Dual Targeting siRNA Agents

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 16/521,422, filed Jul. 24, 2019, pending, which is a continuation of U.S. application Ser. No. 15/724,175, filed Oct. 3, 2017, pending, 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 S1P/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) 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. 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-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, TREBS, 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 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, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.

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

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

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of 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%, 20%, 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

Table 1: AD-9680	Sequence 5′ to 3′	SEQ ID 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
Table 2: AD-10792	Sequence 5′ to 3′	ID 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

Table 3: AD-18038	Sequence 5′ to 3′	SEQ ID NO:
Target sequence	CACCCUGAAUUCAUUGUCU	4153
Sense strand	CACCCUGAAUUCAUUGUCU	4154
Sense strand, modified	cAcccuGAAuucAuuGucudTsdT	4155
Antisense strand	AGACAAUGAAUUCAGGGUG	4156
Antisense strand, modified	AGAcAAUGAAUUcAGGGUGdTsdT	4157

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′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-uridyl-3′-phosphate-5′-uridyl-3′-phosphate); rUsrU (a thiophosphate linker: 5′-uridyl-3′-thiophosphate-5′-uridyl-3′-phosphate); an rUrU linker; dTsdTaa (aadTsdT, 5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-adenyl-3′-phosphate-5′-adenyl-3′-phosphate); dTsdT (5′-2′deoxythyrnidyl-3′-thiophosphate-5′-2′ deoxythymidyl-3′-phosphate); dTsdTuu=uudTsdT=5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-uridyl-3′-phosphate-5′-uridyl-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., 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′deoxythymidyl-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

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. 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 C_m 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 C_m 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, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as 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 αvβ3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

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

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 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

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 R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). However, there are three factors that are important to consider in order to successfully deliver an iRNA molecule in vivo: (a) biological stability of the delivered molecule, (2) preventing non-specific effects, and (3) accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example by direct injection or implantation into a tissue (as a non-limiting example, 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 (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering 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 S H., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

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; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In 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) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.

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

Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-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 mdrl 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 acylcarnitine, an acylcholine, or a C₁-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

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 G_M1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., 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 G_M1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_M1 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 nm, 65 nm, 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)ethylazanediyOdidodecan-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±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 (C12), a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), 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.

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 are 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/US 10/22614, 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:

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.

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.

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:

Synthesis of 515:

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

Synthesis of 516:

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO₃ solution (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 Acid 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 acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the 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, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, 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, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

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

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, 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, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

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, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene olyel ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (M0750), 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, acylcarnitines, acylcholines, C_1-20 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 (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control 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 (Lotto 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-C™ (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.

Methods 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%, 20%, 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 PCSK9 gene 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 mg/dL, 300 mg/dL, or 400 mg/dL.

In general, the dual targeting siRNA agent does not activate the immune system, e.g., it does not increase cytokine levels, such as TNF-alpha or IFN-alpha levels. For example, when measured by an assay, such as an in vitro PBMC assay, such as described herein, the increase in levels of TNF-alpha or IFN-alpha, is less than 30%, 20%, or 10% of control cells treated with a control dsRNA, such as a dsRNA that does not target PCSK9.

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 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 Slo-Niacin. Exemplary antiplatelet agents include, e.g., aspirin (e.g., Bayer's aspirin), clopidogrel (Sanofi-Synthelabo/Bristol-Myers Squibb's Plavix), and ticlopidine (e.g., Sanofi-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{circumflex 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 Pharniaceuticals/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-A1 (ABCA1) (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 Pharmacuticals) 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 are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

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 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 oligonucleotidess 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.

