US20180148722A1

US20180148722A1 - Dual Targeting siRNA Agents

Info

Publication number: US20180148722A1
Application number: US15/724,175
Authority: US
Inventors: Kevin Fitzgerald; Maria Frank-Kamenetsky; Klaus Charisse
Original assignee: Alnylam Pharmaceuticals Inc
Current assignee: Alnylam Pharmaceuticals Inc
Priority date: 2009-09-22
Filing date: 2017-10-03
Publication date: 2018-05-31
Also published as: US20130184324A1; US9187746B2; US20200332292A1; US20160312216A1; US20220213475A1; WO2011038031A1

Abstract

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

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 14/885,342, filed Oct. 16, 2015, (pending) 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 38757_US_sequencelisting.txt, created on Oct. 3, 2017, 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/XXXXXX. This application and the siRNA sequences described therein are incorporated by reference for all purposes.
As used herein, the term “iRNA” refers to an agent that contains RNA and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. The term iRNA includes siRNA.
As described in more detail below, the term “siRNA” and “siRNA agent” refers to a dsRNA that mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway.
A “double-stranded RNA” or “dsRNA,” as used herein, refers to an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having “sense” and “antisense” orientations with respect to a target RNA.
The term “dual targeting siRNA agent” refers to a composition of two siRNAs, e.g., two dsRNAs. One dsRNA includes an antisense strand with a first region of complementarity to a first target gene, e.g., PCSK9. The second dsRNA include an antisense strand with a second region of complementarity to a second target gene. In some embodiments, the first and second target genes are identical, e.g., both are PCSK9 and each dsRNA targets a different region of PCSK9. In other embodiments, the first and second target genes are different, e.g., the first dsRNA targets PCSK9 and the second dsRNA targets a different gene, e.g., XBP-1.
“Covalent linker” refers to a molecule for covalently joining two molecules, e.g., two dsRNAs. As described in more detail below, the term includes, e.g., a nucleic acid linker, a peptide linker, and the like and includes disulfide linkers.
The term “target gene” refers to a gene of interest, e.g., PCSK9 or a second gene, e.g., XBP-1, targeted by an siRNA of the invention for inhibition of expression.
As described in more detail below, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all sub-ranges therebetween.
As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 5, 4, 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
$\frac{(mRNA in control cells) - (mRNA in treated cells)}{(mRNA in control cells)} \cdot 100 %$
Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to target gene expression, e.g., the amount of protein encoded by a target gene, or the number of cells displaying a certain phenotype, e.g., lack of or decreased cytokine production. In principle, target gene silencing may be determined in any cell expressing target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given iRNA inhibits the expression of the target gene by a certain degree and therefore is encompassed by the instant invention, the assays provided in the Examples below shall serve as such reference.
For example, in certain instances, expression of a target gene is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% by administration of an iRNA featured in the invention. In some embodiments, a target gene is suppressed by at least about 60%, 65%, 70%, 75%, or 80% by administration of an iRNA featured in the invention. In some embodiments, a target gene is suppressed by at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more by administration of an iRNA as described herein.
As used herein in the context of target gene expression, the terms “treat,” “treatment,” and the like, refer to relief from or alleviation of pathological processes mediated by target expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by target expression), the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression or anticipated progression of such condition.
By “lower” in the context of a disease marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without such disorder.
As used herein, the phrase “therapeutically effective amount” “refers to an amount that provides a therapeutic benefit in the treatment or management of pathological processes mediated by target gene expression, e.g., PCSK9 and/or a second gene, e.g., XBP-1, or an overt symptom of pathological processes mediated target gene expression. The phrase “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the prevention of pathological processes mediated by target gene expression or an overt symptom of pathological processes mediated by target gene expression. The specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by target gene expression, the patient's history and age, the stage of pathological processes mediated by target gene expression, and the administration of other agents that inhibit pathological processes mediated by target gene expression.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of an iRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an iRNA effective to produce the intended pharmacological or therapeutic result. For example, if a given clinical treatment is considered effective when there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 10% reduction in that parameter.
The term “pharmaceutically carrier” refers to a carrier for administration of a therapeutic agent, e.g., a dual targeting siRNA agent. Carriers are described in more detail below, and include lipid formulations, e.g., LNP09 and SNALP formulations.
Double-Stranded Ribonucleic Acid (dsRNA)
Described herein are dual targeting siRNA agents, e.g., siRNAs that inhibit the expression of a PCSK9 gene and a second gene. The dual targeting siRNA agent includes two siRNA covalently linked via, e.g., a disulfide linker. The first siRNA targets a first region of a PCSK9 gene. The second siRNA targets a second gene, e.g., XBP-1, or, e.g., targets a second region of the PCSK9 gene.
The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Applied Biosystems, Inc. Further descriptions of synthesis are found below and in the examples.
Each siRNA is a dsRNA. A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of a target gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions.
Where the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a “hairpin loop”) between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.
Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a “linker.”
Generally, the duplex structure of the siRNA, e.g., dsRNA, is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Considering a duplex between 9 and 36 base pairs, the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs.
The two siRNAs in the dual targeting siRNA agent can have duplex lengths that are identical or that differ.
The region of complementarity to the target sequence in an siRNA is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive. In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. The region of complementarity can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. As non-limiting examples, the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides. In some embodiments the target sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
The two siRNAs in the dual targeting siRNA agent can have regions of complementarity that are identical in length or that differ in length.
Any of the dsRNA, e.g., siRNA as described herein may include one or more single-stranded nucleotide overhangs. In one embodiment, at least one end of a dsRNA has a single-stranded nucleotide overhang of 1 to 4, or 1 or 2 or 3 or 4 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA can also have a blunt end, generally located at the 5′-end of the antisense strand. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. The two siRNAs in the dual targeting siRNA agent can have different or identical overhangs as described by location, length, and nucleotide.
The dual targeting siRNA agent includes at least a first siRNA targeting a first region of a PCSK9 gene. In one embodiment, a PCSK9 gene is a human PCSK9 gene. In another embodiment the PCSK9 gene is a mouse or a rat PCSK9 gene. Exemplary siRNA targeting PCSK9 are described in U.S. patent application Ser. No. 11/746,864 filed on May 10, 2007 (now U.S. Pat. No. 7,605,251) and International Patent Application No. PCT/US2007/068655 filed May 10, 2007 (published as WO 2007/134161). Additional disclosure can be found in U.S. patent application Ser. No. 12/478,452 filed Jun. 4, 2009 (published as US 2010/0010066) and International Patent Application No. PCT/US2009/032743 filed Jan. 30, 2009 (published as WO 2009/134487). The sequences of the target, sense, and antisense strands are incorporated by reference for all purposes.
Tables 1, 2, and 4-8 disclose sequences of the target, sense strands, and antisense strands of PCSK9 targeting siRNA.
In one embodiment the first siRNA is AD-9680. The dsRNA AD-9680 targets the human PCSK 9 gene at nucleotides 3530-3548 of a human PCSK9 gene (accession number NM_174936).

TABLE 1

AD-9680 siRNA sequences

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

		SEQ
Table 3: AD-18038	Sequence 5′ to 3′	ID NO:

