAUNN 36
     628-646 UUGACUCUUCAGAUUCAGANN 2562 UCUGAAUCUGAAGAGUCAANN 36
     629-647 UGACUCUUCAGAUUCAGAGNN 2563 CUCUGAAUCUGAAGAGUCANN 36
     630-648 GACUCUUCAGAUUCAGAGUNN 2564 ACUCUGAAUCUGAAGAGUCNN 36
     631-649 ACUCUUCAGAUUCAGAGUCNN 2565 GACUCUGAAUCUGAAGAGUNN 36
     632-650 CUCUUCAGAUUCAGAGUCUNN 2566 AGACUCUGAAUCUGAAGAGNN 36
     633-651 UCUUCAGAUUCAGAGUCUGNN 2567 CAGACUCUGAAUCUGAAGANN 36
     634-652 CUUCAGAUUCAGAGUCUGANN 2568 UCAGACUCUGAAUCUGAAGNN 36
     635-653 UUCAGAUUCAGAGUCUGAUNN 2569 AUCAGACUCUGAAUCUGAANN 36
     636-654 UCAGAUUCAGAGUCUGAUANN 2570 UAUCAGACUCUGAAUCUGANN 36
     637-655 CAGAUUCAGAGUCUGAUAUNN 2571 AUAUCAGACUCUGAAUCUGNN 36
     638-656 AGAUUCAGAGUCUGAUAUCNN 2572 GAUAUCAGACUCUGAAUCUNN 36
     639-657 GAUUCAGAGUCUGAUAUCCNN 2573 GGAUAUCAGACUCUGAAUCNN 36
     640-658 AUUCAGAGUCUGAUAUCCUNN 2574 AGGAUAUCAGACUCUGAAUNN 36
     641-659 UUCAGAGUCUGAUAUCCUGNN 2575 CAGGAUAUCAGACUCUGAANN 36
     642-660 UCAGAGUCUGAUAUCCUGUNN 2576 ACAGGAUAUCAGACUCUGANN 36
     643-661 CAGAGUCUGAUAUCCUGUUNN 2577 AACAGGAUAUCAGACUCUGNN 36
     644-662 AGAGUCUGAUAUCCUGUUGNN 2578 CAACAGGAUAUCAGACUCUNN 36
     645-663 GAGUCUGAUAUCCUGUUGGNN 2579 CCAACAGGAUAUCAGACUCNN 36
     646-664 AGUCUGAUAUCCUGUUGGGNN 2580 CCCAACAGGAUAUCAGACUNN 36
     647-665 GUCUGAUAUCCUGUUGGGCNN 2581 GCCCAACAGGAUAUCAGACNN 36
     648-666 UCUGAUAUCCUGUUGGGCANN 2582 UGCCCAACAGGAUAUCAGANN 36
     649-667 CUGAUAUCCUGUUGGGCAUNN 2583 AUGCCCAACAGGAUAUCAGNN 36
     650-668 UGAUAUCCUGUUGGGCAUUNN 2584 AAUGCCCAACAGGAUAUCANN 36
     651-669 GAUAUCCUGUUGGGCAUUCNN 2585 GAAUGCCCAACAGGAUAUCNN 36
     652-670 AUAUCCUGUUGGGCAUUCUNN 2586 AGAAUGCCCAACAGGAUAUNN 36
     653-671 UAUCCUGUUGGGCAUUCUGNN 2587 CAGAAUGCCCAACAGGAUANN 36
     654-672 AUCCUGUUGGGCAUUCUGGNN 2588 CCAGAAUGCCCAACAGGAUNN 36
     655-673 UCCUGUUGGGCAUUCUGGANN 2589 UCCAGAAUGCCCAACAGGANN 36
     656-674 CCUGUUGGGCAUUCUGGACNN 2590 GUCCAGAAUGCCCAACAGGNN 36
     657-675 CUGUUGGGCAUUCUGGACANN 2591 UGUCCAGAAUGCCCAACAGNN 36
     658-676 UGUUGGGCAUUCUGGACAANN 2592 UUGUCCAGAAUGCCCAACANN 36
     659-677 GUUGGGCAUUCUGGACAACNN 2593 GUUGUCCAGAAUGCCCAACNN 36
     660-678 UUGGGCAUUCUGGACAACUNN 2594 AGUUGUCCAGAAUGCCCAANN 36
     661-679 UGGGCAUUCUGGACAACUUNN 2595 AAGUUGUCCAGAAUGCCCANN 36
     662-680 GGGCAUUCUGGACAACUUGNN 2596 CAAGUUGUCCAGAAUGCCCNN 36
     663-681 GGCAUUCUGGACAACUUGGNN 2597 CCAAGUUGUCCAGAAUGCCNN 36
     664-682 GCAUUCUGGACAACUUGGANN 2598 UCCAAGUUGUCCAGAAUGCNN 36
     665-683 CAUUCUGGACAACUUGGACNN 2599 GUCCAAGUUGUCCAGAAUGNN 36
     666-684 AUUCUGGACAACUUGGACCNN 2600 GGUCCAAGUUGUCCAGAAUNN 36
     667-685 UUCUGGACAACUUGGACCCNN 2601 GGGUCCAAGUUGUCCAGAANN 36
     668-686 UCUGGACAACUUGGACCCANN 2602 UGGGUCCAAGUUGUCCAGANN 36
     669-687 CUGGACAACUUGGACCCAGNN 2603 CUGGGUCCAAGUUGUCCAGNN 36
     670-688 UGGACAACUUGGACCCAGUNN 2604 ACUGGGUCCAAGUUGUCCANN 36
     671-689 GGACAACUUGGACCCAGUCNN 2605 GACUGGGUCCAAGUUGUCCNN 36
     672-690 GACAACUUGGACCCAGUCANN 2606 UGACUGGGUCCAAGUUGUCNN 36
     673-691 ACAACUUGGACCCAGUCAUNN 2607 AUGACUGGGUCCAAGUUGUNN 36
     674-692 CAACUUGGACCCAGUCAUGNN 2608 CAUGACUGGGUCCAAGUUGNN 36
     675-693 AACUUGGACCCAGUCAUGUNN 2609 ACAUGACUGGGUCCAAGUUNN 36
     676-694 ACUUGGACCCAGUCAUGUUNN 2610 AACAUGACUGGGUCCAAGUNN 36
     677-695 CUUGGACCCAGUCAUGUUCNN 2611 GAACAUGACUGGGUCCAAGNN 36
     678-696 UUGGACCCAGUCAUGUUCUNN 2612 AGAACAUGACUGGGUCCAANN 36
     679-697 UGGACCCAGUCAUGUUCUUNN 2613 AAGAACAUGACUGGGUCCANN 36
     680-698 GGACCCAGUCAUGUUCUUCNN 2614 GAAGAACAUGACUGGGUCCNN 36
     681-699 GACCCAGUCAUGUUCUUCANN 2615 UGAAGAACAUGACUGGGUCNN 36
     682-700 ACCCAGUCAUGUUCUUCAANN 2616 UUGAAGAACAUGACUGGGUNN 36
     683-701 CCCAGUCAUGUUCUUCAAANN 2617 UUUGAAGAACAUGACUGGGNN 36
     684-702 CCAGUCAUGUUCUUCAAAUNN 2618 AUUUGAAGAACAUGACUGGNN 36
     685-703 CAGUCAUGUUCUUCAAAUGNN 2619 CAUUUGAAGAACAUGACUGNN 36
     686-704 AGUCAUGUUCUUCAAAUGCNN 2620 GCAUUUGAAGAACAUGACUNN 36
     687-705 GUCAUGUUCUUCAAAUGCCNN 2621 GGCAUUUGAAGAACAUGACNN 36
     688-706 UCAUGUUCUUCAAAUGCCCNN 2622 GGGCAUUUGAAGAACAUGANN 36
     689-707 CAUGUUCUUCAAAUGCCCUNN 2623 AGGGCAUUUGAAGAACAUGNN 36
     690-708 AUGUUCUUCAAAUGCCCUUNN 2624 AAGGGCAUUUGAAGAACAUNN 36
     691-709 UGUUCUUCAAAUGCCCUUCNN 2625 GAAGGGCAUUUGAAGAACANN 36
     692-710 GUUCUUCAAAUGCCCUUCCNN 2626 GGAAGGGCAUUUGAAGAACNN 36
     693-711 UUCUUCAAAUGCCCUUCCCNN 2627 GGGAAGGGCAUUUGAAGAANN 36
     694-712 UCUUCAAAUGCCCUUCCCCNN 2628 GGGGAAGGGCAUUUGAAGANN 36
     695-713 CUUCAAAUGCCCUUCCCCANN 2629 UGGGGAAGGGCAUUUGAAGNN 36
     696-714 UUCAAAUGCCCUUCCCCAGNN 2630 CUGGGGAAGGGCAUUUGAANN 36
     697-715 UCAAAUGCCCUUCCCCAGANN 2631 UCUGGGGAAGGGCAUUUGANN 36
     698-716 CAAAUGCCCUUCCCCAGAGNN 2632 CUCUGGGGAAGGGCAUUUGNN 36
     718-736 CUGCCAGCCUGGAGGAGCUNN 2633 AGCUCCUCCAGGCUGGCAGNN 36