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 1×10⁵ 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 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
*Target	Sense strand (5′-3′)1	ID NO:	strand (5′-3′)1	ID 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
993-1011	CAGCGGCCGGGAUGCCGGCTsT	403	GCCGGCAUCCCGG	404	AD-9602
			CCGCUGTsT
993-1011	cAGcGGccGGGAuGccGGcTsT	405	GCCGGcAUCCCGG	406	AD-9728
			CCGCUGTsT
1020-1038	GGGUGCCAGCAUGCGCAGCTT	407	GCUGCGCAUGCUG	408	AD-15386
			GCACCCTT
1038-1056	CCUGCGCGUGCUCAACUGCTsT	409	GCAGUUGAGCACG	410	AD-9580
			CGCAGGTsT
1038-1056	ccuGcGcGuGcucAAcuGcTsT	411	GcAGUUGAGcACG	412	AD-9706
			CGcAGGTsT
1040-1058	UGCGCGUGCUCAACUGCCATsT	413	UGGCAGUUGAGCA	414	AD-9581
			CGCGCATsT
1040-1058	uGcGcGuGcucAAcuGccATsT	415	UGGcAGUUGAGcA	416	AD-9707
			CGCGcATsT
1042-1060	CGCGUGCUCAACUGCCAAGTsT	417	CUUGGCAGUUGAG	418	AD-9543
			CACGCGTsT
1042-1060	cGcGuGcucAAcuGccAAGTsT	419	CUUGGcAGUUGAG	420	AD-9669
			cACGCGTsT
1053-1071	CUGCCAAGGGAAGGGCACGTsT	421	CGUGCCCUUCCCU	422	AD-9574
			UGGCAGTsT
1053-1071	cuGccAAGGGAAGGGcAcGTsT	423	CGUGCCCUUCCCU	424	AD-9700
			UGGcAGTsT
1057-1075	CAAGGGAAGGGCACGGUUATT	425	UAACCGUGCCCUU	426	AD-15320
			CCCUUGTT
1058-1076	AAGGGAAGGGCACGGUUAGTT	427	CUAACCGUGCCCU	428	AD-15321
			UCCCUUTT
1059-1077	AGGGAAGGGCACGGUUAGCTT	429	GCUAACCGUGCCC	430	AD-15199
			UUCCCUTT
1060-1078	GGGAAGGGCACGGUUAGCGTT	431	CGCUAACCGUGCC	432	AD-15167
			CUUCCCTT
1061-1079	GGAAGGGCACGGUUAGCGGTT	433	CCGCUAACCGUGC	434	AD-15164
			CCUUCCTT
1062-1080	GAAGGGCACGGUUAGCGGCTT	435	GCCGCUAACCGUG	436	AD-15166
			CCCUUCTT
1063-1081	AAGGGCACGGUUAGCGGCATT	437	UGCCGCUAACCGU	438	AD-15322
			GCCCUUTT
1064-1082	AGGGCACGGUUAGCGGCACTT	439	GUGCCGCUAACCG	440	AD-15200
			UGCCCUTT
1068-1086	CACGGUUAGCGGCACCCUCTT	441	GAGGGUGCCGCUA	442	AD-15213
			ACCGUGTT
1069-1087	ACGGUUAGCGGCACCCUCATT	443	UGAGGGUGCCGCU	444	AD-15229
			AACCGUTT
1072-1090	GUUAGCGGCACCCUCAUAGTT	445	CUAUGAGGGUGCC	446	AD-15215
			GCUAACTT
1073-1091	UUAGCGGCACCCUCAUAGGTT	447	CCUAUGAGGGUGC	448	AD-15214
			CGCUAATT
1076-1094	GCGGCACCCUCAUAGGCCUTsT	449	AGGCCUAUGAGGG	450	AD-9315
			UGCCGCTsT
1079-1097	GCACCCUCAUAGGCCUGGATsT	451	UCCAGGCCUAUGA	452	AD-9326
			GGGUGCTsT
1085-1103	UCAUAGGCCUGGAGUUUAUTsT	453	AUAAACUCCAGGC	454	AD-9318
			CUAUGATsT
1090-1108	GGCCUGGAGUUUAUUCGGATsT	455	UCCGAAUAAACUC	456	AD-9323
			CAGGCCTsT
1091-1109	GCCUGGAGUUUAUUCGGAATsT	457	UUCCGAAUAAACU	458	AD-9314
			CCAGGCTsT
1091-1109	GccuGGAGuuuAuucGGAATsT	459	UUCCGAAuAAACU	460	AD-10792
			CcAGGCTsT
1091-1109	GccuGGAGuuuAuucGGAATsT	461	UUCCGAAUAACUC	462	AD-10796
			CAGGCTsT
1093-1111	CUGGAGUUUAUUCGGAAAATsT	463	UUUUCCGAAUAAA	464	AD-9638
			CUCCAGTsT
1093-1111	cuGGAGuuuAuucGGAAAATsT	465	UUUUCCGAAuAAA	466	AD-9764
			CUCcAGTsT
1095-1113	GGAGUUUAUUCGGAAAAGCTsT	467	GCUUUUCCGAAUA	468	AD-9525
			AACUCCTsT
1095-1113	GGAGuuuAuucGGAAAAGcTsT	469	GCUUUUCCGAAuA	470	AD-9651
			AACUCCTsT
1096-1114	GAGUUUAUUCGGAAAAGCCTsT	471	GGCUUUUCCGAAU	472	AD-9560
			AAACUCTsT
1096-1114	GAGuuuAuucGGAAAAGccTsT	473	GGCUUUUCCGAAu	474	AD-9686
			AAACUCTsT
1100-1118	UUAUUCGGAAAAGCCAGCUTsT	475	AGCUGGCUUUUCC	476	AD-9536
			GAAUAATsT
1100-1118	uuAuucGGAAAAGccAGcuTsT	477	AGCUGGCUUUUCC	478	AD-9662
			GAAuAATsT
1154-1172	CCCUGGCGGGUGGGUACAGTsT	479	CUGUACCCACCCG	480	AD-9584
			CCAGGGTsT
1154-1172	cccuGGcGGGuGGGuAcAGTsT	481	CUGuACCcACCCG	482	AD-9710
			CcAGGGTsT
1155-1173	CCUGGCGGGUGGGUACAGCTT	483	GCUGUACCCACCC	484	AD-15323
			GCCAGGTT
1157-1175	UGGCGGGUGGGUACAGCCGTsT	485	CGGCUGUACCCAC	486	AD-9551
			CCGCCATsT
1157-1175	uGGcGGGuGGGuAcAGccGTsT	487	CGGCUGuACCcAC	488	AD-9677
			CCGCcATsT
1158-1176	GGCGGGUGGGUACAGCCGCTT	489	GCGGCUGUACCCA	490	AD-15230
			CCCGCCTT
1162-1180	GGUGGGUACAGCCGCGUCCTT	491	GGACGCGGCUGUA	492	AD-15231
			CCCACCTT
1164-1182	UGGGUACAGCCGCGUCCUCTT	493	GAGGACGCGGCUG	494	AD-15285
			UACCCATT
1172-1190	GCCGCGUCCUCAACGCCGCTT	495	GCGGCGUUGAGGA	496	AD-15396
			CGCGGCTT
1173-1191	CCGCGUCCUCAACGCCGCCTT	497	GGCGGCGUUGAGG	498	AD-15397
			ACGCGGTT
1216-1234	GUCGUGCUGGUCACCGCUGTsT	499	CAGCGGUGACCAG	500	AD-9600
			CACGACTsT
1216-1234	GucGuGcuGGucAccGcuGTsT	501	cAGCGGUGACcAG	502	AD-9726
			cACGACTsT
1217-1235	UCGUGCUGGUCACCGCUGCTsT	503	GCAGCGGUGACCA	504	AD-9606
			GCACGATsT
1217-1235	ucGuGcuGGucAccGcuGcTsT	505	GcAGCGGUGACcA	506	AD-9732
			GcACGATsT
1223-1241	UGGUCACCGCUGCCGGCAATsT	507	UUGCCGGCAGCGG	508	AD-9633
			UGACCATsT
1223-1241	uGGucAccGcuGccGGcAATsT	509	UUGCCGGcAGCGG	510	AD-9759
			UGACcATsT
1224-1242	GGUCACCGCUGCCGGCAACTsT	511	GUUGCCGGCAGCG	512	AD-9588
			GUGACCTsT
1224-1242	GGucAccGcuGccGGcAAcTsT	513	GUUGCCGGcAGCG	514	AD-9714
			GUGACCTsT
1227-1245	CACCGCUGCCGGCAACUUCTsT	515	GAAGUUGCCGGCA	516	AD-9589
			GCGGUGTsT
1227-1245	cAccGcuGccGGcAAcuucTsT	517	GAAGUUGCCGGcA	518	AD-9715
			GCGGUGTsT
1229-1247	CCGCUGCCGGCAACUUCCGTsT	519	CGGAAGUUGCCGG	520	AD-9575
			CAGCGGTsT
1229-1247	ccGcuGccGGcAAcuuccGTsT	521	CGGAAGUUGCCGG	522	AD-9701
			cAGCGGTsT
1230-1248	CGCUGCCGGCAACUUCCGGTsT	523	CCGGAAGUUGCCG	524	AD-9563
			GCAGCGTsT
1230-1248	cGcuGccGGcAAcuuccGGTsT	525	CCGGAAGUUGCCG	526	AD-9689
			GcAGCGTsT
1231-1249	GCUGCCGGCAACUUCCGGGTsT	527	CCCGGAAGUUGCC	528	AD-9594
			GGCAGCTsT
1231-1249	GcuGccGGcAAcuuccGGGTsT	529	CCCGGAAGUUGCC	530	AD-9720
			GGcAGCTsT
1236-1254	CGGCAACUUCCGGGACGAUTsT	531	AUCGUCCCGGAAG	532	AD-9585
			UUGCCGTsT
1236-1254	cGGcAAcuuccGGGAcGAuTsT	533	AUCGUCCCGGAAG	534	AD-9711
			UUGCCGTsT
1237-1255	GGCAACUUCCGGGACGAUGTsT	535	CAUCGUCCCGGAA	536	AD-9614
			GUUGCCTsT
1237-1255	GGcAAcuuccGGGAcGAuGTsT	537	cAUCGUCCCGGAA	538	AD-9740
			GUUGCCTsT
1243-1261	UUCCGGGACGAUGCCUGCCTsT	539	GGCAGGCAUCGUC	540	AD-9615
			CCGGAATsT
1243-1261	uuccGGGAcGAuGccuGccTsT	541	GGcAGGcAUCGUC	542	AD-9741
			CCGGAATsT
1248-1266	GGACGAUGCCUGCCUCUACTsT	543	GUAGAGGCAGGCA	544	AD-9534
			UCGUCCTsT
1248-1266	GGACGAUGCCUGCCUCUACTsT	545	GUAGAGGCAGGCA	546	AD-9534
			UCGUCCTsT
1248-1266	GGAcGAuGccuGccucuAcTsT	547	GuAGAGGcAGGcA	548	AD-9660
			UCGUCCTsT
1279-1297	GCUCCCGAGGUCAUCACAGTT	549	CUGUGAUGACCUC	550	AD-15324
			GGGAGCTT
1280-1298	CUCCCGAGGUCAUCACAGUTT	551	ACUGUGAUGACCU	552	AD-15232
			CGGGAGTT
1281-1299	UCCCGAGGUCAUCACAGUUTT	553	AACUGUGAUGACC	554	AD-15233
			UCGGGATT