Target sequence	CACCCUGAAUUCAUUGUCU	4153

Sense strand	CACCCUGAAUUCAUUGUCU	4154

Sense strand,	cAcccuGAAuucAuuGucudTsdT	4155
modified

Antisense strand	AGACAAUGAAUUCAGGGUG	4156

Antisense strand,	AGAcAAUGAAUUcAGGGUGdTsdT	4157
modified

Additional dsRNA
A dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences in Tables 1-13, and differing in their ability to inhibit the expression of a target gene by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated according to the invention.
In addition, the RNAs provided in Tables 1-13 identify a site in the target gene transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of such sequences. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least 15 contiguous nucleotides from one of the sequences provided herein coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a target gene.
While a target sequence is generally 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that may serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, above represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.
Further, it is contemplated that for any sequence identified, e.g., in Tables 1-13, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those and sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.
An iRNA as described in Tables 1-13 can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described in Tables 1-13 contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide iRNA agent RNA strand which is complementary to a region of a PCSK9 gene, the RNA strand generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a PCSK9 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of a PCSK9 gene is important, especially if the particular region of complementarity in a PCSK9 gene is known to have polymorphic sequence variation within the population.
Covalent Linkage
The dual targeting siRNA agents of the invention include two siRNAs joined via a covalent linker. Covalent linkers are well-known to one of skill in the art and include, e.g., a nucleic acid linker, a peptide linker, and the like.
The covalent linker joins the two siRNAs. The covalent linker can join two sense strands, two antisense strands, one sense and one antisense strand, two sense strands and one antisense strand, two antisense strands and one sense strand, or two sense and two antisense strands.
The covalent linker can include RNA and/or DNA and/or a peptide. The linker can be single stranded, double stranded, partially single strands, or partially double stranded. In some embodiments the linker includes a disulfide bond. The linker can be cleavable or non-cleavable.
The covalent linker can be, e.g., dTsdTuu=(5′-2′deoxythymidy 1-3′-thiophosphate-5′-2′deoxythymidy 1-3′-phosphate-5′-uridy 1-3′-phosphate-5′-uridy 1-3′-phosphate); rUsrU (a thiophosphate linker: 5′-uridy 1-3′-thiophosphate-5′-uridy 1-3′-phosphate); an rUrU linker; dTsdTaa (aadTsdT, 5′-2′deoxythymidy 1-3′-thiophosphate-5′-2′deoxythymidy 1-3′-phosphate-5′-adeny 1-3′-phosphate-5′-adeny 1-3′-phosphate); dTsdT (5′-2′deoxythyrnidy 1-3′-thiophosphate-5′-2′ deoxythymidy 1-3′-phosphate); dTsdTuu=uudTsdT=5′-2′deoxythymidy 1-3′-thiophosphate-5′-2′deoxythymidy 1-3′-phosphate-5′-uridy 1-3′-phosphate-5′-uridy 1-3′-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_nsingle 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′deoxythymidy 1-3′-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. No. RE39,464, 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 CH₂component parts.
Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, 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 C₁to C₁₀alkyl or C₂to C₁₀alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_nO]_mCH₃, O(CH₂)._nOCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_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: C₁to C₁₀lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of 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)₂group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, 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. No. 5,252,479; U.S. Pat. No. 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 C_1-20alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.
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 GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).
Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C_1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. 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)ethylazanediyl)didodecan-2-ol (Tech G1, e.g., C12-200), or a mixture thereof.
The cationic lipid may comprise from about 10 mol % to about 70 mol % or about 40 mol % of the total lipid present in the particle. The cationic lipid may comprise 10 mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol %, 70 mol %, 75 mol %, 80 mol %, 85 mol %, 90 mol %, or 95 mol % of the total lipid present in the particle. The cationic lipid may comprise 57.1 mol % or 57.5 mol % of the total lipid present in the particle.
In one embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (XTC) can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.
In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.
The nucleic acid lipid particle generally includes a non-cationic lipid. The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof.
The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle. The non-cationic lipid may be about 5 mol %, 6 mol %, 7 mol %, 7.5 mol %, 7.7 mol %, 8 mol %, 9 mol %, 10 mol %, 11 mol %, 12 mol %, 13 mol %, 14 mol %, 15 mol %, 16 mol %, 17 mol %, 18 mol %, 19 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol %, 70 mol %, 75 mol %, 80 mol %, 85 mol %, 90 mol %, or 95 mol %.
The nucleic acid lipid particle generally includes a conjugated lipid. The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG-distearyloxypropyl (C]₈). The conjugated lipid can be PEG-DMG (PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000); PEG-DSG (PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000); or PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000).
The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0 17.0, 18, 19.0 or 20.0 mol % of the total lipid present in the particle.
In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle. For example, the nucleic acid-lipid particle further includes cholesterol at about 5 mol %, 10 mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol %. The nucleic acid-lipid particle can include cholesterol at about 31.5 mol %, 34.4 mol %, 35 mol %, 38.5 mol %, or 40 mol % of the total lipid present in the particle.
LNP01
In one embodiment, the lipidoid ND98⋅4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is herein incorporated by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.
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 (l,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 Serial No. 61/239,686, filed Sep. 3, 2009, and International patent application no. PCT/US10/22614, filed Jan. 29, 2010, which are hereby incorporated by reference.
MC3 comprising formulations are described, e.g., in U.S. Provisional Serial No. 61/244,834, filed Sep. 22, 2009, and U.S. Provisional Serial 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 Serial 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 R₁and R₂are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R₃and R₄are independently lower alkyl or R₃and R₄can 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 (1×50 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 oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.
Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
Penetration Enhancers
In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
Surfactants:
In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
Fatty Acids:
Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C_1-20alkyl 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 (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine2000™ (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), TransPass^aD1 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 IIb/IIIa inhibitor, aspirin or an aspirin-like compound, an IBAT inhibitor (e.g., S-8921, from Shionogi), a squalene synthase inhibitor, or a monocyte chemoattractant protein (MCP)-I inhibitor. Exemplary HMG-CoA reductase inhibitors include atorvastatin (Pfizer's Lipitor®/Tahor/Sortis/Torvast/Cardyl), pravastatin (Bristol-Myers Squibb's Pravachol, Sankyo's Mevalotin/Sanaprav), simvastatin (Merck's Zocor®/Sinvacor, Boehringer Ingelheim's Denan, Banyu's Lipovas), lovastatin (Merck's Mevacor/Mevinacor, Bexal's Lovastatina, Cepa; Schwarz Pharma's Liposcler), fluvastatin (Novartis' Lescol®/Locol/Lochol, Fujisawa's Cranoc, Solvay's Digaril), cerivastatin (Bayer's Lipobay/GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca's Crestor®), and pitivastatin (itavastatin/risivastatin) (Nissan Chemical, Kowa Kogyo, Sankyo, and Novartis). Exemplary fibrates include, e.g., bezafibrate (e.g., Roche's Befizal®/Cedur®/Bezalip®, Kissei's Bezatol), clofibrate (e.g., Wyeth's Atromid-S®), fenofibrate (e.g., Fournier's Lipidil/Lipantil, Abbott's Tricor®, Takeda's Lipantil, generics), gemfibrozil (e.g., Pfizer's Lopid/Lipur) and ciprofibrate (Sanofi-Synthelabo's Modalim®). Exemplary bile acid sequestrants include, e.g., cholestyramine (Bristol-Myers Squibb's Questran® and Questran Light™), colestipol (e.g., Pharmacia's Colestid), and colesevelam (Genzyme/Sankyo's WelChol™). Exemplary niacin therapies include, e.g., immediate release formulations, such as Aventis' Nicobid, Upsher-Smith's Niacor, Aventis' Nicolar, and Sanwakagaku's Perycit. Niacin extended release formulations include, e.g., Kos Pharmaceuticals' Niaspan and Upsher-Smith's SIo-Niacin. Exemplary antiplatelet agents include, e.g., aspirin (e.g., Bayer's aspirin), clopidogrel (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{acute over (ε)}rieux Pierre Fabre/Eli Lilly), CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito). Exemplary cholesterol absorption inhibitors include, e.g., ezetimibe (Merck/Schering-Plough Pharmaceuticals Zetia®) and Pamaqueside (Pfizer). Exemplary CETP inhibitors include, e.g., Torcetrapib (also called CP-529414, Pfizer), JTT-705 (Japan Tobacco), and CETi-I (Avant Immunotherapeutics). Exemplary microsomal triglyceride transfer protein (MTTP) inhibitors include, e.g., implitapide (Bayer), R-103757 (Janssen), and CP-346086 (Pfizer). Other exemplary cholesterol modulators include, e.g., NO-1886 (Otsuka/TAP Pharmaceutical), CI-1027 (Pfizer), and WAY-135433 (Wyeth-Ayerst). Exemplary bile acid modulators include, e.g., HBS-107 (Hisamitsu/Banyu), Btg-511 (British Technology Group), BARI-1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), and AZD-7806 (AstraZeneca). Exemplary peroxisome proliferation activated receptor (PPAR) agonists include, e.g., tesaglitazar (AZ-242) (AstraZeneca), Netoglitazone (MCC-555) (Mitsubishi/Johnson & Johnson), GW-409544 (Ligand 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 IIb/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{acute over (Å)}, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5′-O-dimethoxytrityl N₆-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N₄-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N₂-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, and 5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) were used for the oligonucleotide synthesis. The 2′-F phosphoramidites, 5′-O-dimethoxytrityl-N4-acetyl-2′-fluro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite and 5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite are purchased from (Promega). All phosphoramidites are used at a concentration of 0.2M in acetonitrile (CH₃CN) except for guanosine which is used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of 16 minutes is used. The activator is 5-ethyl thiotetrazole (0.75M, American International Chemicals); for the PO-oxidation iodine/water/pyridine is used and for the PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) is used.
3′-ligand conjugated strands are synthesized using solid support containing the corresponding ligand. For example, the introduction of cholesterol unit in the sequence is performed from a hydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered to trans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain a hydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore) labeled iRNAs are synthesized from the corresponding Quasar-570 (Cy-3) phosphoramidite are purchased from Biosearch Technologies. Conjugation of ligands to 5′-end and or internal position is achieved by using appropriately protected ligand-phosphoramidite building block. An extended 15 min coupling of 0.1 M solution of phosphoramidite in anhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activator to a solid-support-bound oligonucleotide. Oxidation of the internucleotide phosphite to the phosphate is carried out using standard iodine-water as reported (1) or by treatment with tert-butyl hydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation wait time conjugated oligonucleotide. Phosphorothioate is introduced by the oxidation of phosphite to phosphorothioate by using a sulfur transfer reagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucage reagent. The cholesterol phosphoramidite is synthesized in house and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite is 16 minutes.
Deprotection I (Nucleobase Deprotection)
After completion of synthesis, the support is transferred to a 100 mL glass bottle (VWR). The oligonucleotide is cleaved from the support with simultaneous deprotection of base and phosphate groups with 80 mL of a mixture of ethanolic ammonia [ammonia: ethanol (3:1)] for 6.5 h at 55° C. The bottle is cooled briefly on ice and then the ethanolic ammonia mixture is filtered into a new 250-mL bottle. The CPG is washed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixture is then reduced to ˜30 mL by roto-vap. The mixture is then frozen on dry ice and dried under vacuum on a speed vac.
Deprotection II (Removal of 2′-TBDMS Group)
The dried residue is resuspended in 26 mL of triethylamine, triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60° C. for 90 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reaction is then quenched with 50 mL of 20 mM sodium acetate and the pH is adjusted to 6.5. Oligonucleotide is stored in a freezer until purification.
Analysis
The oligonucleotides are analyzed by high-performance liquid chromatography (HPLC) prior to purification and selection of buffer and column depends on nature of the sequence and or conjugated ligand.
HPLC Purification
The ligand-conjugated oligonucleotides are purified by reverse-phase preparative HPLC. The unconjugated oligonucleotides are purified by anion-exchange HPLC on a TSK gel column packed in house. The buffers are 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractions containing full-length oligonucleotides are pooled, desalted, and lyophilized. Approximately 0.15 OD of desalted 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):

GccuGGAGuuuAuucGGAAdTsdTQ51cAcccuGAAuucAuuGucudTs

dTdTsdTCGGAcCUCAAAuAAGCCUU dTsdTGUGGGAcUUAAGUAAc

AGA

Example 5. Inhibition of PCSK9 and Xbp-1 mRNA Levels by the PCSK9-Xbp1 Dual Targeting siRNA in Primary Mouse Hepatocytes

Primary mouse hepatocytes were transfected with dual targeting AD-23426 or individual siRNAs (AD-10792 and AD-18038) in lipofectamine 2000 (Invitrogen protocol). 48 hours after transfection cells were harvested and lysed. PCSK9, Xbp-1 and GAPDH transcripts were measured via bDNA in cell lysates prepared according to manufacturer's protocol. PCSK9 to GAPDH or Xbp-1 to GAPDH ratios were normalized to control (luciferase) and graphed.
As shown in FIG. 1, the dual targeting siRNA was at least as effective at inhibiting their corresponding target gene as the single siRNAs.

Example 6. Inhibition of PCSK9 and Xbp-1 mRNA Levels and Reduction of Total Serum Cholesterol by the PCSK9-Xbp1 Dual Targeting siRNA in Mice

The dual targeting AD-23426 was formulated in an LNP09 formulation: XTC/DSPC/Cholesterol/PEG-DMG in a % mol ratio of 50/10/38.5/1.5 with a lipid:siRNA ratio of about 10:1. The LNP09-AD-23426 was administered by tail vein injection into C57B6 mice at 6.0 mg/kg, 2.0 mg/kg and 0.6 mg/kg. LNP09 formulated single siRNAs (AD-10792 and AD-18038) were administered each at 3.0 mg/kg, 1.0 mg/kg and 0.3 mg/kg. Livers and plasma were harvested 72 hours post-injection (5 animals per group).
PCSK9, Xbp-1 and GAPDH transcript levels were measured via bDNA in livers prepared according to the manufacturer's protocol. PCSK9 to GAPDH or Xbp-1 to GAPDH ratios were normalized to control (luciferase) and graphed. The results are shown in FIG. 2.
Total cholesterol was measure in serum according to manufacturer's instructions using a cholesterol kit from WAKO Tex.
The results demonstrate that the dual targeting siRNAs were at least as effective at inhibiting their corresponding target as single siRNAs in vivo. The results also show that the dual targeting construct has an additive effect compared to the single siRNAs at reducing total serum cholesterol.

Example 7: No Induction of IFN-α and TNF-α in HuPBMC

The effect of a dual targeting siRNA, AD-23426, on IFN-α and TNF-α in human PBMC was investigated.
Whole Blood anti-coagulated with Sodium Heparin was obtained from healthy donors at Research Blood Components, Inc (Boston, Mass.). Peripheral blood mononuclear cells (PBMC) were isolated by standard Ficoll-Hypaque density centrifugation. Isolated PBMC were seeded at 1×10⁵cells/well in 96 well plates and cultured in RPMI 1640 GlutaMax Medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic/antimycotic (Invitrogen). siRNAs were transfected using DOTAP Transfection Reagent (Roche Applied Science). DOTAP was first diluted in Opti-MEM (Invitrogen) for 5 minutes before mixing with an equal volume of Opti-MEM containing the siRNA. siRNA/transfection reagent complexes were incubated for 15 minutes at room temperature prior to being added to PBMC. siRNAs were transfected at final concentrations of 266 nM, 133 nM or 67 nM using 16 μg/ml, 8 μg/ml or 4 μg/ml DOTAP, respectively. The ratio of siRNA to DOTAP is 16.5 pmol/μg. Transfected PBMC were incubated at 37° C., 5% CO₂for 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-a (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-Xbnl Dual Tart Etine siRNA Humans

A human subject is treated with a pharmaceutical composition, e.g., a nucleic acid-lipid particle having a dual targeting siRNA agent.
At time zero, a suitable first dose of the pharmaceutical composition is subcutaneously administered to the subject. The composition is formulated as described herein. After a period of time, the subject's condition is evaluated, e.g., by measurement of total serum cholesterol. This measurement can be accompanied by a measurement of PCSK9 expression in said subject, and/or the products of the successful siRNA-targeting of PCSK9 mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
After treatment, the subject's condition is compared to the condition existing prior to the treatment, or relative to the condition of a similarly afflicted but untreated subject.
Those skilled in the art are familiar with methods and compositions in addition to those specifically set out in the present disclosure which will allow them to practice this invention to the full scope of the claims hereinafter appended.

TABLE 4

Sequences of siRNA targeted to PCSK9

		SEQ	Antisense	SEQ
	Sense strand	ID	strand	ID
*Target	(5′-3′)¹	NO:	(5′-3′)¹	NO:	Duplex #