     719-737 UGCCAGCCUGGAGGAGCUCNN 2634 GAGCUCCUCCAGGCUGGCANN 36
     720-738 GCCAGCCUGGAGGAGCUCCNN 2635 GGAGCUCCUCCAGGCUGGCNN 36
     721-739 CCAGCCUGGAGGAGCUCCCNN 2636 GGGAGCUCCUCCAGGCUGGNN 36
     722-740 CAGCCUGGAGGAGCUCCCANN 2637 UGGGAGCUCCUCCAGGCUGNN 36
     723-741 AGCCUGGAGGAGCUCCCAGNN 2638 CUGGGAGCUCCUCCAGGCUNN 36
     724-742 GCCUGGAGGAGCUCCCAGANN 2639 UCUGGGAGCUCCUCCAGGCNN 36
     725-743 CCUGGAGGAGCUCCCAGAGNN 2640 CUCUGGGAGCUCCUCCAGGNN 36
     726-744 CUGGAGGAGCUCCCAGAGGNN 2641 CCUCUGGGAGCUCCUCCAGNN 37
     727-745 UGGAGGAGCUCCCAGAGGUNN 2642 ACCUCUGGGAGCUCCUCCANN 37
     728-746 GGAGGAGCUCCCAGAGGUCNN 2643 GACCUCUGGGAGCUCCUCCNN 37
     729-747 GAGGAGCUCCCAGAGGUCUNN 2644 AGACCUCUGGGAGCUCCUCNN 37
     730-748 AGGAGCUCCCAGAGGUCUANN 2645 UAGACCUCUGGGAGCUCCUNN 37
     731-749 GGAGCUCCCAGAGGUCUACNN 2646 GUAGACCUCUGGGAGCUCCNN 37
     732-750 GAGCUCCCAGAGGUCUACCNN 2647 GGUAGACCUCUGGGAGCUCNN 37
     733-751 AGCUCCCAGAGGUCUACCCNN 2648 GGGUAGACCUCUGGGAGCUNN 37
     734-752 GCUCCCAGAGGUCUACCCANN 2649 UGGGUAGACCUCUGGGAGCNN 37
     735-753 CUCCCAGAGGUCUACCCAGNN 2650 CUGGGUAGACCUCUGGGAGNN 37
     736-754 UCCCAGAGGUCUACCCAGANN 2651 UCUGGGUAGACCUCUGGGANN 37
     737-755 CCCAGAGGUCUACCCAGAANN 2652 UUCUGGGUAGACCUCUGGGNN 37
     738-756 CCAGAGGUCUACCCAGAAGNN 2653 CUUCUGGGUAGACCUCUGGNN 37
     739-757 CAGAGGUCUACCCAGAAGGNN 2654 CCUUCUGGGUAGACCUCUGNN 37
     740-758 AGAGGUCUACCCAGAAGGANN 2655 UCCUUCUGGGUAGACCUCUNN 37
     741-759 GAGGUCUACCCAGAAGGACNN 2656 GUCCUUCUGGGUAGACCUCNN 37
     742-760 AGGUCUACCCAGAAGGACCNN 2657 GGUCCUUCUGGGUAGACCUNN 37
     743-761 GGUCUACCCAGAAGGACCCNN 2658 GGGUCCUUCUGGGUAGACCNN 37
     744-762 GUCUACCCAGAAGGACCCANN 2659 UGGGUCCUUCUGGGUAGACNN 37
     745-763 UCUACCCAGAAGGACCCAGNN 2660 CUGGGUCCUUCUGGGUAGANN 37
     746-764 CUACCCAGAAGGACCCAGUNN 2661 ACUGGGUCCUUCUGGGUAGNN 37
     747-765 UACCCAGAAGGACCCAGUUNN 2662 AACUGGGUCCUUCUGGGUANN 37
     748-766 ACCCAGAAGGACCCAGUUCNN 2663 GAACUGGGUCCUUCUGGGUNN 37
     749-767 CCCAGAAGGACCCAGUUCCNN 2664 GGAACUGGGUCCUUCUGGGNN 37
     750-768 CCAGAAGGACCCAGUUCCUNN 2665 AGGAACUGGGUCCUUCUGGNN 37
     751-769 CAGAAGGACCCAGUUCCUUNN 2666 AAGGAACUGGGUCCUUCUGNN 37
     752-770 AGAAGGACCCAGUUCCUUANN 2667 UAAGGAACUGGGUCCUUCUNN 37
     753-771 GAAGGACCCAGUUCCUUACNN 2668 GUAAGGAACUGGGUCCUUCNN 37
     754-772 AAGGACCCAGUUCCUUACCNN 2669 GGUAAGGAACUGGGUCCUUNN 37
     755-773 AGGACCCAGUUCCUUACCANN 2670 UGGUAAGGAACUGGGUCCUNN 37
     756-774 GGACCCAGUUCCUUACCAGNN 2671 CUGGUAAGGAACUGGGUCCNN 37
     757-775 GACCCAGUUCCUUACCAGCNN 2672 GCUGGUAAGGAACUGGGUCNN 37
     758-776 ACCCAGUUCCUUACCAGCCNN 2673 GGCUGGUAAGGAACUGGGUNN 37
     759-777 CCCAGUUCCUUACCAGCCUNN 2674 AGGCUGGUAAGGAACUGGGNN 37
     760-778 CCAGUUCCUUACCAGCCUCNN 2675 GAGGCUGGUAAGGAACUGGNN 37
     761-779 CAGUUCCUUACCAGCCUCCNN 2676 GGAGGCUGGUAAGGAACUGNN 37
     762-780 AGUUCCUUACCAGCCUCCCNN 2677 GGGAGGCUGGUAAGGAACUNN 37
     763-781 GUUCCUUACCAGCCUCCCUNN 2678 AGGGAGGCUGGUAAGGAACNN 37
     764-782 UUCCUUACCAGCCUCCCUUNN 2679 AAGGGAGGCUGGUAAGGAANN 37
     765-783 UCCUUACCAGCCUCCCUUUNN 2680 AAAGGGAGGCUGGUAAGGANN 37
     766-784 CCUUACCAGCCUCCCUUUCNN 2681 GAAAGGGAGGCUGGUAAGGNN 37
     767-785 CUUACCAGCCUCCCUUUCUNN 2682 AGAAAGGGAGGCUGGUAAGNN 37
     768-786 UUACCAGCCUCCCUUUCUCNN 2683 GAGAAAGGGAGGCUGGUAANN 37
     769-787 UACCAGCCUCCCUUUCUCUNN 2684 AGAGAAAGGGAGGCUGGUANN 37
     770-788 ACCAGCCUCCCUUUCUCUGNN 2685 CAGAGAAAGGGAGGCUGGUNN 37
     771-789 CCAGCCUCCCUUUCUCUGUNN 2686 ACAGAGAAAGGGAGGCUGGNN 37
     772-790 CAGCCUCCCUUUCUCUGUCNN 2687 GACAGAGAAAGGGAGGCUGNN 37
     773-791 AGCCUCCCUUUCUCUGUCANN 2688 UGACAGAGAAAGGGAGGCUNN 37
     774-792 GCCUCCCUUUCUCUGUCAGNN 2689 CUGACAGAGAAAGGGAGGCNN 37
     775-793 CCUCCCUUUCUCUGUCAGUNN 2690 ACUGACAGAGAAAGGGAGGNN 37
     776-794 CUCCCUUUCUCUGUCAGUGNN 2691 CACUGACAGAGAAAGGGAGNN 37
     777-795 UCCCUUUCUCUGUCAGUGGNN 2692 CCACUGACAGAGAAAGGGANN 37
     778-796 CCCUUUCUCUGUCAGUGGGNN 2693 CCCACUGACAGAGAAAGGGNN 37
     779-797 CCUUUCUCUGUCAGUGGGGNN 2694 CCCCACUGACAGAGAAAGGNN 37
     780-798 CUUUCUCUGUCAGUGGGGANN 2695 UCCCCACUGACAGAGAAAGNN 37
     781-799 UUUCUCUGUCAGUGGGGACNN 2696 GUCCCCACUGACAGAGAAANN 37
     782-800 UUCUCUGUCAGUGGGGACGNN 2697 CGUCCCCACUGACAGAGAANN 37
     783-801 UCUCUGUCAGUGGGGACGUNN 2698 ACGUCCCCACUGACAGAGANN 37
     784-802 CUCUGUCAGUGGGGACGUCNN 2699 GACGUCCCCACUGACAGAGNN 37
     785-803 UCUGUCAGUGGGGACGUCANN 2700 UGACGUCCCCACUGACAGANN 37
     786-804 CUGUCAGUGGGGACGUCAUNN 2701 AUGACGUCCCCACUGACAGNN 37
     787-805 UGUCAGUGGGGACGUCAUCNN 2702 GAUGACGUCCCCACUGACANN 37
     788-806 GUCAGUGGGGACGUCAUCANN 2703 UGAUGACGUCCCCACUGACNN 37
     789-807 UCAGUGGGGACGUCAUCAGNN 2704 CUGAUGACGUCCCCACUGANN 37
     790-808 CAGUGGGGACGUCAUCAGCNN 2705 GCUGAUGACGUCCCCACUGNN 37