1314-1332	CCAAGACCAGCCGGUGACCTT	555	GGUCACCGGCUGG	556	AD-15234
			UCUUGGTT
1315-1333	CAAGACCAGCCGGUGACCCTT	557	GGGUCACCGGCUG	558	AD-15286
			GUCUUGTT
1348-1366	ACCAACUUUGGCCGCUGUGTsT	559	CACAGCGGCCAAA	560	AD-9590
			GUUGGUTsT
1348-1366	AccAAcuuuGGccGcuGuGTsT	561	cAcAGCGGCcAAA	562	AD-9716
			GUUGGUTsT
1350-1368	CAACUUUGGCCGCUGUGUGTsT	563	CACACAGCGGCCA	564	AD-9632
			AAGUUGTsT
1350-1368	cAAcuuuGGccGcuGuGuGTsT	565	cAcAcAGCGGCcA	566	AD-9758
			AAGUUGTsT
1360-1378	CGCUGUGUGGACCUCUUUGTsT	567	CAAAGAGGUCCAC	568	AD-9567
			ACAGCGTsT
1360-1378	cGcuGuGuGGAccucuuuGTsT	569	cAAAGAGGUCcAc	570	AD-9693
			AcAGCGTsT
1390-1408	GACAUCAUUGGUGCCUCCATsT	571	UGGAGGCACCAAU	572	AD-9586
			GAUGUCTsT
1390-1408	GAcAucAuuGGuGccuccATsT	573	UGGAGGcACcAAU	574	AD-9712
			GAUGUCTsT
1394-1412	UCAUUGGUGCCUCCAGCGATsT	575	UCGCUGGAGGCAC	576	AD-9564
			CAAUGATsT
1394-1412	ucAuuGGuGccuccAGcGATsT	577	UCGCUGGAGGcAC	578	AD-9690
			cAAUGATsT
1417-1435	AGCACCUGCUUUGUGUCACTsT	579	GUGACACAAAGCA	580	AD-9616
			GGUGCUTsT
1417-1435	AGcAccuGcuuuGuGucAcTsT	581	GUGAcAcAAAGcA	582	AD-9742
			GGUGCUTsT
1433-1451	CACAGAGUGGGACAUCACATT	583	UGUGAUGUCCCAC	584	AD-15398
			UCUGUGTT
1486-1504	AUGCUGUCUGCCGAGCCGGTsT	585	CCGGCUCGGCAGA	586	AD-9617
			CAGCAUTsT
1486-1504	AuGcuGucuGccGAGccGGTsT	587	CCGGCUCGGcAGA	588	AD-9743
			cAGcAUTsT
1491-1509	GUCUGCCGAGCCGGAGCUCTsT	589	GAGCUCCGGCUCG	590	AD-9635
			GCAGACTsT
1491-1509	GucuGccGAGccGGAGcucTsT	591	GAGCUCCGGCUCG	592	AD-9761
			GcAGACTsT
1521-1539	GUUGAGGCAGAGACUGAUCTsT	593	GAUCAGUCUCUGC	594	AD-9568
			CUCAACTsT
1521-1539	GuuGAGGcAGAGAcuGAucTsT	595	GAUcAGUCUCUGC	596	AD-9694
			CUcAACTsT
1527-1545	GCAGAGACUGAUCCACUUCTsT	597	GAAGUGGAUCAGU	598	AD-9576
			CUCUGCTsT
1527-1545	GcAGAGAcuGAuccAcuucTsT	599	GAAGUGGAUcAGU	600	AD-9702
			CUCUGCTsT
1529-1547	AGAGACUGAUCCACUUCUCTsT	601	GAGAAGUGGAUCA	602	AD-9627
			GUCUCUTsT
1529-1547	AGAGAcuGAuccAcuucucTsT	603	GAGAAGUGGAUcA	604	AD-9753
			GUCUCUTsT
1543-1561	UUCUCUGCCAAAGAUGUCATsT	605	UGACAUCUUUGGC	606	AD-9628
			AGAGAATsT
1543-1561	uucucuGccAAAGAuGucATsT	607	UGAcAUCUUUGGc	608	AD-9754
			AGAGAATsT
1545-1563	CUCUGCCAAAGAUGUCAUCTsT	609	GAUGACAUCUUUG	610	AD-9631
			GCAGAGTsT
1545-1563	cucuGccAAAGAuGucAucTsT	611	GAUGAcAUCUUUG	612	AD-9757
			GcAGAGTsT
1580-1598	CUGAGGACCAGCGGGUACUTsT	613	AGUACCCGCUGGU	614	AD-9595
			CCUCAGTsT
1580-1598	cuGAGGAccAGcGGGuAcuTsT	615	AGuACCCGCUGGU	616	AD-9721
			CCUcAGTsT
1581-1599	UGAGGACCAGCGGGUACUGTsT	617	CAGUACCCGCUGG	618	AD-9544
			UCCUCATsT
1581-1599	uGAGGAccAGcGGGuAcuGTsT	619	cAGuACCCGCUGG	620	AD-9670
			UCCUcATsT
1666-1684	ACUGUAUGGUCAGCACACUTT	621	AGUGUGCUGACCA	622	AD-15235
			UACAGUTT
1668-1686	UGUAUGGUCAGCACACUCGTT	623	CGAGUGUGCUGAC	624	AD-15236
			CAUACATT
1669-1687	GUAUGGUCAGCACACUCGGTT	625	CCGAGUGUGCUGA	626	AD-15168
			CCAUACTT
1697-1715	GGAUGGCCACAGCCGUCGCTT	627	GCGACGGCUGUGG	628	AD-15174
			CCAUCCTT
1698-1716	GAUGGCCACAGCCGUCGCCTT	629	GGCGACGGCUGUG	630	AD-15325
			GCCAUCTT
1806-1824	CAAGCUGGUCUGCCGGGCCTT	631	GGCCCGGCAGACC	632	AD-15326
			AGCUUGTT
1815-1833	CUGCCGGGCCCACAACGCUTsT	633	AGCGUUGUGGGCC	634	AD-9570
			CGGCAGTsT
1815-1833	cuGccGGGcccAcAAcGcuTsT	635	AGCGUUGUGGGCC	636	AD-9696
			CGGcAGTsT
1816-1834	UGCCGGGCCCACAACGCUUTsT	637	AAGCGUUGUGGGC	638	AD-9566
			CCGGCATsT
1816-1834	uGccGGGcccAcAAcGcuuTsT	639	AAGCGUUGUGGGC	640	AD-9692
			CCGGcATsT
1818-1836	CCGGGCCCACAACGCUUUUTsT	641	AAAAGCGUUGUGG	642	AD-9532
			GCCCGGTsT
1818-1836	ccGGGcccAcAAcGcuuuuTsT	643	AAAAGCGUUGUGG	644	AD-9658
			GCCCGGTsT
1820-1838	GGGCCCACAACGCUUUUGGTsT	645	CCAAAAGCGUUGU	646	AD-9549
			GGGCCCTsT
1820-1838	GGGcccAcAAcGcuuuuGGTsT	647	CcAAAAGCGUUGU	648	AD-9675
			GGGCCCTsT
1840-1858	GGUGAGGGUGUCUACGCCATsT	649	UGGCGUAGACACC	650	AD-9541
			CUCACCTsT
1840-1858	GGuGAGGGuGucuAcGccATsT	651	UGGCGuAGAcACC	652	AD-9667
			CUcACCTsT
1843-1861	GAGGGUGUCUACGCCAUUGTsT	653	CAAUGGCGUAGAC	654	AD-9550
			ACCCUCTsT
1843-1861	GAGGGuGucuAcGccAuuGTsT	655	cAAUGGCGuAGAc	656	AD-9676
			ACCCUCTsT
1861-1879	GCCAGGUGCUGCCUGCUACTsT	657	GUAGCAGGCAGCA	658	AD-9571
			CCUGGCTsT
1861-1879	GccAGGuGcuGccuGcuAcTsT	659	GuAGcAGGcAGcA	660	AD-9697
			CCUGGCTsT
1862-1880	CCAGGUGCUGCCUGCUACCTsT	661	GGUAGCAGGCAGC	662	AD-9572
			ACCUGGTsT
1862-1880	ccAGGuGcuGccuGcuAccTsT	663	GGuAGcAGGcAGc	664	AD-9698
			ACCUGGTsT
2008-2026	ACCCACAAGCCGCCUGUGCTT	665	GCACAGGCGGCUU	666	AD-15327
			GUGGGUTT
2023-2041	GUGCUGAGGCCACGAGGUCTsT	667	GACCUCGUGGCCU	668	AD-9639
			CAGCACTsT
2023-2041	GuGcuGAGGccAcGAGGucTsT	669	GACCUCGUGGCCU	670	AD-9765
			cAGcACTsT
2024-2042	UGCUGAGGCCACGAGGUCATsT	671	UGACCUCGUGGCC	672	AD-9518
			UCAGCATsT
2024-2042	UGCUGAGGCCACGAGGUCATsT	673	UGACCUCGUGGCC	674	AD-9518
			UCAGCATsT
2024-2042	uGcuGAGGccAcGAGGucATsT	675	UGACCUCGUGGCC	676	AD-9644
			UcAGcATsT
2024-2042	UfgCfuGfaGfgCfcAfcGfaG	677	P*uGfaCfcUfcG	678	AD-14672
	fgUfcAfTsT		fuGfgCfcUfcAf
			gCfaTsT
2024-2042	UfGCfUfGAGGCfCfACfGAGG	679	UfGACfCfUfCfG	680	AD-14682
	UfCfATsT		UfGGCfCfUfCfA
			GCfATsT
2024-2042	UgCuGaGgCcAcGaGgUcATsT	681	P*uGfaCfcUfcG	682	AD-14692
			fuGfgCfcUfcAf
			gCfaTsT
2024-2042	UgCuGaGgCcAcGaGgUcATsT	683	UfGACfCfUfCfG	684	AD-14702
			UfGGCfCfUfCfA
			GCfATsT
2024-2042	UfgCfuGfaGfgCfcAfcGfaG	685	UGACCucGUggCC	686	AD-14712
	fgUfcAfTsT		UCAgcaTsT
2024-2042	UfGCfUfGAGGCfCfACfGAGG	687	UGACCucGUggCC	688	AD-14722
	UfCfATsT		UCAgcaTsT
2024-2042	UgCuGaGgCcAcGaGgUcATsT	689	UGACCucGUggCC	690	AD-14732
			UCAgcaTsT
2024-2042	GfuGfgUfcAfgCfgGfcCfgG	691	P*cAfuCfcCfgG	692	AD-15078
	fgAfuGfTsT		fcCfgCfuGfaCf
			cAfcTsT
2024-2042	GUfGGUfCfAGCfGGCfCfGGG	693	CfAUfCfCfCfGG	694	AD-15088
	AUfGTsT		CfCfGCfUfGACf
			CfACfTsT
2024-2042	GuGgUcAgCgGcCgGgAuGTsT	695	P*cAfuCfcCfgG	696	AD-15098
			fcCfgCfuGfaCf
			cAfcTsT
2024-2042	GuGgUcAgCgGcCgGgAuGTsT	697	CfAUfCfCfCfGG	698	AD-15108
			CfCfGCfUfGACf
			CfACfTsT
2024-2042	GfuGfgUfcAfgCfgGfcCfgG	699	CAUCCcgGCcgCU	700	AD-15118
	fgAfuGfTsT		GACcacTsT
2024-2042	GUfGGUfCfAGCfGGCfCfGGG	701	CAUCCcgGCcgCU	702	AD-15128
	AUfGTsT		GACcacTsT
2024-2042	GuGgUcAgCgGcCgGgAuGTsT	703	CAUCCcgGCcgCU	704	AD-15138
			GACcacTsT
2030-2048	GGCCACGAGGUCAGCCCAATT	705	UUGGGCUGACCUC	706	AD-15237
			GUGGCCTT
2035-2053	CGAGGUCAGCCCAACCAGUTT	707	ACUGGUUGGGCUG	708	AD-15287
			ACCUCGTT