2-20	AGCGACGUCGAGGCGCUCATT	1	UGAGCGCCUCGAC	2	AD-15220
			GUCGCUTT

15-33	CGCUCAUGGUUGCAGGCGGTT	3	CCGCCUGCAACCA	4	AD-15275
			UGAGCGTT

16-34	GCUCAUGGUUGCAGGCGGGTT	5	CCCGCCUGCAACC	6	AD-15301
			AUGAGCTT

30-48	GCGGGCGCCGCCGUUCAGUTT	7	ACUGAACGGCGGC	8	AD-15276
			GCCCGCTT

31-49	CGGGCGCCGCCGUUCAGUUTT	9	AACUGAACGGCGG	10	AD-15302
			CGCCCGTT

32-50	GGGCGCCGCCGUUCAGUUCTT	11	GAACUGAACGGCG	12	AD-15303
			GCGCCCTT

40-58	CCGUUCAGUUCAGGGUCUGTT	13	CAGACCCUGAACU	14	AD-15221
			GAACGGTT

43-61	UUCAGUUCAGGGUCUGAGCTT	15	GCUCAGACCCUGA	16	AD-15413
			ACUGAATT

82-100	GUGAGACUGGCUCGGGCGGTT	17	CCGCCCGAGCCAG	18	AD-15304
			UCUCACTT

100-118	GGCCGGGACGCGUCGUUGCTT	19	GCAACGACGCGUC	20	AD-15305
			CCGGCCTT

101-119	GCCGGGACGCGUCGUUGCATT	21	UGCAACGACGCGU	22	AD-15306
			CCCGGCTT

102-120	CCGGGACGCGUCGUUGCAGTT	23	CUGCAACGACGCG	24	AD-15307
			UCCCGGTT

105-123	GGACGCGUCGUUGCAGCAGTT	25	CUGCUGCAACGAC	26	AD-15277
			GCGUCCTT

135-153	UCCCAGCCAGGAUUCCGCGTsT	27	CGCGGAAUCCUGG	28	AD-9526
			CUGGGATsT

135-153	ucccAGccAGGAuuccGcGTsT	29	CGCGGAAUCCUGG	30	AD-9652
			CUGGGATsT

136-154	CCCAGCCAGGAUUCCGCGCTsT	31	GCGCGGAAUCCUG	32	AD-9519
			GCUGGGTsT

136-154	cccAGccAGGAuuccGcGcTsT	33	GCGCGGAAUCCUG	34	AD-9645
			GCUGGGTsT

138-156	CAGCCAGGAUUCCGCGCGCTsT	35	GCGCGCGGAAUCC	36	AD-9523
			UGGCUGTsT

138-156	cAGccAGGAuuccGcGcGcTsT	37	GCGCGCGGAAUCC	38	AD-9649
			UGGCUGTsT

185-203	AGCUCCUGCACAGUCCUCCTsT	39	GGAGGACUGUGCA	40	AD-9569
			GGAGCUTsT

185-203	AGcuccuGcAcAGuccuccTsT	41	GGAGGACUGUGcA	42	AD-9695
			GGAGCUTsT

205-223	CACCGCAAGGCUCAAGGCGTT	43	CGCCUUGAGCCUU	44	AD-15222
			GCGGUGTT

208-226	CGCAAGGCUCAAGGCGCCGTT	45	CGGCGCCUUGAGC	46	AD-15278
			CUUGCGTT

210-228	CAAGGCUCAAGGCGCCGCCTT	47	GGCGGCGCCUUGA	48	AD-15178
			GCCUUGTT

232-250	GUGGACCGCGCACGGCCUCTT	49	GAGGCCGUGCGCG	50	AD-15308
			GUCCACTT

233-251	UGGACCGCGCACGGCCUCUTT	51	AGAGGCCGUGCGC	52	AD-15223
			GGUCCATT

234-252	GGACCGCGCACGGCCUCUATT	53	UAGAGGCCGUGCG	54	AD-15309
			CGGUCCTT

235-253	GACCGCGCACGGCCUCUAGTT	55	CUAGAGGCCGUGC	56	AD-15279
			GCGGUCTT

236-254	ACCGCGCACGGCCUCUAGGTT	57	CCUAGAGGCCGUG	58	AD-15194
			CGCGGUTT

237-255	CCGCGCACGGCCUCUAGGUTT	59	ACCUAGAGGCCGU	60	AD-15310
			GCGCGGTT

238-256	CGCGCACGGCCUCUAGGUCTT	61	GACCUAGAGGCCG	62	AD-15311
			UGCGCGTT

239-257	GCGCACGGCCUCUAGGUCUTT	63	AGACCUAGAGGCC	64	AD-15392
			GUGCGCTT

240-258	CGCACGGCCUCUAGGUCUCTT	65	GAGACCUAGAGGC	66	AD-15312
			CGUGCGTT

248-266	CUCUAGGUCUCCUCGCCAGTT	67	CUGGCGAGGAGAC	68	AD-15313
			CUAGAGTT

249-267	UCUAGGUCUCCUCGCCAGGTT	69	CCUGGCGAGGAGA	70	AD-15280
			CCUAGATT

250-268	CUAGGUCUCCUCGCCAGGATT	71	UCCUGGCGAGGAG	72	AD-15267
			ACCUAGTT

252-270	AGGUCUCCUCGCCAGGACATT	73	UGUCCUGGCGAGG	74	AD-15314
			AGACCUTT

258-276	CCUCGCCAGGACAGCAACCTT	75	GGUUGCUGUCCUG	76	AD-15315
			GCGAGGTT

300-318	CGUCAGCUCCAGGCGGUCCTsT	77	GGACCGCCUGGAG	78	AD-9624
			CUGACGTsT

300-318	cGucAGcuccAGGcGGuccTsT	79	GGACCGCCUGGAG	80	AD-9750
			CUGACGTsT

301-319	GUCAGCUCCAGGCGGUCCUTsT	81	AGGACCGCCUGGA	82	AD-9623
			GCUGACTsT

301-319	GucAGcuccAGGcGGuccuTsT	83	AGGACCGCCUGGA	84	AD-9749
			GCUGACTsT

370-388	GGCGCCCGUGCGCAGGAGGTT	85	CCUCCUGCGCACG	86	AD-15384
			GGCGCCTT

408-426	GGAGCUGGUGCUAGCCUUGTsT	87	CAAGGCUAGCACC	88	AD-9607
			AGCUCCTsT

408-426	GGAGcuGGuGcuAGccuuGTsT	89	cAAGGCuAGcACc	90	AD-9733
			AGCUCCTsT

411-429	GCUGGUGCUAGCCUUGCGUTsT	91	ACGCAAGGCUAGC	92	AD-9524
			ACCAGCTsT

411-429	GcuGGuGcuAGccuuGcGuTsT	93	ACGcAAGGCuAGc	94	AD-9650
			ACcAGCTsT

412-430	CUGGUGCUAGCCUUGCGUUTsT	95	AACGCAAGGCUAG	96	AD-9520
			CACCAGTsT

412-430	CUGGUGCUAGCCUUGCGUUTsT	97	AACGCAAGGCUAG	98	AD-9520
			CACCAGTsT

412-430	cuGGuGcuAGccuuGcGuuTsT	99	AACGcAAGGCuAG	100	AD-9646
			cACcAGTsT

416-434	UGCUAGCCUUGCGUUCCGATsT	101	UCGGAACGCAAGG	102	AD-9608
			CUAGCATsT

416-434	uGcuAGccuuGcGuuccGATsT	103	UCGGAACGcAAGG	104	AD-9734
			CuAGcATsT

419-437	UAGCCUUGCGUUCCGAGGATsT	105	UCCUCGGAACGCA	106	AD-9546
			AGGCUATsT

419-437	uAGccuuGcGuuccGAGGATsT	107	UCCUCGGAACGcA	108	AD-9672
			AGGCuATsT

439-457	GACGGCCUGGCCGAAGCACTT	109	GUGCUUCGGCCAG	110	AD-15385
			GCCGUCTT

447-465	GGCCGAAGCACCCGAGCACTT	111	GUGCUCGGGUGCU	112	AD-15393
			UCGGCCTT

448-466	GCCGAAGCACCCGAGCACGTT	113	CGUGCUCGGGUGC	114	AD-15316
			UUCGGCTT

449-467	CCGAAGCACCCGAGCACGGTT	115	CCGUGCUCGGGUG	116	AD-15317
			CUUCGGTT

458-476	CCGAGCACGGAACCACAGCTT	117	GCUGUGGUUCCGU	118	AD-15318
			GCUCGGTT

484-502	CACCGCUGCGCCAAGGAUCTT	119	GAUCCUUGGCGCA	120	AD-15195
			GCGGUGTT

486-504	CCGCUGCGCCAAGGAUCCGTT	121	CGGAUCCUUGGCG	122	AD-15224
			CAGCGGTT

487-505	CGCUGCGCCAAGGAUCCGUTT	123	ACGGAUCCUUGGC	124	AD-15188
			GCAGCGTT

489-507	CUGCGCCAAGGAUCCGUGGTT	125	CCACGGAUCCUUG	126	AD-15225
			GCGCAGTT

500-518	AUCCGUGGAGGUUGCCUGGTT	127	CCAGGCAACCUCC	128	AD-15281
			ACGGAUTT

509-527	GGUUGCCUGGCACCUACGUTT	129	ACGUAGGUGCCAG	130	AD-15282
			GCAACCTT

542-560	AGGAGACCCACCUCUCGCATT	131	UGCGAGAGGUGGG	132	AD-15319
			UCUCCUTT

543-561	GGAGACCCACCUCUCGCAGTT	133	CUGCGAGAGGUGG	134	AD-15226
			GUCUCCTT

544-562	GAGACCCACCUCUCGCAGUTT	135	ACUGCGAGAGGUG	136	AD-15271
			GGUCUCTT

549-567	CCACCUCUCGCAGUCAGAGTT	137	CUCUGACUGCGAG	138	AD-15283
			AGGUGGTT

552-570	CCUCUCGCAGUCAGAGCGCTT	139	GCGCUCUGACUGC	140	AD-15284
			GAGAGGTT

553-571	CUCUCGCAGUCAGAGCGCATT	141	UGCGCUCUGACUG	142	AD-15189
			CGAGAGTT

554-572	UCUCGCAGUCAGAGCGCACTT	143	GUGCGCUCUGACU	144	AD-15227
			GCGAGATT

555-573	CUCGCAGUCAGAGCGCACUTsT	145	AGUGCGCUCUGAC	146	AD-9547
			UGCGAGTsT

555-573	cucGcAGucAGAGcGcAcuTsT	147	AGUGCGCUCUGAC	148	AD-9673
			UGCGAGTsT

558-576	GCAGUCAGAGCGCACUGCCTsT	149	GGCAGUGCGCUCU	150	AD-9548
			GACUGCTsT

558-576	GcAGucAGAGcGcAcuGccTsT	151	GGcAGUGCGCUCU	152	AD-9674
			GACUGCTsT

606-624	GGGAUACCUCACCAAGAUCTsT	153	GAUCUUGGUGAGG	154	AD-9529
			UAUCCCTsT

606-624	GGGAuAccucAccAAGAucTsT	155	GAUCUUGGUGAGG	156	AD-9655
			uAUCCCTsT

659-677	UGGUGAAGAUGAGUGGCGATsT	157	UCGCCACUCAUCU	158	AD-9605
			UCACCATsT

659-677	uGGuGAAGAuGAGuGGcGATsT	159	UCGCcACUcAUCU	160	AD-9731
			UcACcATsT

663-681	GAAGAUGAGUGGCGACCUGTsT	161	CAGGUCGCCACUC	162	AD-9596
			AUCUUCTsT

663-681	GAAGAuGAGuGGcGAccuGTsT	163	cAGGUCGCcACUc	164	AD-9722
			AUCUUCTsT

704-722	CCCAUGUCGACUACAUCGATsT	165	UCGAUGUAGUCGA	166	AD-9583
			CAUGGGTsT

704-722	cccAuGucGAcuAcAucGATsT	167	UCGAUGuAGUCGA	168	AD-9709
			cAUGGGTsT

718-736	AUCGAGGAGGACUCCUCUGTsT	169	CAGAGGAGUCCUC	170	AD-9579
			CUCGAUTsT

718-736	AucGAGGAGGAcuccucuGTsT	171	cAGAGGAGUCCUC	172	AD-9705
			CUCGAUTsT

758-776	GGAACCUGGAGCGGAUUACTT	173	GUAAUCCGCUCCA	174	AD-15394
			GGUUCCTT

759-777	GAACCUGGAGCGGAUUACCTT	175	GGUAAUCCGCUCC	176	AD-15196
			AGGUUCTT

760-778	AACCUGGAGCGGAUUACCCTT	177	GGGUAAUCCGCUC	178	AD-15197
			CAGGUUTT

777-795	CCCUCCACGGUACCGGGCGTT	179	CGCCCGGUACCGU	180	AD-15198
			GGAGGGTT

782-800	CACGGUACCGGGCGGAUGATsT	181	UCAUCCGCCCGGU	182	AD-9609
			ACCGUGTsT

782-800	cAcGGuAccGGGcGGAuGATsT	183	UcAUCCGCCCGGu	184	AD-9735
			ACCGUGTsT

783-801	ACGGUACCGGGCGGAUGAATsT	185	UUCAUCCGCCCGG	186	AD-9537
			UACCGUTsT

783-801	AcGGuAccGGGcGGAuGAATsT	187	UUcAUCCGCCCGG	188	AD-9663
			uACCGUTsT

784-802	CGGUACCGGGCGGAUGAAUTsT	189	AUUCAUCCGCCCG	190	AD-9528
			GUACCGTsT

784-802	cGGuAccGGGcGGAuGAAuTsT	191	AUUcAUCCGCCCG	192	AD-9654