     791-809 AGUGGGGACGUCAUCAGCCNN 2706 GGCUGAUGACGUCCCCACUNN 37
     792-810 GUGGGGACGUCAUCAGCCANN 2707 UGGCUGAUGACGUCCCCACNN 37
     793-811 UGGGGACGUCAUCAGCCAANN 2708 UUGGCUGAUGACGUCCCCANN 37
     794-812 GGGGACGUCAUCAGCCAAGNN 2709 CUUGGCUGAUGACGUCCCCNN 37
     795-813 GGGACGUCAUCAGCCAAGCNN 2710 GCUUGGCUGAUGACGUCCCNN 37
     796-814 GGACGUCAUCAGCCAAGCUNN 2711 AGCUUGGCUGAUGACGUCCNN 37
     797-815 GACGUCAUCAGCCAAGCUGNN 2712 CAGCUUGGCUGAUGACGUCNN 37
     798-816 ACGUCAUCAGCCAAGCUGGNN 2713 CCAGCUUGGCUGAUGACGUNN 37
     799-817 CGUCAUCAGCCAAGCUGGANN 2714 UCCAGCUUGGCUGAUGACGNN 37
     800-818 GUCAUCAGCCAAGCUGGAANN 2715 UUCCAGCUUGGCUGAUGACNN 37
     801-819 UCAUCAGCCAAGCUGGAAGNN 2716 CUUCCAGCUUGGCUGAUGANN 37
     802-820 CAUCAGCCAAGCUGGAAGCNN 2717 GCUUCCAGCUUGGCUGAUGNN 37
     803-821 AUCAGCCAAGCUGGAAGCCNN 2718 GGCUUCCAGCUUGGCUGAUNN 37
     804-822 UCAGCCAAGCUGGAAGCCANN 2719 UGGCUUCCAGCUUGGCUGANN 37
     805-823 CAGCCAAGCUGGAAGCCAUNN 2720 AUGGCUUCCAGCUUGGCUGNN 37
     806-824 AGCCAAGCUGGAAGCCAUUNN 2721 AAUGGCUUCCAGCUUGGCUNN 37
     807-825 GCCAAGCUGGAAGCCAUUANN 2722 UAAUGGCUUCCAGCUUGGCNN 37
     808-826 CCAAGCUGGAAGCCAUUAANN 2723 UUAAUGGCUUCCAGCUUGGNN 37
     809-827 CAAGCUGGAAGCCAUUAAUNN 2724 AUUAAUGGCUUCCAGCUUGNN 37
     810-828 AAGCUGGAAGCCAUUAAUGNN 2725 CAUUAAUGGCUUCCAGCUUNN 37
     811-829 AGCUGGAAGCCAUUAAUGANN 2726 UCAUUAAUGGCUUCCAGCUNN 37
     812-830 GCUGGAAGCCAUUAAUGAANN 2727 UUCAUUAAUGGCUUCCAGCNN 37
     813-831 CUGGAAGCCAUUAAUGAACNN 2728 GUUCAUUAAUGGCUUCCAGNN 37
     814-832 UGGAAGCCAUUAAUGAACUNN 2729 AGUUCAUUAAUGGCUUCCANN 37
     815-833 GGAAGCCAUUAAUGAACUANN 2730 UAGUUCAUUAAUGGCUUCCNN 37
     816-834 GAAGCCAUUAAUGAACUAANN 2731 UUAGUUCAUUAAUGGCUUCNN 37
     817-835 AAGCCAUUAAUGAACUAAUNN 2732 AUUAGUUCAUUAAUGGCUUNN 37
     818-836 AGCCAUUAAUGAACUAAUUNN 2733 AAUUAGUUCAUUAAUGGCUNN 37
     819-837 GCCAUUAAUGAACUAAUUCNN 2734 GAAUUAGUUCAUUAAUGGCNN 37
     820-838 CCAUUAAUGAACUAAUUCGNN 2735 CGAAUUAGUUCAUUAAUGGNN 37
     821-839 CAUUAAUGAACUAAUUCGUNN 2736 ACGAAUUAGUUCAUUAAUGNN 37
     822-840 AUUAAUGAACUAAUUCGUUNN 2737 AACGAAUUAGUUCAUUAAUNN 37
     823-841 UUAAUGAACUAAUUCGUUUNN 2738 AAACGAAUUAGUUCAUUAANN 37
     824-842 UAAUGAACUAAUUCGUUUUNN 2739 AAAACGAAUUAGUUCAUUANN 37
     825-843 AAUGAACUAAUUCGUUUUGNN 2740 CAAAACGAAUUAGUUCAUUNN 37
     826-844 AUGAACUAAUUCGUUUUGANN 2741 UCAAAACGAAUUAGUUCAUNN 38
     827-845 UGAACUAAUUCGUUUUGACNN 2742 GUCAAAACGAAUUAGUUCANN 38
     828-846 GAACUAAUUCGUUUUGACCNN 2743 GGUCAAAACGAAUUAGUUCNN 38
     829-847 AACUAAUUCGUUUUGACCANN 2744 UGGUCAAAACGAAUUAGUUNN 38
     830-848 ACUAAUUCGUUUUGACCACNN 2745 GUGGUCAAAACGAAUUAGUNN 38
     831-849 CUAAUUCGUUUUGACCACANN 2746 UGUGGUCAAAACGAAUUAGNN 38
     832-850 UAAUUCGUUUUGACCACAUNN 2747 AUGUGGUCAAAACGAAUUANN 38
     833-851 AAUUCGUUUUGACCACAUANN 2748 UAUGUGGUCAAAACGAAUUNN 38
     834-852 AUUCGUUUUGACCACAUAUNN 2749 AUAUGUGGUCAAAACGAAUNN 38
     835-853 UUCGUUUUGACCACAUAUANN 2750 UAUAUGUGGUCAAAACGAANN 38
     836-854 UCGUUUUGACCACAUAUAUNN 2751 AUAUAUGUGGUCAAAACGANN 38
     837-855 CGUUUUGACCACAUAUAUANN 2752 UAUAUAUGUGGUCAAAACGNN 38
     838-856 GUUUUGACCACAUAUAUACNN 2753 GUAUAUAUGUGGUCAAAACNN 38
     839-857 UUUUGACCACAUAUAUACCNN 2754 GGUAUAUAUGUGGUCAAAANN 38
     840-858 UUUGACCACAUAUAUACCANN 2755 UGGUAUAUAUGUGGUCAAANN 38
     841-859 UUGACCACAUAUAUACCAANN 2756 UUGGUAUAUAUGUGGUCAANN 38
     842-860 UGACCACAUAUAUACCAAGNN 2757 CUUGGUAUAUAUGUGGUCANN 38
     843-861 GACCACAUAUAUACCAAGCNN 2758 GCUUGGUAUAUAUGUGGUCNN 38
     844-862 ACCACAUAUAUACCAAGCCNN 2759 GGCUUGGUAUAUAUGUGGUNN 38
     845-863 CCACAUAUAUACCAAGCCCNN 2760 GGGCUUGGUAUAUAUGUGGNN 38
     846-864 CACAUAUAUACCAAGCCCCNN 2761 GGGGCUUGGUAUAUAUGUGNN 38
     847-865 ACAUAUAUACCAAGCCCCUNN 2762 AGGGGCUUGGUAUAUAUGUNN 38
     867-885 GUCUUAGAGAUACCCUCUGNN 2763 CAGAGGGUAUCUCUAAGACNN 38
     868-886 UCUUAGAGAUACCCUCUGANN 2764 UCAGAGGGUAUCUCUAAGANN 38
     869-887 CUUAGAGAUACCCUCUGAGNN 2765 CUCAGAGGGUAUCUCUAAGNN 38
     870-888 UUAGAGAUACCCUCUGAGANN 2766 UCUCAGAGGGUAUCUCUAANN 38
     871-889 UAGAGAUACCCUCUGAGACNN 2767 GUCUCAGAGGGUAUCUCUANN 38
     872-890 AGAGAUACCCUCUGAGACANN 2768 UGUCUCAGAGGGUAUCUCUNN 38
     873-891 GAGAUACCCUCUGAGACAGNN 2769 CUGUCUCAGAGGGUAUCUCNN 38
     874-892 AGAUACCCUCUGAGACAGANN 2770 UCUGUCUCAGAGGGUAUCUNN 38
     875-893 GAUACCCUCUGAGACAGAGNN 2771 CUCUGUCUCAGAGGGUAUCNN 38
     876-894 AUACCCUCUGAGACAGAGANN 2772 UCUCUGUCUCAGAGGGUAUNN 38
     877-895 UACCCUCUGAGACAGAGAGNN 2773 CUCUCUGUCUCAGAGGGUANN 38
     878-896 ACCCUCUGAGACAGAGAGCNN 2774 GCUCUCUGUCUCAGAGGGUNN 38
     879-897 CCCUCUGAGACAGAGAGCCNN 2775 GGCUCUCUGUCUCAGAGGGNN 38
     880-898 CCUCUGAGACAGAGAGCCANN 2776 UGGCUCUCUGUCUCAGAGGNN 38
     881-899 CUCUGAGACAGAGAGCCAANN 2777 UUGGCUCUCUGUCUCAGAGNN 38