2039-2057	GUCAGCCCAACCAGUGCGUTT	709	ACGCACUGGUUGG	710	AD-15238
			GCUGACTT
2041-2059	CAGCCCAACCAGUGCGUGGTT	711	CCACGCACUGGUU	712	AD-15328
			GGGCUGTT
2062-2080	CACAGGGAGGCCAGCAUCCTT	713	GGAUGCUGGCCUC	714	AD-15399
			CCUGUGTT
2072-2090	CCAGCAUCCACGCUUCCUGTsT	715	CAGGAAGCGUGGA	716	AD-9582
			UGCUGGTsT
2072-2090	ccAGcAuccAcGcuuccuGTsT	717	cAGGAAGCGUGGA	718	AD-9708
			UGCUGGTsT
2118-2136	AGUCAAGGAGCAUGGAAUCTsT	719	GAUUCCAUGCUCC	720	AD-9545
			UUGACUTsT
2118-2136	AGucAAGGAGcAuGGAAucTsT	721	GAUUCcAUGCUCC	722	AD-9671
			UUGACUTsT
2118-2136	AfgUfcAfaGfgAfgCfaUfgG	723	P*gAfuUfcCfaU	724	AD-14674
	faAfuCfTsT		fgCfuCfcUfuGf
			aCfuTsT
2118-2136	AGUfCfAAGGAGCfAUfGGAAU	725	GAUfUfCfCfAUf	726	AD-14684
	fCfTsT		GCfUfCfCfUfUf
			GACfUfTsT
2118-2136	AgUcAaGgAgCaUgGaAuCTsT	727	P*gAfuUfcCfaU	728	AD-14694
			fgCfuCfcUfuGf
			aCfuTsT
2118-2136	AgUcAaGgAgCaUgGaAuCTsT	729	GAUfUfCfCfAUf	730	AD-14704
			GCfUfCfCfUfUf
			GACfUfTsT
2118-2136	AfgUfcAfaGfgAfgCfaUfgG	731	GAUUCcaUGcuCC	732	AD-14714
	faAfuCfTsT		UUGacuTsT
2118-2136	AGUfCfAAGGAGCfAUfGGAAU	733	GAUUCcaUGcuCC	734	AD-14724
	fCfTsT		UUGacuTsT
2118-2136	AgUcAaGgAgCaUgGaAuCTsT	735	GAUUCcaUGcuCC	736	AD-14734
			UUGacuTsT
2118-2136	GfcGfgCfaCfcCfuCfaUfaG	737	P*aGfgCfcUfaU	738	AD-15080
	fgCfcUfTsT		fgAfgGfgUfgCf
			cGfcTsT
2118-2136	GCfGGCfACfCfCfUfCfAUfA	739	AGGCfCfUfAUfG	740	AD-15090
	GGCfCfUfTsT		AGGGUfGCfCfGC
			fTsT
2118-2136	GcGgCaCcCuCaUaGgCcUTsT	741	P*aGfgCfcUfaU	742	AD-15100
			fgAfgGfgUfgCf
			cGfcTsT
2118-2136	GcGgCaCcCuCaUaGgCcUTsT	743	AGGCfCfUfAUfG	744	AD-15110
			AGGGUfGCfCfGC
			fTsT
2118-2136	GfcGfgCfaCfcCfuCfaUfaG	745	AGGCCuaUGagGG	746	AD-15120
	fgCfcUfTsT		UGCcgcTsT
2118-2136	GCfGGCfACfCfCfUfCfAUfA	747	AGGCCuaUGagGG	748	AD-15130
	GGCfCfUfTsT		UGCcgcTsT
2118-2136	GcGgCaCcCuCaUaGgCcUTsT	749	AGGCCuaUGagGG	750	AD-15140
			UGCcgcTsT
2122-2140	AAGGAGCAUGGAAUCCCGGTsT	751	CCGGGAUUCCAUG	752	AD-9522
			CUCCUUTsT
2122-2140	AAGGAGcAuGGAAucccGGTsT	753	CCGGGAUUCcAUG	754	AD-9648
			CUCCUUTsT
2123-2141	AGGAGCAUGGAAUCCCGGCTsT	755	GCCGGGAUUCCAU	756	AD-9552
			GCUCCUTsT
2123-2141	AGGAGcAuGGAAucccGGcTsT	757	GCCGGGAUUCcAU	758	AD-9678
			GCUCCUTsT
2125-2143	GAGCAUGGAAUCCCGGCCCTsT	759	GGGCCGGGAUUCC	760	AD-9618
			AUGCUCTsT
2125-2143	GAGcAuGGAAucccGGcccTsT	761	GGGCCGGGAUUCc	762	AD-9744
			AUGCUCTsT
2230-2248	GCCUACGCCGUAGACAACATT	763	UGUUGUCUACGGC	764	AD-15239
			GUAGGCTT
2231-2249	CCUACGCCGUAGACAACACTT	765	GUGUUGUCUACGG	766	AD-15212
			CGUAGGTT
2232-2250	CUACGCCGUAGACAACACGTT	767	CGUGUUGUCUACG	768	AD-15240
			GCGUAGTT
2233-2251	UACGCCGUAGACAACACGUTT	769	ACGUGUUGUCUAC	770	AD-15177
			GGCGUATT
2235-2253	CGCCGUAGACAACACGUGUTT	771	ACACGUGUUGUCU	772	AD-15179
			ACGGCGTT
2236-2254	GCCGUAGACAACACGUGUGTT	773	CACACGUGUUGUC	774	AD-15180
			UACGGCTT
2237-2255	CCGUAGACAACACGUGUGUTT	775	ACACACGUGUUGU	776	AD-15241
			CUACGGTT
2238-2256	CGUAGACAACACGUGUGUATT	777	UACACACGUGUUG	778	AD-15268
			UCUACGTT
2240-2258	UAGACAACACGUGUGUAGUTT	779	ACUACACACGUGU	780	AD-15242
			UGUCUATT
2241-2259	AGACAACACGUGUGUAGUCTT	781	GACUACACACGUG	782	AD-15216
			UUGUCUTT
2242-2260	GACAACACGUGUGUAGUCATT	783	UGACUACACACGU	784	AD-15176
			GUUGUCTT
2243-2261	ACAACACGUGUGUAGUCAGTT	785	CUGACUACACACG	786	AD-15181
			UGUUGUTT
2244-2262	CAACACGUGUGUAGUCAGGTT	787	CCUGACUACACAC	788	AD-15243
			GUGUUGTT
2247-2265	CACGUGUGUAGUCAGGAGCTT	789	GCUCCUGACUACA	790	AD-15182
			CACGUGTT
2248-2266	ACGUGUGUAGUCAGGAGCCTT	791	GGCUCCUGACUAC	792	AD-15244
			ACACGUTT
2249-2267	CGUGUGUAGUCAGGAGCCGTT	793	CGGCUCCUGACUA	794	AD-15387
			CACACGTT
2251-2269	UGUGUAGUCAGGAGCCGGGTT	795	CCCGGCUCCUGAC	796	AD-15245
			UACACATT
2257-2275	GUCAGGAGCCGGGACGUCATsT	797	UGACGUCCCGGCU	798	AD-9555
			CCUGACTsT
2257-2275	GucAGGAGccGGGAcGucATsT	799	UGACGUCCCGGCU	800	AD-9681
			CCUGACTsT
2258-2276	UCAGGAGCCGGGACGUCAGTsT	801	CUGACGUCCCGGC	802	AD-9619
			UCCUGATsT
2258-2276	ucAGGAGccGGGAcGucAGTsT	803	CUGACGUCCCGGC	804	AD-9745
			UCCUGATsT
2259-2277	CAGGAGCCGGGACGUCAGCTsT	805	GCUGACGUCCCGG	806	AD-9620
			CUCCUGTsT
2259-2277	cAGGAGccGGGAcGucAGcTsT	807	GCUGACGUCCCGG	808	AD-9746
			CUCCUGTsT
2263-2281	AGCCGGGACGUCAGCACUATT	809	UAGUGCUGACGUC	810	AD-15288
			CCGGCUTT
2265-2283	CCGGGACGUCAGCACUACATT	811	UGUAGUGCUGACG	812	AD-15246
			UCCCGGTT
2303-2321	CCGUGACAGCCGUUGCCAUTT	813	AUGGCAACGGCUG	814	AD-15289
			UCACGGTT
2317-2335	GCCAUCUGCUGCCGGAGCCTsT	815	GGCUCCGGCAGCA	816	AD-9324
			GAUGGCTsT
2375-2393	CCCAUCCCAGGAUGGGUGUTT	817	ACACCCAUCCUGG	818	AD-15329
			GAUGGGTT
2377-2395	CAUCCCAGGAUGGGUGUCUTT	819	AGACACCCAUCCU	820	AD-15330
			GGGAUGTT
2420-2438	AGCUUMAAAUGGUUCCGATT	821	UCGGAACCAUUUU	822	AD-15169
			AAAGCUTT
2421-2439	GCUUUAAAAUGGUUCCGACTT	823	GUCGGAACCAUUU	824	AD-15201
			UAAAGCTT
2422-2440	CUUMAAAUGGUUCCGACUTT	825	AGUCGGAACCAUU	826	AD-15331
			UUAAAGTT
2423-2441	UUUAAAAUGGUUCCGACUUTT	827	AAGUCGGAACCAU	828	AD-15190
			UUUAAATT
2424-2442	UUAAAAUGGUUCCGACUUGTT	829	CAAGUCGGAACCA	830	AD-15247
			UUUUAATT
2425-2443	UAAAAUGGUUCCGACUUGUTT	831	ACAAGUCGGAACC	832	AD-15248
			AUUUUATT
2426-2444	AAAAUGGUUCCGACUUGUCTT	833	GACAAGUCGGAAC	834	AD-15175
			CAUUUUTT
2427-2445	AAAUGGUUCCGACUUGUCCTT	835	GGACAAGUCGGAA	836	AD-15249
			CCAUUUTT
2428-2446	AAUGGUUCCGACUUGUCCCTT	837	GGGACAAGUCGGA	838	AD-15250
			ACCAUUTT
2431-2449	GGUUCCGACUUGUCCCUCUTT	839	AGAGGGACAAGUC	840	AD-15400
			GGAACCTT
2457-2475	CUCCAUGGCCUGGCACGAGTT	841	CUCGUGCCAGGCC	842	AD-15332
			AUGGAGTT
2459-2477	CCAUGGCCUGGCACGAGGGTT	843	CCCUCGUGCCAGG	844	AD-15388
			CCAUGGTT
2545-2563	GAACUCACUCACUCUGGGUTT	845	ACCCAGAGUGAGU	846	AD-15333
			GAGUUCTT
2549-2567	UCACUCACUCUGGGUGCCUTT	847	AGGCACCCAGAGU	848	AD-15334
			GAGUGATT
2616-2634	UUUCACCAUUCAAACAGGUTT	849	ACCUGUUUGAAUG	850	AD-15335
			GUGAAATT
2622-2640	CAUUCAAACAGGUCGAGCUTT	851	AGCUCGACCUGUU	852	AD-15183
			UGAAUGTT
2623-2641	AUUCAAACAGGUCGAGCUGTT	853	CAGCUCGACCUGU	854	AD-15202
			UUGAAUTT
2624-2642	UUCAAACAGGUCGAGCUGUTT	855	ACAGCUCGACCUG	856	AD-15203
			UUUGAATT
2625-2643	UCAAACAGGUCGAGCUGUGTT	857	CACAGCUCGACCU	858	AD-15272
			GUUUGATT
2626-2644	CAAACAGGUCGAGCUGUGCTT	859	GCACAGCUCGACC	860	AD-15217
			UGUUUGTT
2627-2645	AAACAGGUCGAGCUGUGCUTT	861	AGCACAGCUCGAC	862	AD-15290
			CUGUUUTT