			GuACCGTsT

785-803	GGUACCGGGCGGAUGAAUATsT	193	UAUUCAUCCGCCC	194	AD-9515
			GGUACCTsT

785-803	GGuAccGGGcGGAuGAAuATsT	195	uAUUcAUCCGCCC	196	AD-9641
			GGuACCTsT

786-804	GUACCGGGCGGAUGAAUACTsT	197	GUAUUCAUCCGCC	198	AD-9514
			CGGUACTsT

786-804	GuAccGGGcGGAuGAAuAcTsT	199	GuAUUcAUCCGCC	200	AD-9640
			CGGuACTsT

788-806	ACCGGGCGGAUGAAUACCATsT	201	UGGUAUUCAUCCG	202	AD-9530
			CCCGGUTsT

788-806	AccGGGcGGAuGAAuAccATsT	203	UGGuAUUcAUCCG	204	AD-9656
			CCCGGUTsT

789-807	CCGGGCGGAUGAAUACCAGTsT	205	CUGGUAUUCAUCC	206	AD-9538
			GCCCGGTsT

789-807	ccGGGcGGAuGAAuAccAGTsT	207	CUGGuAUUcAUCC	208	AD-9664
			GCCCGGTsT

825-843	CCUGGUGGAGGUGUAUCUCTsT	209	GAGAUACACCUCC	210	AD-9598
			ACCAGGTsT

825-843	ccuGGuGGAGGuGuAucucTsT	211	GAGAuAcACCUCc	212	AD-9724
			ACcAGGTsT

826-844	CUGGUGGAGGUGUAUCUCCTsT	213	GGAGAUACACCUC	214	AD-9625
			CACCAGTsT

826-844	cuGGuGGAGGuGuAucuccTsT	215	GGAGAuAcACCUC	216	AD-9751
			cACcAGTsT

827-845	UGGUGGAGGUGUAUCUCCUTsT	217	AGGAGAUACACCU	218	AD-9556
			CCACCATsT

827-845	uGGuGGAGGuGuAucuccuTsT	219	AGGAGAuAcACCU	220	AD-9682
			CcACcATsT

828-846	GGUGGAGGUGUAUCUCCUATsT	221	UAGGAGAUACACC	222	AD-9539
			UCCACCTsT

828-846	GGuGGAGGuGuAucuccuATsT	223	uAGGAGAuAcACC	224	AD-9665
			UCcACCTsT

831-849	GGAGGUGUAUCUCCUAGACTsT	225	GUCUAGGAGAUAC	226	AD-9517
			ACCUCCTsT

831-849	GGAGGuGuAucuccuAGAcTsT	227	GUCuAGGAGAuAc	228	AD-9643
			ACCUCCTsT

833-851	AGGUGUAUCUCCUAGACACTsT	229	GUGUCUAGGAGAU	230	AD-9610
			ACACCUTsT

833-851	AGGuGuAucuccuAGAcAcTsT	231	GUGUCuAGGAGAu	232	AD-9736
			AcACCUTsT

833-851	AfgGfuGfuAfuCfuCfcUfaG	233	P*gUfgUfcUfaG	234	AD-14681
	faCfaCfTsT		fgAfgAfuAfcAf
			cCfuTsT

833-851	AGGUfGUfAUfCfUfCfCfUfA	235	GUfGUfCfUfAGG	236	AD-14691
	GACfACfTsT		AGAUfACfACfCf
			UfTsT

833-851	AgGuGuAuCuCcUaGaCaCTsT	237	P*gUfgUfcUfaG	238	AD-14701
			fgAfgAfuAfcAf
			cCfuTsT

833-851	AgGuGuAuCuCcUaGaCaCTsT	239	GUfGUfCfUfAGG	240	AD-14711
			AGAUfACfACfCf
			UfTsT

833-851	AfgGfuGfuAfuCfuCfcUfaG	241	GUGUCuaGGagAU	242	AD-14721
	faCfaCfTsT		ACAccuTsT

833-851	AGGUfGUfAUfCfUfCfCfUfA	243	GUGUCuaGGagAU	244	AD-14731
	GACfACfTsT		ACAccuTsT

833-851	AgGuGuAuCuCcUaGaCaCTsT	245	GUGUCuaGGagAU	246	AD-14741
			ACAccuTsT

833-851	GfcAfcCfcUfcAfuAfgGfcC	247	P*uCfcAfgGfcC	248	AD-15087
	fuGfgAfTsT		fuAfuGfaGfgGf
			uGfcTsT

833-851	GCfACfCfCfUfCfAUfAGGCf	249	UfCfCfAGGCfCf	250	AD-15097
	CfUfGGATsT		UfAUfGAGGGUfG
			CfTsT

833-851	GcAcCcUcAuAgGcCuGgATsT	251	P*uCfcAfgGfcC	252	AD-15107
			fuAfuGfaGfgGf
			uGfcTsT

833-851	GcAcCcUcAuAgGcCuGgATsT	253	UfCfCfAGGCfCf	254	AD-15117
			UfAUfGAGGGUfG
			CfTsT

833-851	GfcAfcCfcUfcAfuAfgGfcC	255	UCCAGgcCUauGA	256	AD-15127
	fuGfgAfTsT		GGGugcTsT

833-851	GCfACfCfCfUfCfAUfAGGCf	257	UCCAGgcCUauGA	258	AD-15137
	CfUfGGATsT		GGGugcTsT

833-851	GcAcCcUcAuAgGcCuGgATsT	259	UCCAGgcCUauGA	260	AD-15147
			GGGugcTsT

836-854	UGUAUCUCCUAGACACCAGTsT	261	CUGGUGUCUAGGA	262	AD-9516
			GAUACATsT

836-854	uGuAucuccuAGAcAccAGTsT	263	CUGGUGUCuAGGA	264	AD-9642
			GAuAcATsT

840-858	UCUCCUAGACACCAGCAUATsT	265	UAUGCUGGUGUCU	266	AD-9562
			AGGAGATsT

840-858	ucuccuAGAcAccAGcAuATsT	267	uAUGCUGGUGUCu	268	AD-9688
			AGGAGATsT

840-858	UfcUfcCfuAfgAfcAfcCfaG	269	P*uAfuGfcUfgG	270	AD-14677
	fcAfuAfTsT		fuGfuCfuAfgGf
			aGfaTsT

840-858	UfCfUfCfCfUfAGACfACfCf	271	UfAUfGCfUfGGU	272	AD-14687
	AGCfAUfATsT		fGUfCfUfAGGAG
			ATsT

840-858	UcUcCuAgAcAcCaGcAuATsT	273	P*uAfuGfcUfgG	274	AD-14697
			fuGfuCfuAfgGf
			aGfaTsT

840-858	UcUcCuAgAcAcCaGcAuATsT	275	UfAUfGCfUfGGU	276	AD-14707
			fGUfCfUfAGGAG
			ATsT

840-858	UfcUfcCfuAafAfcAfcCfaG	277	UAUGCugGUguCU	278	AD-14717
	fcAfuAfTsT		AGGagaTsT

840-858	UfCfUfCfCfUfAGACfACfCf	279	UAUGCugGUguCU	280	AD-14727
	AGCfAUfATsT		AGGagaTsT

840-858	UcUcCuAgAcAcCaGcAuATsT	281	UAUGCugGUguCU	282	AD-14737
			AGGagaTsT

840-858	AfgGfcCfuGfgAfgUfuUfaU	283	P*cCfgAfaUfaA	284	AD-15083
	fuCfgGfTsT		faCfuCfcAfgGf
			cCfuTsT

840-858	AGGCfCfUfGGAGUfUfUfAUf	285	CfCfGAAUfAAAC	286	AD-15093
	UfCfGGTsT		fUfCfCfAGGCfC
			fUfTsT

840-858	AgGcCuGgAgUuUaUuCgGTsT	287	P*cCfgAfaUfaA	288	AD-15103
			faCfuCfcAfgGf
			cCfuTsT

840-858	AgGcCuGgAgUuUaUuCgGTsT	289	CfCfGAAUfAAAC	290	AD-15113
			fUfCfCfAGGCfC
			fUfTsT

840-858	AfgGfcCfuGfgAfgUfuUfaU	291	CCGAAuaAAcuCC	292	AD-15123
	fuCfgGfTsT		AGGccuTsT

840-858	AGGCfCfUfGGAGUfUfUfAUf	293	CCGAAuaAAcuCC	294	AD-15133
	UfCfGGTsT		AGGccuTsT

840-858	AgGcCuGgAgUuUaUuCgGTsT	295	CCGAAuaAAcuCC	296	AD-15143
			AGGccuTsT

841-859	CUCCUAGACACCAGCAUACTsT	297	GUAUGCUGGUGUC	298	AD-9521
			UAGGAGTsT

841-859	cuccuAGAcAccAGcAuAcTsT	299	GuAUGCUGGUGUC	300	AD-9647
			uAGGAGTsT

842-860	UCCUAGACACCAGCAUACATsT	301	UGUAUGCUGGUGU	302	AD-9611
			CUAGGATsT

842-860	uccuAGAcAccAGcAuAcATsT	303	UGuAUGCUGGUGU	304	AD-9737
			CuAGGATsT

843-861	CCUAGACACCAGCAUACAGTsT	305	CUGUAUGCUGGUG	306	AD-9592
			UCUAGGTsT

843-861	ccuAGAcAccAGcAuAcAGTsT	307	CUGuAUGCUGGUG	308	AD-9718
			UCuAGGTsT

847-865	GACACCAGCAUACAGAGUGTsT	309	CACUCUGUAUGCU	310	AD-9561
			GGUGUCTsT

847-865	GAcAccAGcAuAcAGAGuGTsT	311	cACUCUGuAUGCU	312	AD-9687
			GGUGUCTsT

855-873	CAUACAGAGUGACCACCGGTsT	313	CCGGUGGUCACUC	314	AD-9636
			UGUAUGTsT

855-873	cAuAcAGAGuGAccAccGGTsT	315	CCGGUGGUcACUC	316	AD-9762
			UGuAUGTsT

860-878	AGAGUGACCACCGGGAAAUTsT	317	AUUUCCCGGUGGU	318	AD-9540
			CACUCUTsT

860-878	AGAGuGAccAccGGGAAAuTsT	319	AUUUCCCGGUGGU	320	AD-9666
			cACUCUTsT

861-879	GAGUGACCACCGGGAAAUCTsT	321	GAUUUCCCGGUGG	322	AD-9535
			UCACUCTsT

861-879	GAGuGAccAccGGGAAAucTsT	323	GAUUUCCCGGUGG	324	AD-9661
			UcACUCTsT

863-881	GUGACCACCGGGAAAUCGATsT	325	UCGAUUUCCCGGU	326	AD-9559
			GGUCACTsT

863-881	GuGAccAccGGGAAAucGATsT	327	UCGAUUUCCCGGU	328	AD-9685
			GGUcACTsT

865-883	GACCACCGGGAAAUCGAGGTsT	329	CCUCGAUUUCCCG	330	AD-9533
			GUGGUCTsT

865-883	GAccAccGGGAAAucGAGGTsT	331	CCUCGAUUUCCCG	332	AD-9659
			GUGGUCTsT

866-884	ACCACCGGGAAAUCGAGGGTsT	333	CCCUCGAUUUCCC	334	AD-9612
			GGUGGUTsT

866-884	AccAccGGGAAAucGAGGGTsT	335	CCCUCGAUUUCCC	336	AD-9738
			GGUGGUTsT

867-885	CCACCGGGAAAUCGAGGGCTsT	337	GCCCUCGAUUUCC	338	AD-9557
			CGGUGGTsT

867-885	ccAccGGGAAAucGAGGGcTsT	339	GCCCUCGAUUUCC	340	AD-9683
			CGGUGGTsT

875-893	AAAUCGAGGGCAGGGUCAUTsT	341	AUGACCCUGCCCU	342	AD-9531
			CGAUUUTsT

875-893	AAAucGAGGGcAGGGucAuTsT	343	AUGACCCUGCCCU	344	AD-9657
			CGAUUUTsT

875-893	AfaAfuCfgAfgGfgCfaGfgG	345	P*aUfgAfcCfcU	346	AD-14673
	fuCfaUfTsT		fgCfcCfuCfgAf
			uUfuTsT

875-893	AAAUfCfGAGGGCfAGGGUfCf	347	AUfGACfCfCfUf	348	AD-14683
	AUfTsT		GCfCfCfUfCfGA
			UfUfUfTsT

875-893	AaAuCgAgGgCaGgGuCaUTsT	349	P*aUfgAfcCfcU	350	AD-14693
			fgCfcCfuCfgAf
			uUfuTsT

875-893	AaAuCgAgGgCaGgGuCaUTsT	351	AUfGACfCfCfUf	352	AD-14703
			GCfCfCfUfCfGA
			UfUfUfTsT

875-893	AfaAfuCfgAfgGfgCfaGfgG	353	AUGACccUGccCU	354	AD-14713
	fuCfaUfTsT		CGAuuuTsT

875-893	AAAUfCfGAGGGCfAGGGUfCf	355	AUGACccUGccCU	356	AD-14723
	AUfTsT		CGAuuuTsT

875-893	AaAuCgAgGgCaGgGuCaUTsT	357	AUGACccUGccCU	358	AD-14733
			CGAuuuTsT

875-893	CfgGfcAfcCfcUfcAfuAfgG	359	P*cAfgGfcCfuA	360	AD-15079
	fcCfuGfTsT		fuGfaGfgGfuGf
			cCfgTsT

875-893	CfGGCfACfCfCfUfCfAUfAG	361	CfAGGCfCfUfAU	362	AD-15089
	GCfCfUfGTsT		fGAGGGUfGCfCf
			GTsT