     882-900 UCUGAGACAGAGAGCCAAGNN 2778 CUUGGCUCUCUGUCUCAGANN 38
     883-901 CUGAGACAGAGAGCCAAGCNN 2779 GCUUGGCUCUCUGUCUCAGNN 38
     884-902 UGAGACAGAGAGCCAAGCUNN 2780 AGCUUGGCUCUCUGUCUCANN 38
     885-903 GAGACAGAGAGCCAAGCUANN 2781 UAGCUUGGCUCUCUGUCUCNN 38
     886-904 AGACAGAGAGCCAAGCUAANN 2782 UUAGCUUGGCUCUCUGUCUNN 38
     887-905 GACAGAGAGCCAAGCUAAUNN 2783 AUUAGCUUGGCUCUCUGUCNN 38
     888-906 ACAGAGAGCCAAGCUAAUGNN 2784 CAUUAGCUUGGCUCUCUGUNN 38
     889-907 CAGAGAGCCAAGCUAAUGUNN 2785 ACAUUAGCUUGGCUCUCUGNN 38
     890-908 AGAGAGCCAAGCUAAUGUGNN 2786 CACAUUAGCUUGGCUCUCUNN 38
     891-909 GAGAGCCAAGCUAAUGUGGNN 2787 CCACAUUAGCUUGGCUCUCNN 38
     892-910 AGAGCCAAGCUAAUGUGGUNN 2788 ACCACAUUAGCUUGGCUCUNN 38
     893-911 GAGCCAAGCUAAUGUGGUANN 2789 UACCACAUUAGCUUGGCUCNN 38
     894-912 AGCCAAGCUAAUGUGGUAGNN 2790 CUACCACAUUAGCUUGGCUNN 38
     895-913 GCCAAGCUAAUGUGGUAGUNN 2791 ACUACCACAUUAGCUUGGCNN 38
     896-914 CCAAGCUAAUGUGGUAGUGNN 2792 CACUACCACAUUAGCUUGGNN 38
     897-915 CAAGCUAAUGUGGUAGUGANN 2793 UCACUACCACAUUAGCUUGNN 38
     898-916 AAGCUAAUGUGGUAGUGAANN 2794 UUCACUACCACAUUAGCUUNN 38
     899-917 AGCUAAUGUGGUAGUGAAANN 2795 UUUCACUACCACAUUAGCUNN 38
     900-918 GCUAAUGUGGUAGUGAAAANN 2796 UUUUCACUACCACAUUAGCNN 38
     901-919 CUAAUGUGGUAGUGAAAAUNN 2797 AUUUUCACUACCACAUUAGNN 38
     902-920 UAAUGUGGUAGUGAAAAUCNN 2798 GAUUUUCACUACCACAUUANN 38
     903-921 AAUGUGGUAGUGAAAAUCGNN 2799 CGAUUUUCACUACCACAUUNN 38
     904-922 AUGUGGUAGUGAAAAUCGANN 2800 UCGAUUUUCACUACCACAUNN 38
     905-923 UGUGGUAGUGAAAAUCGAGNN 2801 CUCGAUUUUCACUACCACANN 38
     906-924 GUGGUAGUGAAAAUCGAGGNN 2802 CCUCGAUUUUCACUACCACNN 38
     907-925 UGGUAGUGAAAAUCGAGGANN 2803 UCCUCGAUUUUCACUACCANN 38
     908-926 GGUAGUGAAAAUCGAGGAANN 2804 UUCCUCGAUUUUCACUACCNN 38
     909-927 GUAGUGAAAAUCGAGGAAGNN 2805 CUUCCUCGAUUUUCACUACNN 38
     910-928 UAGUGAAAAUCGAGGAAGCNN 2806 GCUUCCUCGAUUUUCACUANN 38
     911-929 AGUGAAAAUCGAGGAAGCANN 2807 UGCUUCCUCGAUUUUCACUNN 38
     912-930 GUGAAAAUCGAGGAAGCACNN 2808 GUGCUUCCUCGAUUUUCACNN 38
     913-931 UGAAAAUCGAGGAAGCACCNN 2809 GGUGCUUCCUCGAUUUUCANN 38
     914-932 GAAAAUCGAGGAAGCACCUNN 2810 AGGUGCUUCCUCGAUUUUCNN 38
     915-933 AAAAUCGAGGAAGCACCUCNN 2811 GAGGUGCUUCCUCGAUUUUNN 38
     916-934 AAAUCGAGGAAGCACCUCUNN 2812 AGAGGUGCUUCCUCGAUUUNN 38
     917-935 AAUCGAGGAAGCACCUCUCNN 2813 GAGAGGUGCUUCCUCGAUUNN 38
     918-936 AUCGAGGAAGCACCUCUCANN 2814 UGAGAGGUGCUUCCUCGAUNN 38
     919-937 UCGAGGAAGCACCUCUCAGNN 2815 CUGAGAGGUGCUUCCUCGANN 38
     920-938 CGAGGAAGCACCUCUCAGCNN 2816 GCUGAGAGGUGCUUCCUCGNN 38
     921-939 GAGGAAGCACCUCUCAGCCNN 2817 GGCUGAGAGGUGCUUCCUCNN 38
     922-940 AGGAAGCACCUCUCAGCCCNN 2818 GGGCUGAGAGGUGCUUCCUNN 38
     923-941 GGAAGCACCUCUCAGCCCCNN 2819 GGGGCUGAGAGGUGCUUCCNN 38
     924-942 GAAGCACCUCUCAGCCCCUNN 2820 AGGGGCUGAGAGGUGCUUCNN 38
     925-943 AAGCACCUCUCAGCCCCUCNN 2821 GAGGGGCUGAGAGGUGCUUNN 38
     926-944 AGCACCUCUCAGCCCCUCANN 2822 UGAGGGGCUGAGAGGUGCUNN 38
     927-945 GCACCUCUCAGCCCCUCAGNN 2823 CUGAGGGGCUGAGAGGUGCNN 38
     928-946 CACCUCUCAGCCCCUCAGANN 2824 UCUGAGGGGCUGAGAGGUGNN 38
     929-947 ACCUCUCAGCCCCUCAGAGNN 2825 CUCUGAGGGGCUGAGAGGUNN 38
     930-948 CCUCUCAGCCCCUCAGAGANN 2826 UCUCUGAGGGGCUGAGAGGNN 38
     931-949 CUCUCAGCCCCUCAGAGAANN 2827 UUCUCUGAGGGGCUGAGAGNN 38
     932-950 UCUCAGCCCCUCAGAGAAUNN 2828 AUUCUCUGAGGGGCUGAGANN 38
     933-951 CUCAGCCCCUCAGAGAAUGNN 2829 CAUUCUCUGAGGGGCUGAGNN 38
     934-952 UCAGCCCCUCAGAGAAUGANN 2830 UCAUUCUCUGAGGGGCUGANN 38
     935-953 CAGCCCCUCAGAGAAUGAUNN 2831 AUCAUUCUCUGAGGGGCUGNN 38
     936-954 AGCCCCUCAGAGAAUGAUCNN 2832 GAUCAUUCUCUGAGGGGCUNN 38
     937-955 GCCCCUCAGAGAAUGAUCANN 2833 UGAUCAUUCUCUGAGGGGCNN 38
     938-956 CCCCUCAGAGAAUGAUCACNN 2834 GUGAUCAUUCUCUGAGGGGNN 38
     939-957 CCCUCAGAGAAUGAUCACCNN 2835 GGUGAUCAUUCUCUGAGGGNN 38
     940-958 CCUCAGAGAAUGAUCACCCNN 2836 GGGUGAUCAUUCUCUGAGGNN 38
     941-959 CUCAGAGAAUGAUCACCCUNN 2837 AGGGUGAUCAUUCUCUGAGNN 38
     942-960 UCAGAGAAUGAUCACCCUGNN 2838 CAGGGUGAUCAUUCUCUGANN 38
     943-961 CAGAGAAUGAUCACCCUGANN 2839 UCAGGGUGAUCAUUCUCUGNN 38
     944-962 AGAGAAUGAUCACCCUGAANN 2840 UUCAGGGUGAUCAUUCUCUNN 38
     945-963 GAGAAUGAUCACCCUGAAUNN 2841 AUUCAGGGUGAUCAUUCUCNN 39
     946-964 AGAAUGAUCACCCUGAAUUNN 2842 AAUUCAGGGUGAUCAUUCUNN 39
     947-965 GAAUGAUCACCCUGAAUUCNN 2843 GAAUUCAGGGUGAUCAUUCNN 39
     948-966 AAUGAUCACCCUGAAUUCANN 2844 UGAAUUCAGGGUGAUCAUUNN 39
     949-967 AUGAUCACCCUGAAUUCAUNN 2845 AUGAAUUCAGGGUGAUCAUNN 39
     950-968 UGAUCACCCUGAAUUCAUUNN 2846 AAUGAAUUCAGGGUGAUCANN 39
     951-969 GAUCACCCUGAAUUCAUUGNN 2847 CAAUGAAUUCAGGGUGAUCNN 39
     952-970 AUCACCCUGAAUUCAUUGUNN 2848 ACAAUGAAUUCAGGGUGAUNN 39
     953-971 UCACCCUGAAUUCAUUGUCNN 2849 GACAAUGAAUUCAGGGUGANN 39
     954-972 CACCCUGAAUUCAUUGUCUNN 2850 AGACAAUGAAUUCAGGGUGNN 39