2628-2646	AACAGGUCGAGCUGUGCUCTT	863	GAGCACAGCUCGA	864	AD-15218
			CCUGUUTT
2630-2648	CAGGUCGAGCUGUGCUCGGTT	865	CCGAGCACAGCUC	866	AD-15389
			GACCUGTT
2631-2649	AGGUCGAGCUGUGCUCGGGTT	867	CCCGAGCACAGCU	868	AD-15336
			CGACCUTT
2633-2651	GUCGAGCUGUGCUCGGGUGTT	869	CACCCGAGCACAG	870	AD-15337
			CUCGACTT
2634-2652	UCGAGCUGUGCUCGGGUGCTT	871	GCACCCGAGCACA	872	AD-15191
			GCUCGATT
2657-2675	AGCUGCUCCCAAUGUGCCGTT	873	CGGCACAUUGGGA	874	AD-15390
			GCAGCUTT
2658-2676	GCUGCUCCCAAUGUGCCGATT	875	UCGGCACAUUGGG	876	AD-15338
			AGCAGCTT
2660-2678	UGCUCCCAAUGUGCCGAUGTT	877	CAUCGGCACAUUG	878	AD-15204
			GGAGCATT
2663-2681	UCCCAAUGUGCCGAUGUCCTT	879	GGACAUCGGCACA	880	AD-15251
			UUGGGATT
2665-2683	CCAAUGUGCCGAUGUCCGUTT	881	ACGGACAUCGGCA	882	AD-15205
			CAUUGGTT
2666-2684	CAAUGUGCCGAUGUCCGUGTT	883	CACGGACAUCGGC	884	AD-15171
			ACAUUGTT
2667-2685	AAUGUGCCGAUGUCCGUGGTT	885	CCACGGACAUCGG	886	AD-15252
			CACAUUTT
2673-2691	CCGAUGUCCGUGGGCAGAATT	887	UUCUGCCCACGGA	888	AD-15339
			CAUCGGTT
2675-2693	GAUGUCCGUGGGCAGAAUGTT	889	CAUUCUGCCCACG	890	AD-15253
			GACAUCTT
2678-2696	GUCCGUGGGCAGAAUGACUTT	891	AGUCAUUCUGCCC	892	AD-15340
			ACGGACTT
2679-2697	UCCGUGGGCAGAAUGACUUTT	893	AAGUCAUUCUGCC	894	AD-15291
			CACGGATT
2683-2701	UGGGCAGAAUGACUUDUAUTT	895	AUAAAAGUCAUUC	896	AD-15341
			UGCCCATT
2694-2712	ACUUUUAUUGAGCUCUUGUTT	897	ACAAGAGCUCAAU	898	AD-15401
			AAAAGUTT
2700-2718	AUUGAGCUCUUGUUCCGUGTT	899	CACGGAACAAGAG	900	AD-15342
			CUCAAUTT
2704-2722	AGCUCUUGUUCCGUGCCAGTT	901	CUGGCACGGAACA	902	AD-15343
			AGAGCUTT
2705-2723	GCUCUUGUUCCGUGCCAGGTT	903	CCUGGCACGGAAC	904	AD-15292
			AAGAGCTT
2710-2728	UGUUCCGUGCCAGGCAUUCTT	905	GAAUGCCUGGCAC	906	AD-15344
			GGAACATT
2711-2729	GUUCCGUGCCAGGCAUUCATT	907	UGAAUGCCUGGCA	908	AD-15254
			CGGAACTT
2712-2730	UUCCGUGCCAGGCAUUCAATT	909	UUGAAUGCCUGGC	910	AD-15345
			ACGGAATT
2715-2733	CGUGCCAGGCAUUCAAUCCTT	911	GGAUUGAAUGCCU	912	AD-15206
			GGCACGTT
2716-2734	GUGCCAGGCAUUCAAUCCUTT	913	AGGAUUGAAUGCC	914	AD-15346
			UGGCACTT
2728-2746	CAAUCCUCAGGUCUCCACCTT	915	GGUGGAGACCUGA	916	AD-15347
			GGAUUGTT
2743-2761	CACCAAGGAGGCAGGAUUCTsT	917	GAAUCCUGCCUCC	918	AD-9577
			UUGGUGTsT
2743-2761	cAccAAGGAGGcAGGAuucTsT	919	GAAUCCUGCCUCC	920	AD-9703
			UUGGUGTsT
2743-2761	CfaCfcAfaGfgAfgGfcAfgG	921	P*gAfaUfcCfuG	922	AD-14678
	faUfuCfTsT		fcCfuCfcUfuGf
			gUfgTsT
2743-2761	CfACfCfAAGGAGGCfAGGAUf	923	GAAUfCfCfUfGC	924	AD-14688
	UfCfTsT		fCfUfCfCfUfUf
			GGUfGTsT
2743-2761	CaCcAaGgAgGcAgGaUuCTsT	925	P*gAfaUfcCfuG	926	AD-14698
			fcCfuCfcUfuGf
			gUfgTsT
2743-2761	CaCcAaGgAgGcAgGaUuCTsT	927	GAAUfCfCfUfGC	928	AD-14708
			fCfUfCfCfUfUf
			GGUfGTsT
2743-2761	CfaCfcAfaGfgAfgGfcAfgG	929	GAAUCcuGCcuCC	930	AD-14718
	faUfuCfTsT		UUGgugTsT
2743-2761	CfACfCfAAGGAGGCfAGGAUf	931	GAAUCcuGCcuCC	932	AD-14728
	UfCfTsT		UUGgugTsT
2743-2761	CaCcAaGgAgGcAgGaUuCTsT	933	GAAUCcuGCcuCC	934	AD-14738
			UUGgugTsT
2743-2761	GfgCfcUfgGfaGfuUfuAfuU	935	P*uCfcGfaAfuA	936	AD-15084
	fcGfgAfTsT		faAfcUfcCfaGf
			gCfcTsT
2743-2761	GGCfCfUfGGAGUfUfUfAUfU	937	UfCfCfGAAUfAA	938	AD-15094
	fCfGGATsT		ACfUfCfCfAGGC
			fCfTsT
2743-2761	GgCcUgGaGuUuAuUcGgATsT	939	P*uCfcGfaAfuA	940	AD-15104
			faAfcUfcCfaGf
			gCfcTsT
2743-2761	GgCcUgGaGuUuAuUcGgATsT	941	UfCfCfGAAUfAA	942	AD-15114
			ACfUfCfCfAGGC
			fCfTsT
2743-2761	GfgCfcUfgGfaGfuUfuAfuU	943	UCCGAauAAacUC	944	AD-15124
	fcGfgAfTsT		CAGgccTsT
2743-2761	GGCfCfUfGGAGUfUfUfAUfU	945	UCCGAauAAacUC	946	AD-15134
	fCfGGATsT		CAGgccTsT
2743-2761	GgCcUgGaGuUuAuUcGgATsT	947	UCCGAauAAacUC	948	AD-15144
			CAGgccTsT
2753-2771	GCAGGAUUCUUCCCAUGGATT	949	UCCAUGGGAAGAA	950	AD-15391
			UCCUGCTT
2794-2812	UGCAGGGACAAACAUCGUUTT	951	AACGAUGUUUGUC	952	AD-15348
			CCUGCATT
2795-2813	GCAGGGACAAACAUCGUUGTT	953	CAACGAUGUUUGU	954	AD-15349
			CCCUGCTT
2797-2815	AGGGACAAACAUCGUUGGGTT	955	CCCAACGAUGUUU	956	AD-15170
			GUCCCUTT
2841-2859	CCCUCAUCUCCAGCUAACUTT	957	AGUUAGCUGGAGA	958	AD-15350
			UGAGGGTT
2845-2863	CAUCUCCAGCUAACUGUGGTT	959	CCACAGUUAGCUG	960	AD-15402
			GAGAUGTT
2878-2896	GCUCCCUGAUUAAUGGAGGTT	961	CCUCCAUUAAUCA	962	AD-15293
			GGGAGCTT
2881-2899	CCCUGAUUAAUGGAGGCUUTT	963	AAGCCUCCAUUAA	964	AD-15351
			UCAGGGTT
2882-2900	CCUGAUUAAUGGAGGCUUATT	965	UAAGCCUCCAUUA	966	AD-15403
			AUCAGGTT
2884-2902	UGAUUAAUGGAGGCUUAGCTT	967	GCUAAGCCUCCAU	968	AD-15404
			UAAUCATT
2885-2903	GAUUAAUGGAGGCUUAGCUTT	969	AGCUAAGCCUCCA	970	AD-15207
			UUAAUCTT
2886-2904	AUUAAUGGAGGCUUAGCUUTT	971	AAGCUAAGCCUCC	972	AD-15352
			AUUAAUTT
2887-2905	UUAAUGGAGGCUUAGCUUUTT	973	AAAGCUAAGCCUC	974	AD-15255
			CAUUAATT
2903-2921	UUUCUGGAUGGCAUCUAGCTsT	975	GCUAGAUGCCAUC	976	AD-9603
			CAGAAATsT
2903-2921	uuucuGGAuGGcAucuAGcTsT	977	GCuAGAUGCcAUC	978	AD-9729
			cAGAAATsT
2904-2922	UUCUGGAUGGCAUCUAGCCTsT	979	GGCUAGAUGCCAU	980	AD-9599
			CCAGAATsT
2904-2922	uucuGGAuGGcAucuAGccTsT	981	GGCuAGAUGCcAU	982	AD-9725
			CcAGAATsT
2905-2923	UCUGGAUGGCAUCUAGCCATsT	983	UGGCUAGAUGCCA	984	AD-9621
			UCCAGATsT
2905-2923	ucuGGAuGGcAucuAGccATsT	985	UGGCuAGAUGCcA	986	AD-9747
			UCcAGATsT
2925-2943	AGGCUGGAGACAGGUGCGCTT	987	GCGCACCUGUCUC	988	AD-15405
			CAGCCUTT
2926-2944	GGCUGGAGACAGGUGCGCCTT	989	GGCGCACCUGUCU	990	AD-15353
			CCAGCCTT
2927-2945	GCUGGAGACAGGUGCGCCCTT	991	GGGCGCACCUGUC	992	AD-15354
			UCCAGCTT
2972-2990	UUCCUGAGCCACCUUUACUTT	993	AGUAAAGGUGGCU	994	AD-15406
			CAGGAATT
2973-2991	UCCUGAGCCACCUUUACUCTT	995	GAGUAAAGGUGGC	996	AD-15407
			UCAGGATT
2974-2992	CCUGAGCCACCUUUACUCUTT	997	AGAGUAAAGGUGG	998	AD-15355
			CUCAGGTT
2976-2994	UGAGCCACCUUUACUCUGCTT	999	GCAGAGUAAAGGU	1000	AD-15356
			GGCUCATT
2978-2996	AGCCACCUUUACUCUGCUCTT	1001	GAGCAGAGUAAAG	1002	AD-15357
			GUGGCUTT
2981-2999	CACCUUUACUCUGCUCUAUTT	1003	AUAGAGCAGAGUA	1004	AD-15269
			AAGGUGTT
2987-3005	UACUCUGCUCUAUGCCAGGTsT	1005	CCUGGCAUAGAGC	1006	AD-9565
			AGAGUATsT
2987-3005	uAcucuGcucuAuGccAGGTsT	1007	CCUGGcAuAGAGc	1008	AD-9691
			AGAGuATsT
2998-3016	AUGCCAGGCUGUGCUAGCATT	1009	UGCUAGCACAGCC	1010	AD-15358
			UGGCAUTT
3003-3021	AGGCUGUGCUAGCAACACCTT	1011	GGUGUUGCUAGCA	1012	AD-15359
			CAGCCUTT
3006-3024	CUGUGCUAGCAACACCCAATT	1013	UUGGGUGUUGCUA	1014	AD-15360
			GCACAGTT
3010-3028	GCUAGCAACACCCAAAGGUTT	1015	ACCUUUGGGUGUU	1016	AD-15219
			GCUAGCTT
3038-3056	GGAGCCAUCACCUAGGACUTT	1017	AGUCCUAGGUGAU	1018	AD-15361