875-893	CgGcAcCcUcAuAgGcCuGTsT	363	P*cAfgGfcCfuA	364	AD-15099
			fuGfaGfgGfuGf
			cCfgTsT

875-893	CgGcAcCcUcAuAgGcCuGTsT	365	CfAGGCfCfUfAU	366	AD-15109
			fGAGGGUfGCfCf
			GTsT

875-893	CfgGfcAfcCfcUfcAfuAfgG	367	CAGGCcuAUgaGG	368	AD-15119
	fcCfuGfTsT		GUGccgTsT

875-893	CfGGCfACfCfCfUfCfAUfAG	369	CAGGCcuAUgaGG	370	AD-15129
	GCfCfUfGTsT		GUGccgTsT

875-893	CgGcAcCcUcAuAgGcCuGTsT	371	CAGGCcuAUgaGG	372	AD-15139
			GUGccgTsT

877-895	AUCGAGGGCAGGGUCAUGGTsT	373	CCAUGACCCUGCC	374	AD-9542
			CUCGAUTsT

877-895	AucGAGGGcAGGGucAuGGTsT	375	CcAUGACCCUGCC	376	AD-9668
			CUCGAUTsT

878-896	cGAGGGcAGGGucAuGGucTsT	377	GACcAUGACCCUG	378	AD-9739
			CCCUCGTsT

880-898	GAGGGCAGGGUCAUGGUCATsT	379	UGACCAUGACCCU	380	AD-9637
			GCCCUCTsT

880-898	GAGGGcAGGGucAuGGucATsT	381	UGACcAUGACCCU	382	AD-9763
			GCCCUCTsT

882-900	GGGCAGGGUCAUGGUCACCTsT	383	GGUGACCAUGACC	384	AD-9630
			CUGCCCTsT

882-900	GGGcAGGGucAuGGucAccTsT	385	GGUGACcAUGACC	386	AD-9756
			CUGCCCTsT

885-903	CAGGGUCAUGGUCACCGACTsT	387	GUCGGUGACCAUG	388	AD-9593
			ACCCUGTsT

885-903	cAGGGucAuGGucAccGAcTsT	389	GUCGGUGACcAUG	390	AD-9719
			ACCCUGTsT

886-904	AGGGUCAUGGUCACCGACUTsT	391	AGUCGGUGACCAU	392	AD-9601
			GACCCUTsT

886-904	AGGGucAuGGucAccGAcuTsT	393	AGUCGGUGACcAU	394	AD-9727
			GACCCUTsT

892-910	AUGGUCACCGACUUCGAGATsT	395	UCUCGAAGUCGGU	396	AD-9573
			GACCAUTsT

892-910	AuGGucAccGAcuucGAGATsT	397	UCUCGAAGUCGGU	398	AD-9699
			GACcAUTsT

899-917	CCGACUUCGAGAAUGUGCCTT	399	GGCACAUUCUCGA	400	AD-15228
			AGUCGGTT

921-939	GGAGGACGGGACCCGCUUCTT	401	GAAGCGGGUCCCG	402	AD-15395
			UCCUCCTT

993-1011	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		uGfgCfcUfcAf
			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	AGCUUUAAAAUGGUUCCGATT	821	UCGGAACCAUUUU	822	AD-15169
			AAAGCUTT

2421-2439	GCUUUAAAAUGGUUCCGACTT	823	GUCGGAACCAUUU	824	AD-15201
			UAAAGCTT

2422-2440	CUUUAAAAUGGUUCCGACUTT	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	UGGGCAGAAUGACUUUUAUTT	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	1065	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		GUfCfUfAGAATsT

3530-3548	UuCuAgAcCuGuUuUgCuUTsT	1235	P*aAfgCfaAfaA	1236	AD-14696
			fcAfgGfuCfuAf
			gAfaTsT

3530-3548	UuCuAgAcCuGuUuUgCuUTsT	1237	AAGCfAAAACfAG	1238	AD-14706
			GUfCfUfAGAATsT

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		GGUfCfUfAGATsT

3531-3549	UcUaGaCcUgUuUuGcUuUTsT	1267	P*aAfaGfcAfaA	1268	AD-14695
			faCfaGfgUfcUf
			aGfaTsT

3531-3549	UcUaGaCcUgUuUuGcUuUTsT	1269	AAAGCfAAAACfA	1270	AD-14705
			GGUfCfUfAGATsT

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 phosphorothiodiester groups; unless denoted by prefix “P*”, oligonucleotides are devoid of a 5′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide.

TABLE 5

Sequences of modified dsRNA targeted to PCSK9

				SEQ
	Sense strand sequence (5′-	SEQ ID	Antisense-strand	ID
Duplex #
	3′)¹	NO:	sequence (5′-3′)¹	NO:

AD-10792	GccuGGAGuuuAuucGGAATsT	1305	UUCCGAAuAAACUCcAGGCT	1306
			sT

AD-10793	GccuGGAGuuuAuucGGAATsT	1307	uUcCGAAuAAACUccAGGCT	1308
			sT

AD-10796	GccuGGAGuuuAuucGGAATsT	1309	UUCCGAAUAAACUCCAGGCT	1310
			sT

AD-12038	GccuGGAGuuuAuucGGAATsT	1311	uUCCGAAUAAACUCCAGGCT	1312
			sT

AD-12039	GccuGGAGuuuAuucGGAATsT	1313	UuCCGAAUAAACUCCAGGCT	1314
			sT

AD-12040	GccuGGAGuuuAuucGGAATsT	1315	UUcCGAAUAAACUCCAGGCT	1316
			sT

AD-12041	GccuGGAGuuuAuucGGAATsT	1317	UUCcGAAUAAACUCCAGGCT	1318
			sT

AD-12042	GCCUGGAGUUUAUUCGGAATsT	1319	uUCCGAAUAAACUCCAGGCT	1320
			sT

AD-12043	GCCUGGAGUUUAUUCGGAATsT	1321	UuCCGAAUAAACUCCAGGCT	1322
			sT

AD-12044	GCCUGGAGUUUAUUCGGAATsT	1323	UUcCGAAUAAACUCCAGGCT	1324
			sT

AD-12045	GCCUGGAGUUUAUUCGGAATsT	1325	UUCcGAAUAAACUCCAGGCT	1326
			sT

AD-12046	GccuGGAGuuuAuucGGAA	1327	UUCCGAAUAAACUCCAGGCs	1328
			csu

AD-12047	GccuGGAGuuuAuucGGAAA	1329	UUUCCGAAUAAACUCCAGGC	1330
			scsu

AD-12048	GccuGGAGuuuAuucGGAAAA	1331	UUUUCCGAAUAAACUCCAGG	1332
			Cscsu

AD-12049	GccuGGAGuuuAuucGGAAAAG	1333	CUUUUCCGAAUAAACUCCAG	1334
			GCscsu

AD-12050	GccuGGAGuuuAuucGGAATTab	1335	UUCCGAAUAAACUCCAGGCT	1336
			Tab

AD-12051	GccuGGAGuuuAuucGGAAATTab	1337	UUUCCGAAuAAACUCCAGGC	1338
			TTab

AD-12052	GccuGGAGuuuAuucGGAAAATTab	1339	UUUUCCGAAUAAACUCCAGG	1340
			CTTab

AD-12053	GccuGGAGuuuAuucGGAAAAGTTab	1341	CUUUUCCGAAUAAACUCCAG	1342
			GCTTab

AD-12054	GCCUGGAGUUUAUUCGGAATsT	1343	UUCCGAAUAAACUCCAGGCs	1344
			csu

AD-12055	GccuGGAGuuuAuucGGAATsT	1345	UUCCGAAUAAACUCCAGGCs	1346
			csu

AD-12056	GcCuGgAgUuUaUuCgGaA	1347	UUCCGAAUAAACUCCAGGCT	1348
			Tab

AD-12057	GcCuGgAgUuUaUuCgGaA	1349	UUCCGAAUAAACUCCAGGCT	1350
			sT

AD-12058	GcCuGgAgUuUaUuCgGaA	1351	UUCCGAAuAAACUCcAGGCT	1352
			sT

AD-12059	GcCuGgAgUuUaUuCgGaA	1353	uUcCGAAuAAACUccAGGCT	1354
			sT

AD-12060	GcCuGgAgUuUaUuCgGaA	1355	UUCCGaaUAaaCUCCAggc	1356

AD-12061	GcCuGgnAgUuUaUuCgGaATsT	1357	UUCCGaaUAaaCUCCAggcT	1358
			sT

AD-12062	GcCuGgAgUuUaUuCgGaATTab	1359	UUCCGaaUAaaCUCCAggcT	1360
			Tab

AD-12063	GcCuGgAgUuUaUuCgGaA	1361	UUCCGaaUAaaCUCCAggcs	1362
			csu

AD-12064	GcCuGgnAgUuUaUuCgGaATsT	1363	UUCCGAAuAAACUCcAGGCT	1364
			sT

AD-12065	GcCuGgAgUuUaUuCgGaATTab	1365	UUCCGAAuAAACUCcAGGCT	1366
			Tab

AD-12066	GcCuGgAgUuUaUuCgGaA	1367	UUCCGAAuAAACUCcAGGCs	1368
			csu

AD-12067	GcCuGgnAgUuUaUuCgGaATsT	1369	UUCCGAAUAAACUCCAGGCT	1370
			sT

AD-12068	GcCuGgAgUuUaUuCgGaATTab	1371	UUCCGAAUAAACUCCAGGCT	1372
			Tab

AD-12069	GcCuGgAgUuUaUuCgGaA	1373	UUCCGAAUAAACUCCAGGCs	1374
			csu

AD-12338	GfcCfuGfgAfgUfuUfaUfuCfgGfaAf	1375	P*uUfcCfgAfaUfaAfaCf	1376
			uCfcAfgGfc

AD-12339	GcCuGgAgUuUaUuCgGaA	1377	P*uUfcCfgAfaUfaAfaCf	1378
			uCfcAfgGfc

AD-12340	GccuGGAGuuuAuucGGAA	1379	P*uUfcCfgAfaUfaAfaCf	1380
			uCfcAfgGfc

AD-12341	GfcCfuGfgAfgUfuUfaUfuCfgGfaA	1381	P*uUfcCfgAfaUfaAfaCf	1382
	fTsT		uCfcAfgGfcTsT

AD-12342	GfcCfuGfgAfgUfuUfaUfuCfgGfaA	1383	UUCCGAAuAAACUCcAGGCT	1384
	fTsT		sT

AD-12343	GfcCfuGfgAfgUfuUfaUfuCfgGfaA	1385	uUcCGAAuAAACUccAGGCT	1386
	fTsT		sT

AD-12344	GfcCfuGfgAfgUfuUfaUfuCfgGfaA	1387	UUCCGAAUAAACUCCAGGCT	1388
	fTsT		sT

AD-12345	GfcCfuGfgAfgUfuUfaUfuCfgGfaA	1389	UUCCGAAUAAACUCCAGGCs	1390
	fTsT		csu

AD-12346	GfcCfuGfgAfgUfuUfaUfuCfgGfaA	1391	UUCCGaaUAaaCUCCAggcs	1392
	fTsT		csu

AD-12347	GCCUGGAGUUUAUUCGGAATsT	1393	P*uUfcCfgAfaUfaAfaCf	1394
			uCfcAfgGfcTsT

AD-12348	GccuGGAGuuuAuucGGAATsT	1395	P*uUfcCfgAfaUfaAfaCf	1396
			uCfcAfgGfcTsT

AD-12349	GcCuGgnAgUuUaUuCgGaATsT	1397	P*uUfcCfgAfaUfaAfaCf	1398
			uCfcAfgGfcTsT

AD-12350	GfcCfuGfgAfgUfuUfaUfuCfgGfaA	1399	P*uUfcCfgAfaUfaAfaCf	1400
	fTTab		uCfcAfgGfcTTab

AD-12351	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	GfcCfuGfgAfgUfuUfaUfuCfgGfaA	1491	P*UfUfCfCfGAAUfAAACf	1492
	fTsT		UfCfCfAGGCfsCfsUf

AD-12399	GcCuGgnAgUuUaUuCgGaATsT	1493	P*UfUfCfCfGAAUfAAACf	1494
			UfCfCfAGGCfsCfsUf

AD-12400	GCCUGGAGUUUAUUCGGAATsT	1495	P*UfUfCfCfGAAUfAAACf	1496
			UfCfCfAGGCfsCfsUf

AD-12401	GccuGGAGuuuAuucGGAATsT	1497	P*UfUfCfCfGAAUfAAACf	1498
			UfCfCfAGGCfsCfsUf