     955-973 ACCCUGAAUUCAUUGUCUCNN 2851 GAGACAAUGAAUUCAGGGUNN 39
     956-974 CCCUGAAUUCAUUGUCUCANN 2852 UGAGACAAUGAAUUCAGGGNN 39
     957-975 CCUGAAUUCAUUGUCUCAGNN 2853 CUGAGACAAUGAAUUCAGGNN 39
     958-976 CUGAAUUCAUUGUCUCAGUNN 2854 ACUGAGACAAUGAAUUCAGNN 39
     959-977 UGAAUUCAUUGUCUCAGUGNN 2855 CACUGAGACAAUGAAUUCANN 39
     960-978 GAAUUCAUUGUCUCAGUGANN 2856 UCACUGAGACAAUGAAUUCNN 39
     961-979 AAUUCAUUGUCUCAGUGAANN 2857 UUCACUGAGACAAUGAAUUNN 39
     962-980 AUUCAUUGUCUCAGUGAAGNN 2858 CUUCACUGAGACAAUGAAUNN 39
     963-981 UUCAUUGUCUCAGUGAAGGNN 2859 CCUUCACUGAGACAAUGAANN 39
     964-982 UCAUUGUCUCAGUGAAGGANN 2860 UCCUUCACUGAGACAAUGANN 39
     965-983 CAUUGUCUCAGUGAAGGAANN 2861 UUCCUUCACUGAGACAAUGNN 39
     966-984 AUUGUCUCAGUGAAGGAAGNN 2862 CUUCCUUCACUGAGACAAUNN 39
     967-985 UUGUCUCAGUGAAGGAAGANN 2863 UCUUCCUUCACUGAGACAANN 39
     968-986 UGUCUCAGUGAAGGAAGAANN 2864 UUCUUCCUUCACUGAGACANN 39
     969-987 GUCUCAGUGAAGGAAGAACNN 2865 GUUCUUCCUUCACUGAGACNN 39
     970-988 UCUCAGUGAAGGAAGAACCNN 2866 GGUUCUUCCUUCACUGAGANN 39
     971-989 CUCAGUGAAGGAAGAACCUNN 2867 AGGUUCUUCCUUCACUGAGNN 39
     972-990 UCAGUGAAGGAAGAACCUGNN 2868 CAGGUUCUUCCUUCACUGANN 39
     973-991 CAGUGAAGGAAGAACCUGUNN 2869 ACAGGUUCUUCCUUCACUGNN 39
     974-992 AGUGAAGGAAGAACCUGUANN 2870 UACAGGUUCUUCCUUCACUNN 39
     975-993 GUGAAGGAAGAACCUGUAGNN 2871 CUACAGGUUCUUCCUUCACNN 39
     976-994 UGAAGGAAGAACCUGUAGANN 2872 UCUACAGGUUCUUCCUUCANN 39
     977-995 GAAGGAAGAACCUGUAGAANN 2873 UUCUACAGGUUCUUCCUUCNN 39
     978-996 AAGGAAGAACCUGUAGAAGNN 2874 CUUCUACAGGUUCUUCCUUNN 39
     979-997 AGGAAGAACCUGUAGAAGANN 2875 UCUUCUACAGGUUCUUCCUNN 39
     980-998 GGAAGAACCUGUAGAAGAUNN 2876 AUCUUCUACAGGUUCUUCCNN 39
     981-999 GAAGAACCUGUAGAAGAUGNN 2877 CAUCUUCUACAGGUUCUUCNN 39
     982-1000 AAGAACCUGUAGAAGAUGANN 2878 UCAUCUUCUACAGGUUCUUNN 39
     983-1001 AGAACCUGUAGAAGAUGACNN 2879 GUCAUCUUCUACAGGUUCUNN 39
     984-1002 GAACCUGUAGAAGAUGACCNN 2880 GGUCAUCUUCUACAGGUUCNN 39
     985-1003 AACCUGUAGAAGAUGACCUNN 2881 AGGUCAUCUUCUACAGGUUNN 39
     986-1004 ACCUGUAGAAGAUGACCUCNN 2882 GAGGUCAUCUUCUACAGGUNN 39
  • TABLE 13
    Sequences of dsRNA targeting both mouse and rhesus monkey XBP-1.
    *Target refers location of target sequence in NM_013842 (Mus musculis
    XPB1 mRNA). Sense and antisense sequences are described with optional
    dinucleotide (NN) overhangs.
    SEQ ID SEQ ID
    *Target sense (5′-3′) NO antisense (5′-3′) NO
     369-387 AGAAAACUCACGGCCUUGUNN 3942 ACAAGGCCGUGAGUUUUCUNN 4042
     237-255 AACUGAAAAACAGAGUAGCNN 3943 GCUACUCUGUUUUUCAGUUNN 4043
     491-509 GGGUCUGCUGAGUCCGCAGNN 3944 CUGCGGACUCAGCAGACCCNN 4044
     917-935 AUCACCCUGAAUUCAUUGUNN 3945 ACAAUGAAUUCAGGGUGAUNN 4045
     923-941 CUGAAUUCAUUGUCUCAGUNN 3946 ACUGAGACAAUGAAUUCAGNN 4046
     702-720 CCCAGAGGUCUACCCAGAANN 3947 UUCUGGGUAGACCUCUGGGNN 4047
     926-944 AAUUCAUUGUCUCAGUGAANN 3948 UUCACUGAGACAAUGAAUUNN 4048
     391-409 UGAGAACCAGGAGUUAAGANN 3949 UCUUAACUCCUGGUUCUCANN 4049
     775-793 AAGCUGGAAGCCAUUAAUGNN 3950 CAUUAAUGGCUUCCAGCUUNN 4050
    1150-1168 CCCCAGCUGAUUAGUGUCUNN 3951 AGACACUAAUCAGCUGGGGNN 4051
     776-794 AGCUGGAAGCCAUUAAUGANN 3952 UCAUUAAUGGCUUCCAGCUNN 4052
     921-939 CCCUGAAUUCAUUGUCUCANN 3953 UGAGACAAUGAAUUCAGGGNN 4053
     777-795 GCUGGAAGCCAUUAAUGAANN 3954 UUCAUUAAUGGCUUCCAGCNN 4054
     539-557 GUGCAGGCCCAGUUGUCACNN 3955 GUGACAACUGGGCCUGCACNN 4055
     731-749 CCUUACCAGCCUCCCUUUCNN 3956 GAAAGGGAGGCUGGUAAGGNN 4056
     924-942 UGAAUUCAUUGUCUCAGUGNN 3957 CACUGAGACAAUGAAUUCANN 4057
    1151-1169 CCCAGCUGAUUAGUGUCUANN 3958 UAGACACUAAUCAGCUGGGNN 4058
    1152-1170 CCAGCUGAUUAGUGUCUAANN 3959 UUAGACACUAAUCAGCUGGNN 4059
    1718-1736 ACUAUGUAAAUGCUUGAUGNN 3960 CAUCAAGCAUUUACAUAGUNN 4060
     368-386 GAGAAAACUCACGGCCUUGNN 3961 CAAGGCCGUGAGUUUUCUCNN 4061
     489-507 CCGGGUCUGCUGAGUCCGCNN 3962 GCGGACUCAGCAGACCCGGNN 4062
     238-256 ACUGAAAAACAGAGUAGCANN 3963 UGCUACUCUGUUUUUCAGUNN 4063
     240-258 UGAAAAACAGAGUAGCAGCNN 3964 GCUGCUACUCUGUUUUUCANN 4064
     390-408 UUGAGAACCAGGAGUUAAGNN 3965 CUUAACUCCUGGUUCUCAANN 4065
     487-505 GGCCGGGUCUGCUGAGUCCNN 3966 GGACUCAGCAGACCCGGCCNN 4066
     741-759 CUCCCUUUCUCUGUCAGUGNN 3967 CACUGACAGAGAAAGGGAGNN 4067
     918-936 UCACCCUGAAUUCAUUGUCNN 3968 GACAAUGAAUUCAGGGUGANN 4068
     919-937 CACCCUGAAUUCAUUGUCUNN 3969 AGACAAUGAAUUCAGGGUGNN 4069
    1130-1148 CUUUUGCCAAUGAACUUUUNN 3970 AAAAGUUCAUUGGCAAAAGNN 4070
    1712-1730 AAAUUUACUAUGUAAAUGCNN 3971 GCAUUUACAUAGUAAAUUUNN 4071
    1714-1732 AUUUACUAUGUAAAUGCUUNN 3972 AAGCAUUUACAUAGUAAAUNN 4072
    1717-1735 UACUAUGUAAAUGCUUGAUNN 3973 AUCAAGCAUUUACAUAGUANN 4073
    1719-1737 CUAUGUAAAUGCUUGAUGGNN 3974 CCAUCAAGCAUUUACAUAGNN 4074