			GGCUCCTT
3046-3064	CACCUAGGACUGACUCGGCTT	1019	GCCGAGUCAGUCC	1020	AD-15273
			UAGGUGTT
3051-3069	AGGACUGACUCGGCAGUGUTT	1021	ACACUGCCGAGUC	1022	AD-15362
			AGUCCUTT
3052-3070	GGACUGACUCGGCAGUGUGTT	1023	CACACUGCCGAGU	1024	AD-15192
			CAGUCCTT
3074-3092	UGGUGCAUGCACUGUCUCATT	1025	UGAGACAGUGCAU	1026	AD-15256
			GCACCATT
3080-3098	AUGCACUGUCUCAGCCAACTT	1027	GUUGGCUGAGACA	1028	AD-15363
			GUGCAUTT
3085-3103	CUGUCUCAGCCAACCCGCUTT	1029	AGCGGGUUGGCUG	1030	AD-15364
			AGACAGTT
3089-3107	CUCAGCCAACCCGCUCCACTsT	1031	GUGGAGCGGGUUG	1032	AD-9604
			GCUGAGTsT
3089-3107	cucAGccAAcccGcuccAcTsT	1033	GUGGAGCGGGUUG	1034	AD-9730
			GCUGAGTsT
3093-3111	GCCAACCCGCUCCACUACCTsT	1035	GGUAGUGGAGCGG	1036	AD-9527
			GUUGGCTsT
3093-3111	GccAAcccGcuccAcuAccTsT	1037	GGuAGUGGAGCGG	1038	AD-9653
			GUUGGCTsT
3096-3114	AACCCGCUCCACUACCCGGTT	1039	CCGGGUAGUGGAG	1040	AD-15365
			CGGGUUTT
3099-3117	CCGCUCCACUACCCGGCAGTT	1041	CUGCCGGGUAGUG	1042	AD-15294
			GAGCGGTT
3107-3125	CUACCCGGCAGGGUACACATT	1043	UGUGUACCCUGCC	1044	AD-15173
			GGGUAGTT
3108-3126	UACCCGGCAGGGUACACAUTT	1045	AUGUGUACCCUGC	1046	AD-15366
			CGGGUATT
3109-3127	ACCCGGCAGGGUACACAUUTT	1047	AAUGUGUACCCUG	1048	AD-15367
			CCGGGUTT
3110-3128	CCCGGCAGGGUACACAUUCTT	1049	GAAUGUGUACCCU	1050	AD-15257
			GCCGGGTT
3112-3130	CGGCAGGGUACACAUUCGCTT	1051	GCGAAUGUGUACC	1052	AD-15184
			CUGCCGTT
3114-3132	GCAGGGUACACAUUCGCACTT	1053	GUGCGAAUGUGUA	1054	AD-15185
			CCCUGCTT
3115-3133	CAGGGUACACAUUCGCACCTT	1055	GGUGCGAAUGUGU	1056	AD-15258
			ACCCUGTT
3116-3134	AGGGUACACAUUCGCACCCTT	1057	GGGUGCGAAUGUG	1058	AD-15186
			UACCCUTT
3196-3214	GGAACUGAGCCAGAAACGCTT	1059	GCGUUUCUGGCUC	1060	AD-15274
			AGUUCCTT
3197-3215	GAACUGAGCCAGAAACGCATT	1061	UGCGUUUCUGGCU	1062	AD-15368
			CAGUUCTT
3198-3216	AACUGAGCCAGAAACGCAGTT	1063	CUGCGUUUCUGGC	1064	AD-15369
			UCAGUUTT
3201-3219	UGAGCCAGAAACGCAGAUUTT	1064	AAUCUGCGUUUCU	1066	AD-15370
			GGCUCATT
3207-3225	AGAAACGCAGAUUGGGCUGTT	1067	CAGCCCAAUCUGC	1068	AD-15259
			GUUUCUTT
3210-3228	AACGCAGAUUGGGCUGGCUTT	1069	AGCCAGCCCAAUC	1070	AD-15408
			UGCGUUTT
3233-3251	AGCCAAGCCUCUUCUUACUTsT	1071	AGUAAGAAGAGGC	1072	AD-9597
			UUGGCUTsT
3233-3251	AGccAAGccucuucuuAcuTsT	1073	AGuAAGAAGAGGC	1074	AD-9723
			UUGGCUTsT
3233-3251	AfgCfcAfaGfcCfuCfuUfcU	1075	P*aGfuAfaGfaA	1076	AD-14680
	fuAfcUfTsT		fgAfgGfcUfuGf
			gCfuTsT
3233-3251	AGCfCfAAGCfCfUfCfUfUfC	1077	AGUfAAGAAGAGG	1078	AD-14690
	fUfUfACfUfTsT		CfUfUfGGCfUfT
			sT
3233-3251	AgCcAaGcCuCuUcUuAcUTsT	1079	P*aGfuAfaGfaA	1080	AD-14700
			fgAfgGfcUfuGf
			gCfuTsT
3233-3251	AgCcAaGcCuCuUcUuAcUTsT	1081	AGUfAAGAAGAGG	1082	AD-14710
			CfUfUfGGCfUfT
			sT
3233-3251	AfgCfcAfaGfcCfuCfuUfcU	1083	AGUAAgaAGagGC	1084	AD-14720
	fuAfcUfTsT		UUGgcuTsT
3233-3251	AGCfCfAAGCfCfUfCfUfUfC	1085	AGUAAgaAGagGC	1086	AD-14730
	fUfUfACfUfTsT		UUGgcuTsT
3233-3251	AgCcAaGcCuCuUcUuAcUTsT	1087	AGUAAgaAGagGC	1088	AD-14740
			UUGgcuTsT
3233-3251	UfgGfuUfcCfcUfgAfgGfaC	1089	P*gCfuGfgUfcC	1090	AD-15086
	fcAfgCfTsT		fuCfaGfgGfaAf
			cCfaTsT
3233-3251	UfGGUfUfCfCfCfUfGAGGAC	1091	GCfUfGGUfCfCf	1092	AD-15096
	fCfAGCfTsT		UfCfAGGGAACfC
			fATsT
3233-3251	UgGuUcCcUgAgGaCcAgCTsT	1093	P*gCfuGfgUfcC	1094	AD-15106
			fuCfaGfgGfaAf
			cCfaTsT
3233-3251	UgGuUcCcUgAgGaCcAgCTsT	1095	GCfUfGGUfCfCf	1096	AD-15116
			UfCfAGGGAACfC
			fATsT
3233-3251	UfgGfuUfcCfcUfgAfgGfaC	1097	GCUGGucCUcaGG	1098	AD-15126
	fcAfgCfTsT		GAAccaTsT
3233-3251	UfGGUfUfCfCfCfUfGAGGAC	1099	GCUGGucCUcaGG	1100	AD-15136
	fCfAGCfTsT		GAAccaTsT
3233-3251	UgGuUcCcUgAgGaCcAgCTsT	1101	GCUGGucCUcaGG	1102	AD-15146
			GAAccaTsT
3242-3260	UCUUCUUACUUCACCCGGCTT	1103	GCCGGGUGAAGUA	1104	AD-15260
			AGAAGATT
3243-3261	CUUCUUACUUCACCCGGCUTT	1105	AGCCGGGUGAAGU	1106	AD-15371
			AAGAAGTT
3244-3262	UUCUUACUUCACCCGGCUGTT	1107	CAGCCGGGUGAAG	1108	AD-15372
			UAAGAATT
3262-3280	GGGCUCCUCAUUUUUACGGTT	1109	CCGUAAAAAUGAG	1110	AD-15172
			GAGCCCTT
3263-3281	GGCUCCUCAUUUUUACGGGTT	1111	CCCGUAAAAAUGA	1112	AD-15295
			GGAGCCTT
3264-3282	GCUCCUCAUUUUUACGGGUTT	1113	ACCCGUAAAAAUG	1114	AD-15373
			AGGAGCTT
3265-3283	CUCCUCAUUUUUACGGGUATT	1115	UACCCGUAAAAAU	1116	AD-15163
			GAGGAGTT
3266-3284	UCCUCAUUUUUACGGGUAATT	1117	UUACCCGUAAAAA	1118	AD-15165
			UGAGGATT
3267-3285	CCUCAUUUUUACGGGUAACTT	1119	GUUACCCGUAAAA	1120	AD-15374
			AUGAGGTT
3268-3286	CUCAUUUUUACGGGUAACATT	1121	UGUUACCCGUAAA	1122	AD-15296
			AAUGAGTT
3270-3288	CAUUUUUACGGGUAACAGUTT	1123	ACUGUUACCCGUA	1124	AD-15261
			AAAAUGTT
3271-3289	AUUUUUACGGGUAACAGUGTT	1125	CACUGUUACCCGU	1126	AD-15375
			AAAAAUTT
3274-3292	UUUACGGGUAACAGUGAGGTT	1127	CCUCACUGUUACC	1128	AD-15262
			CGUAAATT
3308-3326	CAGACCAGGAAGCUCGGUGTT	1129	CACCGAGCUUCCU	1130	AD-15376
			GGUCUGTT
3310-3328	GACCAGGAAGCUCGGUGAGTT	1131	CUCACCGAGCUUC	1132	AD-15377
			CUGGUCTT
3312-3330	CCAGGAAGCUCGGUGAGUGTT	1133	CACUCACCGAGCU	1134	AD-15409
			UCCUGGTT
3315-3333	GGAAGCUCGGUGAGUGAUGTT	1135	CAUCACUCACCGA	1136	AD-15378
			GCUUCCTT
3324-3342	GUGAGUGAUGGCAGAACGATT	1137	UCGUUCUGCCAUC	1138	AD-15410
			ACUCACTT
3326-3344	GAGUGAUGGCAGAACGAUGTT	1139	CAUCGUUCUGCCA	1140	AD-15379
			UCACUCTT
3330-3348	GAUGGCAGAACGAUGCCUGTT	1141	CAGGCAUCGUUCU	1142	AD-15187
			GCCAUCTT
3336-3354	AGAACGAUGCCUGCAGGCATT	1143	UGCCUGCAGGCAU	1144	AD-15263
			CGUUCUTT
3339-3357	ACGAUGCCUGCAGGCAUGGTT	1145	CCAUGCCUGCAGG	1146	AD-15264
			CAUCGUTT
3348-3366	GCAGGCAUGGAACUUUUUCTT	1147	GAAAAAGUUCCAU	1148	AD-15297
			GCCUGCTT
3356-3374	GGAACUUUUUCCGUUAUCATT	1149	UGAUAACGGAAAA	1150	AD-15208
			AGUUCCTT
3357-3375	GAACUUUUUCCGUUAUCACTT	1151	GUGAUAACGGAAA	1152	AD-15209
			AAGUUCTT
3358-3376	AACUUUUUCCGUUAUCACCTT	1153	GGUGAUAACGGAA	1154	AD-15193
			AAAGUUTT
3370-3388	UAUCACCCAGGCCUGAUUCTT	1155	GAAUCAGGCCUGG	1156	AD-15380
			GUGAUATT
3378-3396	AGGCCUGAUUCACUGGCCUTT	1157	AGGCCAGUGAAUC	1158	AD-15298
			AGGCCUTT
3383-3401	UGAUUCACUGGCCUGGCGGTT	1159	CCGCCAGGCCAGU	1160	AD-15299
			GAAUCATT
3385-3403	AUUCACUGGCCUGGCGGAGTT	1161	CUCCGCCAGGCCA	1162	AD-15265
			GUGAAUTT
3406-3424	GCUUCUAAGGCAUGGUCGGTT	1163	CCGACCAUGCCUU	1164	AD-15381
			AGAAGCTT
3407-3425	CUUCUAAGGCAUGGUCGGGTT	1165	CCCGACCAUGCCU	1166	AD-15210
			UAGAAGTT
3429-3447	GAGGGCCAACAACUGUCCCTT	1167	GGGACAGUUGUUG	1168	AD-15270
			GCCCUCTT