AD-12402	GccuGGAGuuuAuucGGAA	1499	P*UfUfCfCfGAAUfAAACf	1500
			UfCfCfAGGCfsCfsUf

AD-12403	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 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 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	uucuAGAccY1GuuuuGcuuTsT
	AS	1546	AAGcAAAAcAGGUCuAGAATsT

AD-3273	S	1547	uucuAGAcCY1GuuuuGcuuTsT
	AS	1548	AAGcAAAAcAGGUCuAGAATsT

AD-3274	S	1549	uucuAGAccuY1uuuuGcuuTsT
	AS	1550	AAGcAAAAcAGGUCuAGAATsT

AD-3275	S	1551	uucuAGAcCUY1uuuuGcuuTsT
	AS	1552	AAGcAAAAcAGGUCuAGAATsT

AD-14676	S	1553	UfuCfuAfgAfcCfuGfuUfuUfgCfuUfTsT
	AS	1554	P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

AD-3276	S	1555	UfuCfuAfgAfcCuGfuUfuUfgCfuUfTsT
	AS	1556	P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

AD-3277	S	1557	UfuCfuAfgAfcCfUGfuUfuUfgCfuUfTsT
	AS	1558	P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

AD-3278	S	1559	UfuCfuAfgAfcCUGfuUfuUfgCfuUfTsT
	AS	1560	P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

AD-3279	S	1561	UfuCfuAfgAfcY1uGfuUfuUfgCfuUfTsT
	AS	1562	P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

AD-3280	S	1563	UfuCfuAfgAfcY1UGfuUfuUfgCfuUfTsT
	AS	1564	P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

AD-3281	S	1565	UfuCfuAfgAfcCfY1GfuUfuUfgCfuUfTsT
	AS	1566	P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

AD-3282	S	1567	UfuCfuAfgAfcCY1GfuUfuUfgCfuUfTsT
	AS	1568	P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

AD-3283	S	1569	UfuCfuAfgAfcCfuY1uUfuUfgCfuUfTsT
	AS	1570	P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

AD-3284	S	1571	UfuCfuAfgAfcCUY1uUfuUfgCfuUfTsT
	AS	1572	P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

AD-10792	S	459	GccuGGAGuuuAuucGGAATsT
	AS	460	UUCCGAAuAAACUCcAGGCTsT

AD-3254	S	1573	GccuGGAGuY1uAuucGGAATsT
	AS	1574	UUCCGAAuAAACUCcAGGCTsT

AD-3255	S	1575	GccuGGAGUY1uAuucGGAATsT
	AS	1576	UUCCGAAuAAACUCcAGGCTsT

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′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide

TABLE 7

Sequences of unmodified siRNA flankine AD-9680

Duplex #	Strand	Sequence (5′ to 3′)	*Target	SEQ ID NO:

AD-22169-b1	sense	CAGCCAACUUUUCUAGACCdTsdT	3520	1577
	antis	GGUCUAGAAAAGUUGGCUGdTsdT	3520	1578

AD-22170-b1	sense	AGCCAACUUUUCUAGACCUdTsdT	3521	1579
	antis	AGGUCUAGAAAAGUUGGCUdTsdT	3521	1580

AD-22171-b1	sense	GCCAACUUUUCUAGACCUGdTsdT	3522	1581
	antis	CAGGUCUAGAAAAGUUGGCdTsdT	3522	1582

AD-22172-b1	sense	CCAACUUUUCUAGACCUGUdTsdT	3523	1583
	antis	ACAGGUCUAGAAAAGUUGGdTsdT	3523	1584

AD-22173-b1	sense	CAACUUUUCUAGACCUGUUdTsdT	3524	1585
	antis	AACAGGUCUAGAAAAGUUGdTsdT	3524	1586

AD-22174-b1	sense	AACUUUUCUAGACCUGUUUdTsdT	3525	1587
	antis	AAACAGGUCUAGAAAAGUUdTsdT	3525	1588

AD-22175-b1	sense	ACUUUUCUAGACCUGUUUUdTsdT	3526	1589
	antis	AAAACAGGUCUAGAAAAGUdTsdT	3526	1590

AD-22176-b1	sense	CUUUUCUAGACCUGUUUUGdTsdT	3527	1591
	antis	CAAAACAGGUCUAGAAAAGdTsdT	3527	1592

AD-22177-b1	sense	UUUUCUAGACCUGUUUUGCdTsdT	3528	1593
	antis	GCAAAACAGGUCUAGAAAAdTsdT	3528	1594

AD-22178-b1	sense	UUUCUAGACCUGUUUUGCUdTsdT	3529	1595
	antis	AGCAAAACAGGUCUAGAAAdTsdT	3529	1596

AD-22179-b1	sense	UCUAGACCUGUUUUGCUUUdTsdT	3531	1597
	antis	AAAGCAAAACAGGUCUAGAdTsdT	3531	1598

AD-22180-b1	sense	CUAGACCUGUUUUGCUUUUdTsdT	3532	1599
	antis	AAAAGCAAAACAGGUCUAGdTsdT	3532	1600

AD-22181-b1	sense	UAGACCUGUUUUGCUUUUGdTsdT	3533	1601
	antis	CAAAAGCAAAACAGGUCUAdTsdT	3533	1602

AD-22182-b1	sense	AGACCUGUUUUGCUUUUGUdTsdT	3534	1603
	antis	ACAAAAGCAAAACAGGUCUdTsdT	3534	1604

AD-22183-b1	sense	GACCUGUUUUGCUUUUGUAdTsdT	3535	1605
	antis	UACAAAAGCAAAACAGGUCdTsdT	3535	1606

AD-22184-b1	sense	ACCUGUUUUGCUUUUGUAAdTsdT	3536	1607
	antis	UUACAAAAGCAAAACAGGUdTsdT	3536	1608

AD-22185-b1	sense	CCUGUUUUGCUUUUGUAACdTsdT	3537	1609
	antis	GUUACAAAAGCAAAACAGGdTsdT	3537	1610

AD-22186-b1	sense	CUGUUUUGCUUUUGUAACUdTsdT	3538	1611
	antis	AGUUACAAAAGCAAAACAGdTsdT	3538	1612

AD-22187-b1	sense	UGUUUUGCUUUUGUAACUUdTsdT	3539	1613
	antis	AAGUUACAAAAGCAAAACAdTsdT	3539	1614

AD-22188-b1	sense	GUUUUGCUUUUGUAACUUGdTsdT	3540	1615
	antis	CAAGUUACAAAAGCAAAACdTsdT	3540	1616

AD-22189-b1	sense	UUUUGCUUUUGUAACUUGAdTsdT	3541	1617
	antis	UCAAGUUACAAAAGCAAAAdTsdT	3541	1618

AD-22190-b1	sense	UUUGCUUUUGUAACUUGAAdTsdT	3542	1619
	antis	UUCAAGUUACAAAAGCAAAdTsdT	3542	1620

AD-22191-b1	sense	UUGCUUUUGUAACUUGAAGdTsdT	3543	1621
	antis	CUUCAAGUUACAAAAGCAAdTsdT	3543	1622

AD-22192-b1	sense	UGCUUUUGUAACUUGAAGAdTsdT	3544	1623
	antis	UCUUCAAGUUACAAAAGCAdTsdT	3544	1624

AD-22193-b1	sense	GCUUUUGUAACUUGAAGAUdTsdT	3545	1625
	antis	AUCUUCAAGUUACAAAAGCdTsdT	3545	1626

AD-22194-b1	sense	CUUUUGUAACUUGAAGAUAdTsdT	3546	1627
	antis	UAUCUUCAAGUUACAAAAGdTsdT	3546	1628

AD-22195-b1	sense	UUUUGUAACUUGAAGAUAUdTsdT	3547	1629
	antis	AUAUCUUCAAGUUACAAAAdTsdT	3547	1630

AD-22196-b1	sense	UUUGUAACUUGAAGAUAUUdTsdT	3548	1631
	antis	AAUAUCUUCAAGUUACAAAdTsdT	3548	1632

AD-22197-b1	sense	UUGUAACUUGAAGAUAUUUdTsdT	3549	1633
	antis	AAAUAUCUUCAAGUUACAAdTsdT	3549	1634

AD-22198-b1	sense	UGUAACUUGAAGAUAUUUAdTsdT	3550	1635
	antis	UAAABAUCUUCAAGUUACAdTsdT	3550	1636

AD-22199-b1	sense	GUAACUUGAAGAUAUUUAUdTsdT	3551	1637
	antis	AUAAAUAUCUUCAAGUUACdTsdT	3551	1638

AD-22200-b1	sense	UAACUUGAAGAUAUUUAUUdTsdT	3552	1639
	antis	AAUAAAUAUCUUCAAGUUAdTsdT	3552	1640

AD-22201-b1	sense	AACUUGAAGAUAUUUAUUCdTsdT	3553	1641
	antis	GAAUAAAUAUCUUCAAGUUdTsdT	3553	1642

AD-22202-b1	sense	ACUUGAAGAUAUUUAUUCUdTsdT	3554	1643
	antis	AGAAUAAAUAUCUUCAAGUdTsdT	3554	1644

AD-22203-b1	sense	CUUGAAGAUAUUUAUUCUGdTsdT	3555	1645
	antis	CAGAAUAAAUAUCUUCAAGdTsdT	3555	1646

AD-22204-b1	sense	UUGAAGAUAUUUAUUCUGGdTsdT	3556	1647
	antis	CCAGAAUAAAUAUCUUCAAdTsdT	3556	1648

AD-22205-b1	sense	UGAAGAUAUUUAUUCUGGGdTsdT	3557	1649
	antis	CCCAGAAUAAAUAUCUUCAdTsdT	3557	1650

AD-22206-b1	sense	GAAGAUAUUUAUUCUGGGUdTsdT	3558	1651
	antis	ACCCAGAAUAAAUAUCUUCdTsdT	3558	1652

*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

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

NM_001004210	1729	CCCAGCUGAUUAGUGUCUA	1753	UAGACACUAAUCAGCUGGG
1128-1146

NM_001004210	1730	CCAGCUGAUUAGUGUCUAA	1754	UUAGACACUAAUCAGCUGG
1129-1147

NM_001004210	1731	CUCCCAGAGGUCUACCCAG	1755	CUGGGUAGACCUCUGGGAG
677-695

NM_001004210	1732	GAUCACCCUGAAUUCAUUG	1756	CAAUGAAUUCAGGGUGAUC
893-911

NM_001004210	1733	UCACCCUGAAUUCAUUGUC	1757	GACAAUGAAUUCAGGGUGA
895-913

NM_001004210	1734	CCCCAGCUGAUUAGUGUCU	1758	AGACACUAAUCAGCUGGGG
1127-1145

NM_001004210	1735	AUCACCCUGAAUUCAUUGU	1759	ACAAUGAAUUCAGGGUGAU
894-912

NM_001004210	1736	CAUUUAUUUAAAACUACCC	1760	GGGUAGUUUUAAAUAAAUG
1760-1778

NM_001004210	1737	ACUGAAAAACAGAGUAGCA	1761	UGCUACUCUGUUUUUCAGU
215-233

NM_001004210	1738	CCAUUUAUUUAAAACUACC	1762	GGUAGUUUUAAAUAAAUGG
1759-1777

NM_001004210	1739	UUGAGAACCAGGAGUUAAG	1763	CUUAACUCCUGGUUCUCAA
367-385

NM_001004210	1740	CACCCUGAAUUCAUUGUCU	1764	AGACAAUGAAUUCAGGGUG
896-914

NM_001004210	1741	AACUGAAAAACAGAGUAGC	1765	GCUACUCUGUUUUUCAGUU
214-232

NM_001004210	1742	CUGAAAAACAGAGUAGCAG	1766	CUGCUACUCUGUUUUUCAG
216-234

XM_001103095	1743	AGAAAAUCAGCUUUUACGA	1767	UCGUAAAAGCUGAUUUUCU
387-405

XM_001103095	1744	UCCCCAGCUGAUUAGUGUC	1768	GACACUAAUCAGCUGGGGA
1151-1169

XM_001103095	1745	UACUUAUUAUGUAAGGGUC	1769	GACCCUUACAUAAUAAGUA
1466-1484

XM_001103095	1746	UAUCUUAAAAGGGUGGUAG	1770	CUACCACCCUUUUAAGAUA
1435-1453

XM_001103095	1747	CCAUGGAUUCUGGCGGUAU	1771	AUACCGCCAGAAUCCAUGG
577-595

XM_001103095	1748	UUAAUGAACUAAUUCGUUU	1772	AAACGAAUUAGUUCAUUAA
790-808

XM_001103095	1749	AGGGUCAUUAGACAAAUGU	1773	ACAUUUGUCUAAUGACCCU
1479-1497

XM_001103095	1750	UGAACUAAUUCGUUUUGAC	1774	GUCAAAACGAAUUAGUUCA
794-812

XM_001103095	1751	UUCCCCAGCUGAUUAGUGU	1775	ACACUAAUCAGCUGGGGAA
1150-1168

XM_001103095	1752	UAUGUAAGGGUCAUUAGAC	1776	GUCUAAUGACCCUUACAUA
1473-1491

*Target refers to target gene and location of target sequence. NM_001004210 is the gene for rat XBP-1. XM_001103095 is the sequence for Macaca mulatta (rhesus monkey) XBP-1.