    1775-1793 CCAUUUAUUUAAAACUACCNN 3975 GGUAGUUUUAAAUAAAUGGNN 4075
    1776-1794 CAUUUAUUUAAAACUACCCNN 3976 GGGUAGUUUUAAAUAAAUGNN 4076
     239-257 CUGAAAAACAGAGUAGCAGNN 3977 CUGCUACUCUGUUUUUCAGNN 4077
     347-365 CUAGAAAAUCAGCUUUUACNN 3978 GUAAAAGCUGAUUUUCUAGNN 4078
     348-366 UAGAAAAUCAGCUUUUACGNN 3979 CGUAAAAGCUGAUUUUCUANN 4079
     485-503 GUGGCCGGGUCUGCUGAGUNN 3980 ACUCAGCAGACCCGGCCACNN 4080
     486-504 UGGCCGGGUCUGCUGAGUCNN 3981 GACUCAGCAGACCCGGCCANN 4081
     488-506 GCCGGGUCUGCUGAGUCCGNN 3982 CGGACUCAGCAGACCCGGCNN 4082
     540-558 UGCAGGCCCAGUUGUCACCNN 3983 GGUGACAACUGGGCCUGCANN 4083
     703-721 CCAGAGGUCUACCCAGAAGNN 3984 CUUCUGGGUAGACCUCUGGNN 4084
     705-723 AGAGGUCUACCCAGAAGGANN 3985 UCCUUCUGGGUAGACCUCUNN 4085
     730-748 UCCUUACCAGCCUCCCUUUNN 3986 AAAGGGAGGCUGGUAAGGANN 4086
     742-760 UCCCUUUCUCUGUCAGUGGNN 3987 CCACUGACAGAGAAAGGGANN 4087
     744-762 CCUUUCUCUGUCAGUGGGGNN 3988 CCCCACUGACAGAGAAAGGNN 4088
     767-785 CAUCAGCCAAGCUGGAAGCNN 3989 GCUUCCAGCUUGGCUGAUGNN 4089
     771-789 AGCCAAGCUGGAAGCCAUUNN 3990 AAUGGCUUCCAGCUUGGCUNN 4090
     916-934 GAUCACCCUGAAUUCAUUGNN 3991 CAAUGAAUUCAGGGUGAUCNN 4091
     920-938 ACCCUGAAUUCAUUGUCUCNN 3992 GAGACAAUGAAUUCAGGGUNN 4092
     922-940 CCUGAAUUCAUUGUCUCAGNN 3993 CUGAGACAAUGAAUUCAGGNN 4093
     925-943 GAAUUCAUUGUCUCAGUGANN 3994 UCACUGAGACAAUGAAUUCNN 4094
    1720-1738 UAUGUAAAUGCUUGAUGGANN 3995 UCCAUCAAGCAUUUACAUANN 4095
     232-250 GAGGAAACUGAAAAACAGANN 3996 UCUGUUUUUCAGUUUCCUCNN 4096
     236-254 AAACUGAAAAACAGAGUAGNN 3997 CUACUCUGUUUUUCAGUUUNN 4097
     728-746 GUUCCUUACCAGCCUCCCUNN 3998 AGGGAGGCUGGUAAGGAACNN 4098
     729-747 UUCCUUACCAGCCUCCCUUNN 3999 AAGGGAGGCUGGUAAGGAANN 4099
     745-763 CUUUCUCUGUCAGUGGGGANN 4000 UCCCCACUGACAGAGAAAGNN 4100
     766-784 UCAUCAGCCAAGCUGGAAGNN 4001 CUUCCAGCUUGGCUGAUGANN 4101
     927-945 AUUCAUUGUCUCAGUGAAGNN 4002 CUUCACUGAGACAAUGAAUNN 4102
     234-252 GGAAACUGAAAAACAGAGUNN 4003 ACUCUGUUUUUCAGUUUCCNN 4103
     235-253 GAAACUGAAAAACAGAGUANN 4004 UACUCUGUUUUUCAGUUUCNN 4104
     346-364 GCUAGAAAAUCAGCUUUUANN 4005 UAAAAGCUGAUUUUCUAGCNN 4105
     490-508 CGGGUCUGCUGAGUCCGCANN 4006 UGCGGACUCAGCAGACCCGNN 4106
     700-718 CUCCCAGAGGUCUACCCAGNN 4007 CUGGGUAGACCUCUGGGAGNN 4107
    1715-1733 UUUACUAUGUAAAUGCUUGNN 4008 CAAGCAUUUACAUAGUAAANN 4108
     734-752 UACCAGCCUCCCUUUCUCUNN 4009 AGAGAAAGGGAGGCUGGUANN 4109
     773-791 CCAAGCUGGAAGCCAUUAANN 4010 UUAAUGGCUUCCAGCUUGGNN 4110
     778-796 CUGGAAGCCAUUAAUGAACNN 4011 GUUCAUUAAUGGCUUCCAGNN 4111
     779-797 UGGAAGCCAUUAAUGAACUNN 4012 AGUUCAUUAAUGGCUUCCANN 4112
    1774-1792 UCCAUUUAUUUAAAACUACNN 4013 GUAGUUUUAAAUAAAUGGANN 4113
     704-722 CAGAGGUCUACCCAGAAGGNN 4014 CCUUCUGGGUAGACCUCUGNN 4114
    1716-1734 UUACUAUGUAAAUGCUUGANN 4015 UCAAGCAUUUACAUAGUAANN 4115
    1713-1731 AAUUUACUAUGUAAAUGCUNN 4016 AGCAUUUACAUAGUAAAUUNN 4116
     768-786 AUCAGCCAAGCUGGAAGCCNN 4017 GGCUUCCAGCUUGGCUGAUNN 4117
    1129-1147 ACUUUUGCCAAUGAACUUUNN 4018 AAAGUUCAUUGGCAAAAGUNN 4118
     389-407 GUUGAGAACCAGGAGUUAANN 4019 UUAACUCCUGGUUCUCAACNN 4119
     701-719 UCCCAGAGGUCUACCCAGANN 4020 UCUGGGUAGACCUCUGGGANN 4120
     706-724 GAGGUCUACCCAGAAGGACNN 4021 GUCCUUCUGGGUAGACCUCNN 4121
     707-725 AGGUCUACCCAGAAGGACCNN 4022 GGUCCUUCUGGGUAGACCUNN 4122
     727-745 AGUUCCUUACCAGCCUCCCNN 4023 GGGAGGCUGGUAAGGAACUNN 4123
     733-751 UUACCAGCCUCCCUUUCUCNN 4024 GAGAAAGGGAGGCUGGUAANN 4124
     736-754 CCAGCCUCCCUUUCUCUGUNN 4025 ACAGAGAAAGGGAGGCUGGNN 4125
     738-756 AGCCUCCCUUUCUCUGUCANN 4026 UGACAGAGAAAGGGAGGCUNN 4126
     743-761 CCCUUUCUCUGUCAGUGGGNN 4027 CCCACUGACAGAGAAAGGGNN 4127
     769-787 UCAGCCAAGCUGGAAGCCANN 4028 UGGCUUCCAGCUUGGCUGANN 4128
     772-790 GCCAAGCUGGAAGCCAUUANN 4029 UAAUGGCUUCCAGCUUGGCNN 4129
     774-792 CAAGCUGGAAGCCAUUAAUNN 4030 AUUAAUGGCUUCCAGCUUGNN 4130
     231-249 GGAGGAAACUGAAAAACAGNN 4031 CUGUUUUUCAGUUUCCUCCNN 4131
     233-251 AGGAAACUGAAAAACAGAGNN 4032 CUCUGUUUUUCAGUUUCCUNN 4132
     735-753 ACCAGCCUCCCUUUCUCUGNN 4033 CAGAGAAAGGGAGGCUGGUNN 4133
     737-755 CAGCCUCCCUUUCUCUGUCNN 4034 GACAGAGAAAGGGAGGCUGNN 4134
     739-757 GCCUCCCUUUCUCUGUCAGNN 4035 CUGACAGAGAAAGGGAGGCNN 4135
     740-758 CCUCCCUUUCUCUGUCAGUNN 4036 ACUGACAGAGAAAGGGAGGNN 4136
     746-764 UUUCUCUGUCAGUGGGGACNN 4037 GUCCCCACUGACAGAGAAANN 4137
     770-788 CAGCCAAGCUGGAAGCCAUNN 4038 AUGGCUUCCAGCUUGGCUGNN 4138
      26-44 GCUAUGGUGGUGGUGGCAGNN 4039 CUGCCACCACCACCAUAGCNN 4139
      27-45 CUAUGGUGGUGGUGGCAGCNN 4040 GCUGCCACCACCACCAUAGNN 4140
     732-750 CUUACCAGCCUCCCUUUCUNN 4041 AGAAAGGGAGGCUGGUAAGNN 4141

Claims (28)

1. A dual targeting siRNA agent comprising a first dsRNA targeting a PCSK9 gene and a second dsRNA targeting a second gene, wherein the first dsRNA and the second dsRNA are linked with a covalent linker.