3440-3458	ACUGUCCCUCCUUGAGCACTsT	1169	GUGCUCAAGGAGG	1170	AD-9591
			GACAGUTsT
3440-3458	AcuGucccuccuuGAGcAcTsT	1171	GUGCUcAAGGAGG	1172	AD-9717
			GAcAGUTsT
3441-3459	CUGUCCCUCCUUGAGCACCTsT	1173	GGUGCUCAAGGAG	1174	AD-9622
			GGACAGTsT
3441-3459	cuGucccuccuuGAGcAccTsT	1175	GGUGCUcAAGGAG	1176	AD-9748
			GGAcAGTsT
3480-3498	ACAUUUAUCUUUUGGGUCUTsT	1177	AGACCCAAAAGAU	1178	AD-9587
			AAAUGUTsT
3480-3498	AcAuuuAucuuuuGGGucuTsT	1179	AGACCcAAAAGAu	1180	AD-9713
			AAAUGUTsT
3480-3498	AfcAfuUfuAfuCfuUfuUfgG	1181	P*aGfaCfcCfaA	1182	AD-14679
	fgUfcUfTsT		faAfgAfuAfaAf
			uGfuTsT
3480-3498	ACfAUfUfUfAUfCfUfUfUfU	1183	AGACfCfCfAAAA	1184	AD-14689
	fGGGUfCfUfTsT		GAUfAAAUfGUfT
			sT
3480-3498	AcAuUuAuCuUuUgGgUcUTsT	1185	P*aGfaCfcCfaA	1186	AD-14699
			faAfgAfuAfaAf
			uGfuTsT
3480-3498	AcAuUuAuCuUuUgGgUcUTsT	1187	AGACfCfCfAAAA	1188	AD-14709
			GAUfAAAUfGUfT
			sT
3480-3498	AfcAfuUfuAfuCfuUfuUfgG	1189	AGACCcaAAagAU	1190	AD-14719
	fgUfcUfTsT		AAAuguTsT
3480-3498	ACfAUfUfUfAUfCfUfUfUfU	1191	AGACCcaAAagAU	1192	AD-14729
	fGGGUfCfUfTsT		AAAuguTsT
3480-3498	AcAuUuAuCuUuUgGgUcUTsT	1193	AGACCcaAAagAU	1194	AD-14739
			AAAuguTsT
3480-3498	GfcCfaUfcUfgCfuGfcCfgG	1195	P*gGfcUfcCfgG	1196	AD-15085
	faGfcCfTsT		fcAfgCfaGfaUf
			gGfcTsT
3480-3498	GCfCfAUfCfUfGCfUfGCfCf	1197	GGCfUfCfCfGGC	1198	AD-15095
	GGAGCfCfTsT		fAGCfAGAUfGGC
			fTsT
3480-3498	GcCaUcUgCuGcCgGaGcCTsT	1199	P*gGfcUfcCfgG	1200	AD-15105
			fcAfgCfaGfaUf
			gGfcTsT
3480-3498	GcCaUcUgCuGcCgGaGcCTsT	1201	GGCfUfCfCfGGC	1202	AD-15115
			fAGCfAGAUfGGC
			fTsT
3480-3498	GfcCfaUfcUfgCfuGfcCfgG	1203	GGCUCauGCagCA	1204	AD-15125
	faGfcCfTsT		GAUggcTsT
3480-3498	GCfCfAUfCfUfGCfUfGCfCf	1205	GGCUCauGCagCA	1206	AD-15135
	GGAGCfCfTsT		GAUggcTsT
3480-3498	GcCaUcUgCuGcCgGaGcCTsT	1207	GGCUCauGCagCA	1208	AD-15145
			GAUggcTsT
3481-3499	CAUUUAUCUUUUGGGUCUGTsT	1209	CAGACCCAAAAGA	1210	AD-9578
			UAAAUGTsT
3481-3499	cAuuuAucuuuuGGGucuGTsT	1211	cAGACCcAAAAGA	1212	AD-9704
			uAAAUGTsT
3485-3503	UAUCUUUUGGGUCUGUCCUTsT	1213	AGGACAGACCCAA	1214	AD-9558
			AAGAUATsT
3485-3503	uAucuuuuGGGucuGuccuTsT	1215	AGGAcAGACCcAA	1216	AD-9684
			AAGAuATsT
3504-3522	CUCUGUUGCCUUUUUACAGTsT	1217	CUGUAAAAAGGCA	1218	AD-9634
			ACAGAGTsT
3504-3522	cucuGuuGccuuuuuAcAGTsT	1219	CUGuAAAAAGGcA	1220	AD-9760
			AcAGAGTsT
3512-3530	CCUUUUUACAGCCAACUUUTT	1221	AAAGUUGGCUGUA	1222	AD-15411
			AAAAGGTT
3521-3539	AGCCAACUUUUCUAGACCUTT	1223	AGGUCUAGAAAAG	1224	AD-15266
			UUGGCUTT
3526-3544	ACUUUUCUAGACCUGUUUUTT	1225	AAAACAGGUCUAG	1226	AD-15382
			AAAAGUTT
3530-3548	UUCUAGACCUGUUUUGCUUTsT	1227	AAGCAAAACAGGU	1228	AD-9554
			CUAGAATsT
3530-3548	uucuAGAccuGuuuuGcuuTsT	1229	AAGcAAAAcAGGU	1230	AD-9680
			CuAGAATsT
3530-3548	UfuCfuAfgAfcCfuGfuUfuU	1231	P*aAfgCfaAfaA	1232	AD-14676
	fgCfuUfTsT		fcAfgGfuCfuAf
			gAfaTsT
3530-3548	UfUfCfUfAGACfCfUfGUfUf	1233	AAGCfAAAACfAG	1234	AD-14686
	UfUfGCfUfUfTsT		GUfCfUfAGAATs
			T
3530-3548	UuCuAgAcCuGuUuUgCuUTsT	1235	P*aAfgCfaAfaA	1236	AD-14696
			fcAfgGfuCfuAf
			gAfaTsT
3530-3548	UuCuAgAcCuGuUuUgCuUTsT	1237	AAGCfAAAACfAG	1238	AD-14706
			GUfCfUfAGAATs
			T
3530-3548	UfuCfuAfgAfcCfuGfuUfuU	1239	AAGcAaaACagGU	1240	AD-14716
	ffCfuUfTsT		CUAgaaTsT
3530-3548	UfUfCfUfAGACfCfUfGUfUf	1241	AAGcAaaACagGU	1242	AD-14726
	UfUfGCfUfUfTsT		CUAgaaTsT
3530-3548	UuCuAgAcCuGuUuUgCuUTsT	1243	AAGcAaaACagGU	1244	AD-14736
			CUAgaaTsT
3530-3548	CfaUfaGfgCfcUfgGfaGfuU	1245	P*aAfuAfaAfcU	1246	AD-15082
	fuAfuUfTsT		fcCfaGfgCfcUf
			aUfgTsT
3530-3548	CfAUfAGGCfCfUfGGAGUfUf	1247	AAUfAAACfUfCf	1248	AD-15092
	UfAUfUfTsT		CfAGGCfCfUfAU
			fGTsT
3530-3548	CaUaGgCcUgGaGuUuAuUTsT	1249	P*aAfuAfaAfcU	1250	AD-15102
			fcCfaGfgCfcUf
			aUfgTsT
3530-3548	CaUaGgCcUgGaGuUuAuUTsT	1251	AAUfAAACfUfCf	1252	AD-15112
			CfAGGCfCfUfAU
			fGTsT
3530-3548	CfaUfaGfgCfcUfgGfaGfuU	1253	AAUAAacUCcaGG	1254	AD-15122
	fuAfuUfTsT		CCUaugTsT
3530-3548	CfAUfAGGCfCfUfGGAGUfUf	1255	AAUAAacUCcaGG	1256	AD-15132
	UfAUfUfTsT		CCUaugTsT
3530-3548	CaUaGgCcUgGaGuUuAuUTsT	1257	AAUAAacUCcaGG	1258	AD-15142
			CCUaugTsT
3531-3549	UCUAGACCUGUUUUGCUUUTsT	1259	AAAGCAAAACAGG	1260	AD-9553
			UCUAGATsT
3531-3549	ucuAGAccuGuuuuGcuuuTsT	1261	AAAGcAAAAcAGG	1262	AD-9679
			UCuAGATsT
3531-3549	UfcUfaGfaCfcUfgUfuUfuG	1263	P*aAfaGfcAfaA	1264	AD-14675
	fcUfuUfTsT		faCfaGfgUfcUf
			aGfaTsT
3531-3549	UfCfUfAGACfCfUfGUfUfUf	1265	AAAGCfAAAACfA	1266	AD-14685
	UfGCfUfUfUfTsT		GGUfCfUfAGATs
			T
3531-3549	UcUaGaCcUgUuUuGcUuUTsT	1267	P*aAfaGfcAfaA	1268	AD-14695
			faCfaGfgUfcUf
			aGfaTsT
3531-3549	UcUaGaCcUgUuUuGcUuUTsT	1269	AAAGCfAAAACfA	1270	AD-14705
			GGUfCfUfAGATs
			T
3531-3549	UfcUfaGfaCfcUfgUfuUfuG	1271	AAAGCaaAAcaGG	1272	AD-14715
	fcUfuUfTsT		UCUagaTsT
3531-3549	UfCfUfAGACfCfUfGUfUfUf	1273	AAAGCaaAAcaGG	1274	AD-14725
	UfGCfUfUfUfTsT		UCUagaTsT
3531-3549	UcUaGaCcUgUuUuGcUuUTsT	1275	AAAGCaaAAcaGG	1276	AD-14735
			UCUagaTsT
3531-3549	UfcAfuAfgGfcCfuGfgAfgU	1277	P*aUfaAfaCfuC	1278	AD-15081
	fuUfaUfTsT		fcAfgGfcCfuAf
			uGfaTsT
3531-3549	UfCfAUfAGGCfCfUfGGAGUf	1279	AUfAAACfUfCfC	1280	AD-15091
	UfUfAUfTsT		fAGGCfCfUfAUf
			GATsT
3531-3549	UcAuAgGcCuGgAgUuUaUTsT	1281	P*aUfaAfaCfuC	1282	AD-15101
			fcAfgGfcCfuAf
			uGfaTsT
3531-3549	UcAuAgGcCuGgAgUuUaUTsT	1283	AUfAAACfUfCfC	1284	AD-15111
			fAGGCfCfUfAUf
			GATsT
3531-3549	UfcAfuAfgGfcCfuGfgAfgU	1285	AUAAAcuCCagGC	1286	AD-15121
	fuUfaUfTsT		CUAugaTsT
3531-3549	UfCfAUfAGGCfCfUfGGAGUf	1287	AUAAAcuCCagGC	1288	AD-15131
	UfUfAUfTsT		CUAugaTsT
3531-3549	UcAuAgGcCuGgAgUuUaUTsT	1289	AUAAAcuCCagGC	1290	AD-15141
			CUAugaTsT
3557-3575	UGAAGAUAUUUAUUCUGGGTsT	1291	CCCAGAAUAAAUA	1292	AD-9626
			UCUUCATsT
3557-3575	uGAAGAuAuuuAuucuGGGTsT	1293	CCcAGAAuAAAuA	1294	AD-9752
			UCUUcATsT
3570-3588	UCUGGGUUUUGUAGCAUUUTsT	1295	AAAUGCUACAAAA	1296	AD-9629
			CCCAGATsT
3570-3588	ucuGGGuuuuGuAGcAuuuTsT	1297	AAAUGCuAcAAAA	1298	AD-9755
			CCcAGATsT
3613-3631	AUAAAAACAAACAAACGUUTT	1299	AACGUUUGUUUGU	1300	AD-15412
			UUUUAUTT
3617-3635	AAACAAACAAACGUUGUCCTT	1301	GGACAACGUUUGU	1302	AD-15211
			UUGUUUTT
3618-3636	AACAAACAAACGUUGUCCUTT	1303	AGGACAACGUUUG	1304	AD-15300
			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 phosphorodiester 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