TABLE 10

Target gene name and target sequence
location for dsRNA targeting XBP-1

	Duplex #	Target gene and location of target sequence

	AD18027	NM_001004210_1128-1146
	AD18028	NM_001004210_1129-1147
	AD18029	NM_001004210_677-695
	AD18030	NM_001004210_893-911
	AD18031	NM_001004210_895-913
	AD18032	NM_001004210_1127-1145
	AD18033	NM_001004210_894-912
	AD18034	NM_001004210_1760-1778
	AD18035	NM_001004210_215-233
	AD18036	NM_001004210_1759-1777
	AD18037	NM_001004210_367-385
	AD18038	NM_001004210_896-914
	AD18039	NM_001004210_214-232
	AD18040	NM_001004210_216-234
	AD18041	XM_001103095_387-405
	AD18042	XM_001103095_1151-1169
	AD18043	XM_001103095_1466-1484
	AD18044	XM_001103095_1435-1453
	AD18045	XM_001103095_577-595
	AD18046	XM_001103095_790-808
	AD18047	XM_001103095_1479-1497
	AD18048	XM_001103095_794-812
	AD18049	XM_001103095_1150-1168
	AD18050	XM_001103095_1473-1491

	Target refers to target gene and location of target sequence. NM_001004210 is the gene for rat XBP-1. XM_001103095 is the sequence for Macaca mulatta* (rhesus monkey) XBP-1.

TABLE 11

Sequences of dsRNA targeting XBP-1, with
Endolight chemistry modifications

	SEQ ID		SEQ 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

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.

TABLE 12

Sequences of dsRNA targeting both human and
rhesus monkeyXBP-1.