2. A dual targeting siRNA agent comprising a first dsRNA AD-10792 targeting a PCSK9 gene and a second dsRNA AD-18038 targeting an XBP-1 gene, wherein the AD-10792 sense strand and the AD-18038 sense strand are covalently linked with a disulfide linker.
3. The dual targeting siRNA agent of claim 1, wherein the second gene is selected from the group consisting of XBP-1, PCSK9, PCSK5, ApoC3, SCAP, and MIG12.
4. (canceled)
5. The dual targeting siRNA agent of claim 1, wherein the first dsRNA comprises at least 15 contiguous nucleotides of an antisense strand of one of Tables 1, 2, or 4-8, or comprises an antisense strand of one of Tables 1, 2, or 4-8, or comprises a sense strand and an antisense strand of one of Tables 1, 2, or 4-8.
6. The dual targeting siRNA agent of claim 1, wherein the first dsRNA comprises AD-9680 or AD-10792.
7. The dual targeting siRNA agent of claim 1, wherein the second dsRNA comprises at least 15 contiguous nucleotides of an antisense strand of one of Tables 3 or 9-13, or comprises an antisense strand of one of Tables 3 or 9-13, or comprises a sense strand and an antisense strand of one of Tables 3 or 9-13.
8. The dual targeting siRNA agent of claim 1, wherein the second dsRNA comprises AD-18038.
9. The dual targeting siRNA agent of claim 1, wherein the first and second dsRNA comprises at least one modified nucleotide.
10. The dual targeting siRNA agent of claim 9, wherein the modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
11. The dual targeting siRNA agent of claim 9, wherein the modified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
12. The dual targeting siRNA agent of claim 1, wherein each strand of each dsRNA is 19-23 bases in length.
13. The dual targeting siRNA agent of claim 1,
(a) wherein the first and second dsRNAs are linked with a disulfide linker;
(b) wherein the covalent linker links the sense strand of the first dsRNA to the sense strand of the second dsRNA; and/or
(c) wherein the covalent linker links the antisense strand of the first dsRNA to the antisense strand of the second dsRNA.
14. (canceled)
15. (canceled)
16. The dual targeting siRNA agent of claim 1, further comprising a ligand.
17. The dual targeting siRNA agent of claim 1,
(a) wherein administration of the dual targeting siRNA agent to a cell inhibits expression of the PCSK9 gene and the second gene at a level equivalent to inhibition of expression of both genes obtained by the administration of each siRNA individually; or
(b) wherein administration of the dual targeting siRNA agent to a subject results in a greater reduction of total serum cholesterol that that obtained by administration of each siRNA individually.
18. (canceled)
19. A pharmaceutical composition comprising the dual targeting siRNA agent of claim 1 and a pharmaceutical carrier.
20. The pharmaceutical composition of claim 19, wherein the pharmaceutical carrier is
(a) a lipid formulation;
(b) a lipid formulation comprising cationic lipid DLinDMA or cationic lipid XTC;
(c) a lipid formulation described in Table A: or
(d) a lipid formulation comprising XTC/DSPC/Cholesterol/PEG-DMG at % mol ratios of 50/10/38.5/1.5.
21-23. (canceled)
24. A method of inhibiting expression of the PCSK9 gene and a second gene in a cell, the method comprising (a) introducing into the cell the dual targeting siRNA agent of claim 1; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the PCSK9 gene and the second gene, thereby inhibiting expression of the PCSK9 gene and the second gene in the cell.
25. A method of treating a disorder mediated by PCSK9 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of the pharmaceutical composition of claim 19.
26. The method of claim 25, wherein the disorder is hyperlipidemia.
27. A method of reducing total serum cholesterol in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 19.
28. An isolated cell comprising the dual targeting siRNA agent of claim 1.
29. A vector encoding at least one strand of the first dsRNA and at least one strand of the second dsRNA of the dual targeting siRNA agent of claim 1.
30. A cell comprising the vector of claim 29.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024059881A3 (en) * 2022-09-16 2024-05-16 Sirnaomics, Inc. Products and compositions
WO2024138136A1 (en) * 2022-12-22 2024-06-27 Sirnaomics, Inc. Products and compositions
WO2025124364A1 (en) * 2023-12-11 2025-06-19 Hangzhou Dinovate Biotech Co., Ltd. Sirna molecule for inhibiting pcsk9 expression and use thereof

Families Citing this family (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010083615A1 (en) 2009-01-26 2010-07-29 Protiva Biotherapeutics, Inc. Compositions and methods for silencing apolipoprotein c-iii expression
WO2010147992A1 (en) 2009-06-15 2010-12-23 Alnylam Pharmaceuticals, Inc. Methods for increasing efficacy of lipid formulated sirna
EP3533873A1 (en) * 2011-09-14 2019-09-04 Translate Bio MA, Inc. Multimeric oligonucleotide compounds
US9255154B2 (en) 2012-05-08 2016-02-09 Alderbio Holdings, Llc Anti-PCSK9 antibodies and use thereof
CA2901983A1 (en) * 2013-03-01 2014-09-04 National University Corporation Tokyo Medical And Dental University Chimeric single-stranded antisense polynucleotides and double-stranded antisense agent
WO2017007886A2 (en) * 2015-07-08 2017-01-12 Idera Pharmaceuticals, Inc. Compositions for inhibiting dux4 gene expression and uses thereof
CA3205381A1 (en) * 2015-07-17 2017-01-26 Alnylam Pharmaceuticals, Inc. Multi-targeted single entity conjugates
KR20190058477A (en) 2016-08-17 2019-05-29 솔스티스 바이올로직스, 리미티드 Polynucleotide construct
US20190175755A1 (en) * 2016-08-23 2019-06-13 The Johns Hopkins University Immunoswitch nanoparticles for reprogrammed t cell responses
AU2017368050A1 (en) 2016-11-29 2019-06-20 Puretech Lyt, Inc. Exosomes for delivery of therapeutic agents
KR102401796B1 (en) 2017-04-13 2022-05-25 카딜라 핼쓰캐어 리미티드 Novel peptide-based PCSK9 vaccine
WO2019006455A1 (en) 2017-06-30 2019-01-03 Solstice Biologics, Ltd. Chiral phosphoramidite auxiliaries and methods of their use
AU2018301477A1 (en) * 2017-07-13 2020-02-27 Alnylam Pharmaceuticals Inc. Lactate dehydrogenase a (LDHA) iRNA compositions and methods of use thereof
KR102834361B1 (en) 2017-12-01 2025-07-17 쑤저우 리보 라이프 사이언스 컴퍼니, 리미티드 Nucleic acids, compositions and conjugates containing them, and methods for their preparation and use
US11414665B2 (en) 2017-12-01 2022-08-16 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof
WO2019105418A1 (en) 2017-12-01 2019-06-06 苏州瑞博生物技术有限公司 Double-stranded oligonucleotide, composition and conjugate comprising double-stranded oligonucleotide, preparation method therefor and use thereof
US11414661B2 (en) 2017-12-01 2022-08-16 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate containing nucleic acid, preparation method therefor and use thereof
JP7365052B2 (en) 2017-12-01 2023-10-19 スーチョウ リボ ライフ サイエンス カンパニー、リミテッド Nucleic acids, compositions and complexes containing the nucleic acids, and preparation methods and uses
CA3087106A1 (en) 2017-12-29 2019-07-04 Suzhou Ribo Life Science Co., Ltd. Conjugates and preparation and use thereof
CA3095040A1 (en) * 2018-04-06 2019-10-10 Camp4 Therapeutics Corporation Treating diseases via targeted modulation of gene signaling networks
WO2020010186A1 (en) * 2018-07-06 2020-01-09 Derek Klarin Pcsk9 variants
JP2021533800A (en) 2018-08-21 2021-12-09 スーチョウ リボ ライフ サイエンス カンパニー、リミテッドSuzhou Ribo Life Science Co., Ltd. Nucleic acid, drug compositions and complexes containing the nucleic acid, and their use