	Sense strand sequence	SEQ ID	Antisense-strand	SEQ ID
Duplex #	(5′-3′)1	NO:	sequence (5′-3′)1	NO:
AD-10792	GccuGGAGuuuAuucGGAATsT	1305	UUCCGAAuAAACUCcAGGCTsT	1306
AD-10793	GccuGGAGuuuAuucGGAATsT	1307	uUcCGAAuAAACUccAGGCTsT	1308
AD-10796	GccuGGAGuuuAuucGGAATsT	1309	UUCCGAAUAAACUCCAGGCTsT	1310
AD-12038	GccuGGAGuuuAuucGGAATsT	1311	UUCCGAAUAAACUCCAGGCTsT	1312
AD-12039	GccuGGAGuuuAuucGGAATsT	1313	UUCCGAAUAAACUCCAGGCTsT	1314
AD-12040	GccuGGAGuuuAuucGGAATsT	1315	UUcCGAAUAAACUCCAGGCTsT	1316
AD-12041	GccuGGAGuuuAuucGGAATsT	1317	UUCCGAAUAAACUCCAGGCTsT	1318
AD-12042	GCCUGGAGUUUAUUCGGAATsT	1319	UUCCGAAUAAACUCCAGGCTsT	1320
AD-12043	GCCUGGAGUUUAUUCGGAATsT	1321	UUCCGAAUAAACUCCAGGCTsT	1322
AD-12044	GCCUGGAGUUUAUUCGGAATsT	1323	UUCCGAAUAAACUCCAGGCTsT	1324
AD-12045	GCCUGGAGUUUAUUCGGAATsT	1325	UUCCGAAUAAACUCCAGGCTsT	1326
AD-12046	GccuGGAGuuuAuucGGAA	1327	UUCCGAAUAAACUCCAGGCscsu	1328
AD-12047	GccuGGAGuuuAuucGGAAA	1329	UUUCCGAAUAAACUCCAGGCscsu	1330
AD-12048	GccuGGAGuuuAuuGGAAAA	1331	UUUUCCGAAUAAACUCCAGGCscsu	1332
AD-12049	GccuGGAGuuuAuucGGAAAAG	1333	CUUUUCCGAAUAAACUCCAGGCscsu	1334
AD-12050	GccuGGAGuuuAuucGGAATTab	1335	UUCCGAAUAAACUCCAGGCTTab	1336
AD-12051	GccuGGAGuuuAuucGGAAATTab	1337	UUUCCGAAUAAACUCCAGGCTTab	1338
AD-12052	GccuGGAGuuuAuucGGAAAATTab	1339	UUUUCCGAAUAAACUCCAGGCTTab	1340
AD-12053	GccuGGAGuuuAuucGGAAAAGTTab	1341	CUUUUCCGAAUAAACUCCAGGCTTab	1342
AD-12054	GCCUGGAGUUUAUUCGGAATsT	1343	UUCCGAAUAAACUCCAGGCscsu	1344
AD-12055	GccuGGAGuuuAuucGGAATsT	1345	UUCCGAAUAAACUCCAGGCscsu	1346
AD-12056	GcCuGgAgUuUaUuCgGaA	1347	UUCCGAAUAAACUCCAGGCTTab	1348
AD-12057	GcCuGgAgUuUaUuCgGaA	1349	UUCCGAAUAAACUCCAGGCTsT	1350
AD-12058	GcCuGgAgUuUaUuCgGaA	1351	UUCCGAAUAAACUCCAGGCTsT	1352
AD-12059	GcCuGgAgUuUaUuCgGaA	1353	uUcCGAAuAAACUccAGGCTsT	1354
AD-12060	GcCuGgAgUuUaUuCgGaA	1355	UUCCGaaUAaaCUCCAggc	1356
AD-12061	GcCuGgnAgUuUaUuCgGaATsT	1357	UUCCGaaUAaaCUCCAggcTsT	1358
AD-12062	GcCuGgAgUuUaUuCgGaATTab	1359	UUCCGaaUAaaCUCCAggcTTab	1360
AD-12063	GcCuGgAgUuUaUuCgGaA	1361	UUCCGaaUAaaCUCCAggcscsu	1362
AD-12064	GcCuGgnAgUuUaUuCgGaATsT	1363	UUCCGAAuAAACUCcAGGCTsT	1364
AD-12065	GcCuGgAgUuUaUuCgGaATTab	1365	UUCCGAAuAAACUCcAGGCTTab	1366
AD-12066	GcCuGgAgUuUaUuCgGaA	1367	UUCCGAAuAAACUCcAGGCscsu	1368
AD-12067	GcCuGgnAgUuUaUuCgGaATsT	1369	UUCCGAAUAAACUCCAGGCTsT	1370
AD-12068	GcCuGgAgUuUaUuCgGaATTab	1371	UUCCGAAUAAACUCCAGGCTTab	1372
AD-12069	GcCuGgAgUuUaUuCgGaA	1373	UUCCGAAUAAACUCCAGGCScsu	1374
AD-12338	GfcCfuGfgAfgUfuUfaUfuCfgGfaAf	1375	P*uUfcCfgAfaUfaAfaCf	1376
			uCfcAfgGfc
AD-12339	GcCuGgAgUuUaUuCgGaA	1377	P*uUfcCfgAfaUfaAfaCf	1378
			uCfcAfgGfc
AD-12340	GccuGGAGuuuAuucGGAA	1379	P*uUfcCfgAfaUfaAfaCf	1380
			uCfcAfgGfc
AD-12341	GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT	1381	P*uUfcCfgAfaUfaAfaCf	1382
			uCfcAfgGfcTsT
AD-12342	GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT	1383	UUCCGAAuAAACUCcAGGCTsT	1384
AD-12343	GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT	1385	uUcCGAAuAAACUccAGGCTsT	1386
AD-12344	GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT	1387	UUCCGAAUAAACUCCAGGCTsT	1388
AD-12345	GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT	1389	UUCCGAAUAAACUCCAGGCScsu	1390
AD-12346	GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT	1391	UUCCGaaUAaaCUCCAggcscsu	1392
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	GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTTab	1399	P*uUfcCfgAfaUfaAfaCf	1400
			uCfcAfgGfcTTab
AD-12351	GfcCfuGfgAfgUfuUfaUfuCfgGfaAf	1401	P*uUfcCfgAfaUfaAfaCf	1402
			uCfcAfgGfcsCfsu
AD-12352	GfcCfuGfgAfgUfuUfaUfuCfgGfaAf	1403	UUCCGaaUAaaCUCCAggcs	1404
			csu
AD-12354	GfcCfuGfgAfgUfuUfaUfuCfgGfaAf	1405	UUCCGAAUAAACUCCAGGCs	1406
			csu
AD-12355	GfcCfuGfgAfgUfuUfaUfuCfgGfaAf	1407	UUCCGAAUAAACUCCAGGCT	1408
			sT
AD-12356	GfcCfuGfgAfgUfuUfaUfuCfgGfaAf	1409	uUcCGAAuAAACUccAGGCT	1410
			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	GfcCfuGfgAfgUfuUfaUfuCfgGfaAf	1467	P*UfUfCfCfGAAUfAAACf	1468
			UfCfCfAGGCfTsT
AD-12387	GCfCfUfGGAGGUfUfUfAUfUfCfGGAA	1469	UfUfCfCfGAAUfAAACfUf	1470
			CfCfAGGCfsCfsUf
AD-12388	GCfCfUfGGAGGUfUfUfAUfUfCfGGAA	1471	P*uUfcCfgAfaUfaAfaCf	1472
			uCfcAfgGfc
AD-12389	GCfCfUfGGAGGUfUfUfAUfUfCfGGAA	1473	P*uUfcCfgAfaUfaAfaCf	1474
			uCfcAfgGfcsCfsu
AD-12390	GCfCfUfGGAGGUfUfUfAUfUfCfGGAA	1475	UUCCGAAUAAACUCCAGGCS	1476
			csu
AD-12391	GCfCfUfGGAGGUfUfUfAUfUfCfGGAA	1477	UUCCGaaUAaaCUCCAggc	1478
AD-12392	GCfCfUfGGAGGUfUfUfAUfUfCfGGAA	1479	UUCCGAAUAAACUCCAGGCT	1480
			sT
AD-12393	GCfCfUfGGAGGUfUfUfAUfUfCfGGAA	1481	UUCCGAAuAAACUCcAGGCT	1482
			sT
AD-12394	GCfCfUfGGAGGUfUfUfAUfUfCfGGAA	1483	uUcCGAAuAAACUccAGGCT	1484
			sT
AD-12395	GmocCmouGmogAmogUmouUmoaUmou	1485	P*UfUfCfCfGAAUfAAACf	1486
	CmogGmoaATsT		UfCfCfAGGCfsCfsUf
AD-12396	GmocCmouGmogAm02gUmouUmoaUmo	1487	P*UfUfCfCfGAAUfAAACf	1488
	uCmogGmoaA		UfCfCfAGGCfsCfsUf
AD-12397	GfcCfuGfgAfgUfuUfaUfuCfgGfaAf	1489	P*UfUfCfCfGAAUfAAACf	1490
			UfCfCfAGGCfsCfsUf
AD-12398	GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT	1491	P*UfUfCfCfGAAUfAAACf	1492
			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	GCfCfUfGGAGGUfUfUfAUfUfCfGGAA	1501	P*UfUfCfCfGAAUfAAACf	1502
			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
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 inteijected “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 6
dsRNA targeted to PCSK9: mismatches and modifications

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	uucuAGAccYIGuuuuGcuuTsT
	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	GccuGGAGuYIuAuucGGAATsT
	AS	1574	UUCCGAAuAAACUCcAGGCTsT
AD-3255	S	1575	GccuGGAGUY1uAuucGGAATsT
	AS	1576	UUCCGAAuAAACUCcAGGCTsT
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′ 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′-phospltate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide

TABLE 7
Sequences of unmodified siRNA flanking AD-9680

				SEQ
Duplex #	Strand	Sequence (5′ to 3′)	*Target	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	UAAAUAUCUUCAAGUUACAdTsdT	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
*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.

TABLE 8
Sequences of modified siRNA flanking AD-9680

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
*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 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 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	AAACGANUUAGUUCAUUAA
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
*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.

Duplex	Target gene and location
#	of target sequence
D18027	NM_001004210_1128-1146
D18028	NM_001004210_1129-1147
D18029	NM_001004210_677-695
D18030	NM_001004210_893-911
D18031	NM_001004210_895-913
D18032	NM_001004210_1127-1145
D18033	NM_001004210_894-912
D18034	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

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
Target*	sense (5′-3′)	NO	antisense (5′-3′)	ID 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