CN108964051B (en) * 2018-08-27 2021-10-12 广西大学 Method for constructing preventive scheduling and recovery scheduling coordination optimization strategy of power system
CN111655297A (en) 2018-09-30 2020-09-11 苏州瑞博生物技术有限公司 siRNA conjugate and preparation method and application thereof
US11504390B2 (en) 2019-03-20 2022-11-22 Regeneron Pharmaceuticals, Inc. Treatment of increased lipid levels with sterol regulatory element binding transcription factor 1 (SREBF1) inhibitors
EP3974530A4 (en) * 2019-05-22 2023-07-12 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, pharmaceutical composition, conjugate, preparation method, and use
CN110638788A (en) * 2019-10-25 2020-01-03 广州医科大学 siRNA capable of silencing Pcsk9 protein, nano delivery system and application thereof
EP4069838A1 (en) * 2019-12-03 2022-10-12 Beam Therapeutics Inc. Synthetic guide rna, compositions, methods, and uses thereof
WO2021188611A1 (en) 2020-03-18 2021-09-23 Alnylam Pharmaceuticals, Inc. Compositions and methods for treating subjects having a heterozygous alanine-glyoxylate aminotransferase gene (agxt) variant
WO2022221736A2 (en) * 2021-04-16 2022-10-20 Genentech, Inc. Optimized tlr7 ligands and uses thereof
CA3221985A1 (en) * 2021-06-21 2022-12-29 Chad PECOT Inverted chimeric sirna molecules and methods of use thereof
JP2024528659A (en) 2021-07-19 2024-07-30 アルナイラム ファーマシューティカルズ， インコーポレイテッド Methods and compositions for treating subjects having or at risk of developing a non-primary hyperoxaluric disease or disorder - Patents.com
IL313660A (en) 2021-12-22 2024-08-01 Camp4 Therapeutics Corp Modulation of gene transcription using antisense oligonucleotides targeting regulatory rnas
WO2023240249A1 (en) * 2022-06-11 2023-12-14 Sirnaomics, Inc. Products and compositions
WO2024188174A1 (en) * 2023-03-10 2024-09-19 苏州瑞博生物技术股份有限公司 Pharmaceutical composition and use thereof
WO2025031485A1 (en) * 2023-08-10 2025-02-13 石药集团中奇制药技术（石家庄）有限公司 Dsrna molecule inhibiting hbv gene expression and use thereof

Family Cites Families (41)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1261620A2 (en) 2000-02-07 2002-12-04 Roche Diagnostics Corporation Cationic amphiphiles for use in nucleic acid transfection
WO2003070918A2 (en) 2002-02-20 2003-08-28 Ribozyme Pharmaceuticals, Incorporated Rna interference by modified short interfering nucleic acid
US20070026394A1 (en) 2000-02-11 2007-02-01 Lawrence Blatt Modulation of gene expression associated with inflammation proliferation and neurite outgrowth using nucleic acid based technologies
RU2322500C2 (en) 2000-12-01 2008-04-20 Макс-Планк-Гезелльшафт Цур Фердерунг Дер Виссеншафтен Е.Ф. Small rna molecules mediating rna interference
US20030170891A1 (en) 2001-06-06 2003-09-11 Mcswiggen James A. RNA interference mediated inhibition of epidermal growth factor receptor gene expression using short interfering nucleic acid (siNA)
US20070173473A1 (en) * 2001-05-18 2007-07-26 Sirna Therapeutics, Inc. RNA interference mediated inhibition of proprotein convertase subtilisin Kexin 9 (PCSK9) gene expression using short interfering nucleic acid (siNA)
US7923547B2 (en) 2002-09-05 2011-04-12 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
DK2284266T3 (en) 2002-11-14 2014-01-13 Thermo Fisher Scient Biosciences Inc SIRNA MOLECULE MOD TP53
US7250496B2 (en) 2002-11-14 2007-07-31 Rosetta Genomics Ltd. Bioinformatically detectable group of novel regulatory genes and uses thereof
EP3450559A1 (en) 2003-03-07 2019-03-06 Alnylam Pharmaceuticals, Inc. Therapeutic compositions
EP1608735A4 (en) 2003-04-03 2008-11-05 Alnylam Pharmaceuticals Irna conjugates
EP2336317B1 (en) 2003-06-13 2019-09-11 Alnylam Europe AG Double-stranded ribonucleic acid with increased effectiveness in an organism
SG190613A1 (en) 2003-07-16 2013-06-28 Protiva Biotherapeutics Inc Lipid encapsulated interfering rna
JP4764426B2 (en) 2004-06-07 2011-09-07 プロチバ バイオセラピューティクス インコーポレイティッド Cationic lipids and methods of use
AU2005252273B2 (en) 2004-06-07 2011-04-28 Arbutus Biopharma Corporation Lipid encapsulated interfering RNA
WO2007086883A2 (en) 2005-02-14 2007-08-02 Sirna Therapeutics, Inc. Cationic lipids and formulated molecular compositions containing them
US7427605B2 (en) 2005-03-31 2008-09-23 Calando Pharmaceuticals, Inc. Inhibitors of ribonucleotide reductase subunit 2 and uses thereof
US7915230B2 (en) 2005-05-17 2011-03-29 Molecular Transfer, Inc. Reagents for transfection of eukaryotic cells
JP5639338B2 (en) 2005-07-27 2014-12-10 プロチバ バイオセラピューティクス インコーポレイティッド Liposome production system and production method
US8101741B2 (en) 2005-11-02 2012-01-24 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
US8362229B2 (en) 2006-02-08 2013-01-29 Quark Pharmaceuticals, Inc. Tandem siRNAS
WO2007115168A2 (en) 2006-03-31 2007-10-11 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of eg5 gene
SG171676A1 (en) 2006-05-11 2011-06-29 Alnylam Pharmaceuticals Inc Compositions and methods for inhibiting expression of the pcsk9 gene
US8598333B2 (en) 2006-05-26 2013-12-03 Alnylam Pharmaceuticals, Inc. SiRNA silencing of genes expressed in cancer
AU2007275365A1 (en) 2006-07-17 2008-01-24 Sirna Therapeutics Inc. RNA interference mediated inhibition of Proprotein Convertase Subtilisin Kexin 9 (PCSK9) gene expression using short interfering nucleic acid (siNA)
WO2008036638A2 (en) 2006-09-18 2008-03-27 Alnylam Pharmaceuticals, Inc. Rnai modulation of scap and therapeutic uses thereof
CA2927045A1 (en) 2006-10-03 2008-04-10 Muthiah Manoharan Lipid containing formulations
CA2710713C (en) 2007-12-27 2017-09-19 Protiva Biotherapeutics, Inc. Silencing of polo-like kinase expression using interfering rna
US20110117125A1 (en) 2008-01-02 2011-05-19 Tekmira Pharmaceuticals Corporation Compositions and methods for the delivery of nucleic acids
US8227184B2 (en) 2008-01-14 2012-07-24 President And Fellows Of Harvard College Methods for modulating de novo hepatic lipogenesis by modulating XBP-1 activity
EP2245039A4 (en) 2008-01-31 2012-06-06 Alnylam Pharmaceuticals Inc Optimized methods for delivery of dsrna targeting the pcsk9 gene
US8058069B2 (en) 2008-04-15 2011-11-15 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
WO2009129465A2 (en) 2008-04-17 2009-10-22 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of xbp-1 gene
CN105152939A (en) 2008-11-10 2015-12-16 阿尔尼拉姆医药品有限公司 Lipids and compositions for the delivery of therapeutics
EP3243504A1 (en) 2009-01-29 2017-11-15 Arbutus Biopharma Corporation Improved lipid formulation
WO2010099341A1 (en) 2009-02-26 2010-09-02 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of mig-12 gene
KR20210031549A (en) 2009-05-05 2021-03-19 알닐람 파마슈티칼스 인코포레이티드 Lipid compositions
KR20230098713A (en) 2009-06-10 2023-07-04 알닐람 파마슈티칼스 인코포레이티드 Improved lipid formulation
EP2442792A4 (en) 2009-06-15 2015-12-23 Alnylam Pharmaceuticals Inc DOUBLE STRANDED RNA IN LIPID FORMULATION TARGETING THE PCSK9 GENE
WO2010147992A1 (en) 2009-06-15 2010-12-23 Alnylam Pharmaceuticals, Inc. Methods for increasing efficacy of lipid formulated sirna
WO2011028938A1 (en) 2009-09-02 2011-03-10 Alnylam Pharmaceuticals, Inc. Methods for lowering serum cholestrol in a subject using inhibition of pcsk9

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024059881A3 (en) * 2022-09-16 2024-05-16 Sirnaomics, Inc. Products and compositions
WO2024138136A1 (en) * 2022-12-22 2024-06-27 Sirnaomics, Inc. Products and compositions
WO2025124364A1 (en) * 2023-12-11 2025-06-19 Hangzhou Dinovate Biotech Co., Ltd. Sirna molecule for inhibiting pcsk9 expression and use thereof

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