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

100-118	CUGCUUCUGUCGGGGCAGCNN	1824	GCUGCCCCGACAGAAGCAGNN	28

1011-1029	GAGCUGGGUAUCUCAAAUCNN	1825	GAUUUGAGAUACCCAGCUCNN	28

101-119	UGCUUCUGUCGGGGCAGCCNN	1826	GGCUGCCCCGACAGAAGCANN	28

1012-1030	AGCUGGGUAUCUCAAAUCUNN	1827	AGAUUUGAGAUACCCAGCUNN	28

1013-1031	GCUGGGUAUCUCAAAUCUGNN	1828	CAGAUUUGAGAUACCCAGCNN	28

1014-1032	CUGGGUAUCUCAAAUCUGCNN	1829	GCAGAUUUGAGAUACCCAGNN	28

1015-1033	UGGGUAUCUCAAAUCUGCUNN	1830	AGCAGAUUUGAGAUACCCANN	28

1016-1034	GGGUAUCUCAAAUCUGCUUNN	1831	AAGCAGAUUUGAGAUACCCNN	28

1017-1035	GGUAUCUCAAAUCUGCUUUNN	1832	AAAGCAGAUUUGAGAUACCNN	28

1018-1036	GUAUCUCAAAUCUGCUUUCNN	1833	GAAAGCAGAUUUGAGAUACNN	28

1019-1037	UAUCUCAAAUCUGCUUUCANN	1834	UGAAAGCAGAUUUGAGAUANN	28

1020-1038	AUCUCAAAUCUGCUUUCAUNN	1835	AUGAAAGCAGAUUUGAGAUNN	28

1021-1039	UCUCAAAUCUGCUUUCAUCNN	1836	GAUGAAAGCAGAUUUGAGANN	28

102-120	GCUUCUGUCGGGGCAGCCCNN	1837	GGGCUGCCCCGACAGAAGCNN	28

1022-1040	CUCAAAUCUGCUUUCAUCCNN	1838	GGAUGAAAGCAGAUUUGAGNN	28

1023-1041	UCAAAUCUGCUUUCAUCCANN	1839	UGGAUGAAAGCAGAUUUGANN	28

1024-1042	CAAAUCUGCUUUCAUCCAGNN	1840	CUGGAUGAAAGCAGAUUUGNN	28

1025-1043	AAAUCUGCUUUCAUCCAGCNN	1841	GCUGGAUGAAAGCAGAUUUNN	29

1026-1044	AAUCUGCUUUCAUCCAGCCNN	1842	GGCUGGAUGAAAGCAGAUUNN	29

1027-1045	AUCUGCUUUCAUCCAGCCANN	1843	UGGCUGGAUGAAAGCAGAUNN	29

1028-1046	UCUGCUUUCAUCCAGCCACNN	1844	GUGGCUGGAUGAAAGCAGANN	29

1029-1047	CUGCUUUCAUCCAGCCACUNN	1845	AGUGGCUGGAUGAAAGCAGNN	29

1030-1048	UGCUUUCAUCCAGCCACUGNN	1846	CAGUGGCUGGAUGAAAGCANN	29

1031-1049	GCUUUCAUCCAGCCACUGCNN	1847	GCAGUGGCUGGAUGAAAGCNN	29

103-121	CUUCUGUCGGGGCAGCCCGNN	1848	CGGGCUGCCCCGACAGAAGNN	29

1032-1050	CUUUCAUCCAGCCACUGCCNN	1849	GGCAGUGGCUGGAUGAAAGNN	29

1033-1051	UUUCAUCCAGCCACUGCCCNN	1850	GGGCAGUGGCUGGAUGAAANN	29

104-122	UUCUGUCGGGGCAGCCCGCNN	1851	GCGGGCUGCCCCGACAGAANN	29

105-123	UCUGUCGGGGCAGCCCGCCNN	1852	GGCGGGCUGCCCCGACAGANN	29

1056-1074	CCAUCUUCCUGCCUACUGGNN	1853	CCAGUAGGCAGGAAGAUGGNN	29

1057-1075	CAUCUUCCUGCCUACUGGANN	1854	UCCAGUAGGCAGGAAGAUGNN	29

1058-1076	AUCUUCCUGCCUACUGGAUNN	1855	AUCCAGUAGGCAGGAAGAUNN	29

1059-1077	UCUUCCUGCCUACUGGAUGNN	1856	CAUCCAGUAGGCAGGAAGANN	29

1060-1078	CUUCCUGCCUACUGGAUGCNN	1857	GCAUCCAGUAGGCAGGAAGNN	29

1061-1079	UUCCUGCCUACUGGAUGCUNN	1858	AGCAUCCAGUAGGCAGGAANN	29

106-124	CUGUCGGGGCAGCCCGCCUNN	1859	AGGCGGGCUGCCCCGACAGNN	29

1062-1080	UCCUGCCUACUGGAUGCUUNN	1860	AAGCAUCCAGUAGGCAGGANN	29

1063-1081	CCUGCCUACUGGAUGCUUANN	1861	UAAGCAUCCAGUAGGCAGGNN	29

1064-1082	CUGCCUACUGGAUGCUUACNN	1862	GUAAGCAUCCAGUAGGCAGNN	29

1065-1083	UGCCUACUGGAUGCUUACANN	1863	UGUAAGCAUCCAGUAGGCANN	29

1066-1084	GCCUACUGGAUGCUUACAGNN	1864	CUGUAAGCAUCCAGUAGGCNN	29

1067-1085	CCUACUGGAUGCUUACAGUNN	1865	ACUGUAAGCAUCCAGUAGGNN	29

1068-1086	CUACUGGAUGCUUACAGUGNN	1866	CACUGUAAGCAUCCAGUAGNN	29

1069-1087	UACUGGAUGCUUACAGUGANN	1867	UCACUGUAAGCAUCCAGUANN	29

1070-1088	ACUGGAUGCUUACAGUGACNN	1868	GUCACUGUAAGCAUCCAGUNN	29

1071-1089	CUGGAUGCUUACAGUGACUNN	1869	AGUCACUGUAAGCAUCCAGNN	29

107-125	UGUCGGGGCAGCCCGCCUCNN	1870	GAGGCGGGCUGCCCCGACANN	29

1072-1090	UGGAUGCUUACAGUGACUGNN	1871	CAGUCACUGUAAGCAUCCANN	29

1073-1091	GGAUGCUUACAGUGACUGUNN	1872	ACAGUCACUGUAAGCAUCCNN	29

1074-1092	GAUGCUUACAGUGACUGUGNN	1873	CACAGUCACUGUAAGCAUCNN	29

1075-1093	AUGCUUACAGUGACUGUGGNN	1874	CCACAGUCACUGUAAGCAUNN	29

1076-1094	UGCUUACAGUGACUGUGGANN	1875	UCCACAGUCACUGUAAGCANN	29

1077-1095	GCUUACAGUGACUGUGGAUNN	1876	AUCCACAGUCACUGUAAGCNN	29

1078-1096	CUUACAGUGACUGUGGAUANN	1877	UAUCCACAGUCACUGUAAGNN	29

108-126	GUCGGGGCAGCCCGCCUCCNN	1878	GGAGGCGGGCUGCCCCGACNN	29

109-127	UCGGGGCAGCCCGCCUCCGNN	1879	CGGAGGCGGGCUGCCCCGANN	29

110-128	CGGGGCAGCCCGCCUCCGCNN	1880	GCGGAGGCGGGCUGCCCCGNN	29

111-129	GGGGCAGCCCGCCUCCGCCNN	1881	GGCGGAGGCGGGCUGCCCCNN	29

1116-1134	UUCAGUGACAUGUCCUCUCNN	1882	GAGAGGACAUGUCACUGAANN	29

112-130	GGGCAGCCCGCCUCCGCCGNN	1883	CGGCGGAGGCGGGCUGCCCNN	29

113-131	GGCAGCCCGCCUCCGCCGCNN	1884	GCGGCGGAGGCGGGCUGCCNN	29

1136-1154	GCUUGGUGUAAACCAUUCUNN	1885	AGAAUGGUUUACACCAAGCNN	29

1137-1155	CUUGGUGUAAACCAUUCUUNN	1886	AAGAAUGGUUUACACCAAGNN	29

1138-1156	UUGGUGUAAACCAUUCUUGNN	1887	CAAGAAUGGUUUACACCAANN	29

1139-1157	UGGUGUAAACCAUUCUUGGNN	1888	CCAAGAAUGGUUUACACCANN	29

1140-1158	GGUGUAAACCAUUCUUGGGNN	1889	CCCAAGAAUGGUUUACACCNN	29

1141-1159	GUGUAAACCAUUCUUGGGANN	1890	UCCCAAGAAUGGUUUACACNN	29

114-132	GCAGCCCGCCUCCGCCGCCNN	1891	GGCGGCGGAGGCGGGCUGCNN	29

1142-1160	UGUAAACCAUUCUUGGGAGNN	1892	CUCCCAAGAAUGGUUUACANN	29

1143-1161	GUAAACCAUUCUUGGGAGGNN	1893	CCUCCCAAGAAUGGUUUACNN	29

1144-1162	UAAACCAUUCUUGGGAGGANN	1894	UCCUCCCAAGAAUGGUUUANN	29

1145-1163	AAACCAUUCUUGGGAGGACNN	1895	GUCCUCCCAAGAAUGGUUUNN	29

1146-1164	AACCAUUCUUGGGAGGACANN	1896	UGUCCUCCCAAGAAUGGUUNN	29

1147-1165	ACCAUUCUUGGGAGGACACNN	1897	GUGUCCUCCCAAGAAUGGUNN	29

1148-1166	CCAUUCUUGGGAGGACACUNN	1898	AGUGUCCUCCCAAGAAUGGNN	29

1149-1167	CAUUCUUGGGAGGACACUUNN	1899	AAGUGUCCUCCCAAGAAUGNN	29

1150-1168	AUUCUUGGGAGGACACUUUNN	1900	AAAGUGUCCUCCCAAGAAUNN	29

1151-1169	UUCUUGGGAGGACACUUUUNN	1901	AAAAGUGUCCUCCCAAGAANN	29

115-133	CAGCCCGCCUCCGCCGCCGNN	1902	CGGCGGCGGAGGCGGGCUGNN	29

1152-1170	UCUUGGGAGGACACUUUUGNN	1903	CAAAAGUGUCCUCCCAAGANN	29

1153-1171	CUUGGGAGGACACUUUUGCNN	1904	GCAAAAGUGUCCUCCCAAGNN	29

1154-1172	UUGGGAGGACACUUUUGCCNN	1905	GGCAAAAGUGUCCUCCCAANN	29

1155-1173	UGGGAGGACACUUUUGCCANN	1906	UGGCAAAAGUGUCCUCCCANN	29

1156-1174	GGGAGGACACUUUUGCCAANN	1907	UUGGCAAAAGUGUCCUCCCNN	29

1157-1175	GGAGGACACUUUUGCCAAUNN	1908	AUUGGCAAAAGUGUCCUCCNN	29

1158-1176	GAGGACACUUUUGCCAAUGNN	1909	CAUUGGCAAAAGUGUCCUCNN	29

1159-1177	AGGACACUUUUGCCAAUGANN	1910	UCAUUGGCAAAAGUGUCCUNN	29

1160-1178	GGACACUUUUGCCAAUGAANN	1911	UUCAUUGGCAAAAGUGUCCNN	29

1161-1179	GACACUUUUGCCAAUGAACNN	1912	GUUCAUUGGCAAAAGUGUCNN	29

116-134	AGCCCGCCUCCGCCGCCGGNN	1913	CCGGCGGCGGAGGCGGGCUNN	29

1162-1180	ACACUUUUGCCAAUGAACUNN	1914	AGUUCAUUGGCAAAAGUGUNN	29

117-135	GCCCGCCUCCGCCGCCGGANN	1915	UCCGGCGGCGGAGGCGGGCNN	29

118-136	CCCGCCUCCGCCGCCGGAGNN	1916	CUCCGGCGGCGGAGGCGGGNN	29

1182-1200	UUUCCCCAGCUGAUUAGUGNN	1917	CACUAAUCAGCUGGGGAAANN	29

1183-1201	UUCCCCAGCUGAUUAGUGUNN	1918	ACACUAAUCAGCUGGGGAANN	29

1184-1202	UCCCCAGCUGAUUAGUGUCNN	1919	GACACUAAUCAGCUGGGGANN	29

1185-1203	CCCCAGCUGAUUAGUGUCUNN	1920	AGACACUAAUCAGCUGGGGNN	29

1186-1204	CCCAGCUGAUUAGUGUCUANN	1921	UAGACACUAAUCAGCUGGGNN	29

1187-1205	CCAGCUGAUUAGUGUCUAANN	1922	UUAGACACUAAUCAGCUGGNN	29

1188-1206	CAGCUGAUUAGUGUCUAAGNN	1923	CUUAGACACUAAUCAGCUGNN	29

1189-1207	AGCUGAUUAGUGUCUAAGGNN	1924	CCUUAGACACUAAUCAGCUNN	29

1190-1208	GCUGAUUAGUGUCUAAGGANN	1925	UCCUUAGACACUAAUCAGCNN	29

1191-1209	CUGAUUAGUGUCUAAGGAANN	1926	UUCCUUAGACACUAAUCAGNN	29

119-137	CCGCCUCCGCCGCCGGAGCNN	1927	GCUCCGGCGGCGGAGGCGGNN	29

1192-1210	UGAUUAGUGUCUAAGGAAUNN	1928	AUUCCUUAGACACUAAUCANN	29

1193-1211	GAUUAGUGUCUAAGGAAUGNN	1929	CAUUCCUUAGACACUAAUCNN	29

1194-1212	AUUAGUGUCUAAGGAAUGANN	1930	UCAUUCCUUAGACACUAAUNN	29

1195-1213	UUAGUGUCUAAGGAAUGAUNN	1931	AUCAUUCCUUAGACACUAANN	29

1196-1214	UAGUGUCUAAGGAAUGAUCNN	1932	GAUCAUUCCUUAGACACUANN	29

1197-1215	AGUGUCUAAGGAAUGAUCCNN	1933	GGAUCAUUCCUUAGACACUNN	29

1198-1216	GUGUCUAAGGAAUGAUCCANN	1934	UGGAUCAUUCCUUAGACACNN	29

120-138	CGCCUCCGCCGCCGGAGCCNN	1935	GGCUCCGGCGGCGGAGGCGNN	29

121-139	GCCUCCGCCGCCGGAGCCCNN	1936	GGGCUCCGGCGGCGGAGGCNN	29

1218-1236	UACUGUUGCCCUUUUCCUUNN	1937	AAGGAAAAGGGCAACAGUANN	29

1219-1237	ACUGUUGCCCUUUUCCUUGNN	1938	CAAGGAAAAGGGCAACAGUNN	29

1220-1238	CUGUUGCCCUUUUCCUUGANN	1939	UCAAGGAAAAGGGCAACAGNN	29

1221-1239	UGUUGCCCUUUUCCUUGACNN	1940	GUCAAGGAAAAGGGCAACANN	29

122-140	CCUCCGCCGCCGGAGCCCCNN	1941	GGGGCUCCGGCGGCGGAGGNN	30

1222-1240	GUUGCCCUUUUCCUUGACUNN	1942	AGUCAAGGAAAAGGGCAACNN	30

1223-1241	UUGCCCUUUUCCUUGACUANN	1943	UAGUCAAGGAAAAGGGCAANN	30

1224-1242	UGCCCUUUUCCUUGACUAUNN	1944	AUAGUCAAGGAAAAGGGCANN	30

1225-1243	GCCCUUUUCCUUGACUAUUNN	1945	AAUAGUCAAGGAAAAGGGCNN	30

1226-1244	CCCUUUUCCUUGACUAUUANN	1946	UAAUAGUCAAGGAAAAGGGNN	30

1227-1245	CCUUUUCCUUGACUAUUACNN	1947	GUAAUAGUCAAGGAAAAGGNN	30

1228-1246	CUUUUCCUUGACUAUUACANN	1948	UGUAAUAGUCAAGGAAAAGNN	30

1229-1247	UUUUCCUUGACUAUUACACNN	1949	GUGUAAUAGUCAAGGAAAANN	30

1230-1248	UUUCCUUGACUAUUACACUNN	1950	AGUGUAAUAGUCAAGGAAANN	30

1231-1249	UUCCUUGACUAUUACACUGNN	1951	CAGUGUAAUAGUCAAGGAANN	30

123-141	CUCCGCCGCCGGAGCCCCGNN	1952	CGGGGCUCCGGCGGCGGAGNN	30

1232-1250	UCCUUGACUAUUACACUGCNN	1953	GCAGUGUAAUAGUCAAGGANN	30

1233-1251	CCUUGACUAUUACACUGCCNN	1954	GGCAGUGUAAUAGUCAAGGNN	30

1234-1252	CUUGACUAUUACACUGCCUNN	1955	AGGCAGUGUAAUAGUCAAGNN	30

1235-1253	UUGACUAUUACACUGCCUGNN	1956	CAGGCAGUGUAAUAGUCAANN	30

1236-1254	UGACUAUUACACUGCCUGGNN	1957	CCAGGCAGUGUAAUAGUCANN	30

1237-1255	GACUAUUACACUGCCUGGANN	1958	UCCAGGCAGUGUAAUAGUCNN	30

1238-1256	ACUAUUACACUGCCUGGAGNN	1959	CUCCAGGCAGUGUAAUAGUNN	30

1239-1257	CUAUUACACUGCCUGGAGGNN	1960	CCUCCAGGCAGUGUAAUAGNN	30

1240-1258	UAUUACACUGCCUGGAGGANN	1961	UCCUCCAGGCAGUGUAAUANN	30

1241-1259	AUUACACUGCCUGGAGGAUNN	1962	AUCCUCCAGGCAGUGUAAUNN	30

124-142	UCCGCCGCCGGAGCCCCGGNN	1963	CCGGGGCUCCGGCGGCGGANN	30

1242-1260	UUACACUGCCUGGAGGAUANN	1964	UAUCCUCCAGGCAGUGUAANN	30

1243-1261	UACACUGCCUGGAGGAUAGNN	1965	CUAUCCUCCAGGCAGUGUANN	30

1244-1262	ACACUGCCUGGAGGAUAGCNN	1966	GCUAUCCUCCAGGCAGUGUNN	30

1245-1263	CACUGCCUGGAGGAUAGCANN	1967	UGCUAUCCUCCAGGCAGUGNN	30

1246-1264	ACUGCCUGGAGGAUAGCAGNN	1968	CUGCUAUCCUCCAGGCAGUNN	30

125-143	CCGCCGCCGGAGCCCCGGCNN	1969	GCCGGGGCTCCGGCGGCGGNN	30

126-144	CGCCGCCGGAGCCCCGGCCNN	1970	GGCCGGGGCTCCGGCGGCGNN	30

127-145	GCCGCCGGAGCCCCGGCCGNN	1971	CGGCCGGGGCTCCGGCGGCNN	30

1280-1298	CUUCAUUCAAAAAGCCAAANN	1972	UUUGGCUUUUUGAAUGAAGNN	30

1281-1299	UUCAUUCAAAAAGCCAAAANN	1973	UUUUGGCUUUUUGAAUGAANN	30

128-146	CCGCCGGAGCCCCGGCCGGNN	1974	CCGGCCGGGGCTCCGGCGGNN	30

1282-1300	UCAUUCAAAAAGCCAAAAUNN	1975	AUUUUGGCUUUUUGAAUGANN	30

1283-1301	CAUUCAAAAAGCCAAAAUANN	1976	UAUUUUGGCUUUUUGAAUGNN	30

1284-1302	AUUCAAAAAGCCAAAAUAGNN	1977	CUAUUUUGGCUUUUUGAAUNN	30

1285-1303	UUCAAAAAGCCAAAAUAGANN	1978	UCUAUUUUGGCUUUUUGAANN	30

1286-1304	UCAAAAAGCCAAAAUAGAGNN	1979	CUCUAUUUUGGCUUUUUGANN	30

1287-1305	CAAAAAGCCAAAAUAGAGANN	1980	UCUCUAUUUUGGCUUUUUGNN	30

1288-1306	AAAAAGCCAAAAUAGAGAGNN	1981	CUCUCUAUUUUGGCUUUUUNN	30

1289-1307	AAAAGCCAAAAUAGAGAGUNN	1982	ACUCUCUAUUUUGGCUUUUNN	30

1290-1308	AAAGCCAAAAUAGAGAGUANN	1983	UACUCUCUAUUUUGGCUUUNN	30

129-147	CGCCGGAGCCCCGGCCGGCNN	1984	GCCGGCCGGGGCTCCGGCGNN	30

130-148	GCCGGAGCCCCGGCCGGCCNN	1985	GGCCGGCCGGGGCTCCGGCNN	30

1310-1328	ACAGUCCUAGAGAAUUCCUNN	1986	AGGAAUUCUCUAGGACUGUNN	30

131-149	CCGGAGCCCCGGCCGGCCANN	1987	TGGCCGGCCGGGGCTCCGGNN	30

132-150	CGGAGCCCCGGCCGGCCAGNN	1988	CTGGCCGGCCGGGGCTCCGNN	30

1330-1348	UAUUUGUUCAGAUCUCAUANN	1989	UAUGAGAUCUGAACAAAUANN	30

1331-1349	AUUUGUUCAGAUCUCAUAGNN	1990	CUAUGAGAUCUGAACAAAUNN	30

133-151	GGAGCCCCGGCCGGCCAGGNN	1991	CCTGGCCGGCCGGGGCTCCNN	30

1332-1350	UUUGUUCAGAUCUCAUAGANN	1992	UCUAUGAGAUCUGAACAAANN	30

1333-1351	UUGUUCAGAUCUCAUAGAUNN	1993	AUCUAUGAGAUCUGAACAANN	30

1334-1352	UGUUCAGAUCUCAUAGAUGNN	1994	CAUCUAUGAGAUCUGAACANN	30

1335-1353	GUUCAGAUCUCAUAGAUGANN	1995	UCAUCUAUGAGAUCUGAACNN	30

134-152	GAGCCCCGGCCGGCCAGGCNN	1996	GCCTGGCCGGCCGGGGCTCNN	30

135-153	AGCCCCGGCCGGCCAGGCCNN	1997	GGCCTGGCCGGCCGGGGCTNN	30

136-154	GCCCCGGCCGGCCAGGCCCNN	1998	GGGCCTGGCCGGCCGGGGCNN	30

1365-1383	UGUCUUUUGACAUCCAGCANN	1999	UGCUGGAUGUCAAAAGACANN	30

1366-1384	GUCUUUUGACAUCCAGCAGNN	2000	CUGCUGGAUGUCAAAAGACNN	30

1367-1385	UCUUUUGACAUCCAGCAGUNN	2001	ACUGCUGGAUGUCAAAAGANN	30

1368-1386	CUUUUGACAUCCAGCAGUCNN	2002	GACUGCUGGAUGUCAAAAGNN	30

1369-1387	UUUUGACAUCCAGCAGUCCNN	2003	GGACUGCUGGAUGUCAAAANN	30

1370-1388	UUUGACAUCCAGCAGUCCANN	2004	UGGACUGCUGGAUGUCAAANN	30

1371-1389	UUGACAUCCAGCAGUCCAANN	2005	UUGGACUGCUGGAUGUCAANN	30

137-155	CCCCGGCCGGCCAGGCCCUNN	2006	AGGGCCUGGCCGGCCGGGGNN	30

138-156	CCCGGCCGGCCAGGCCCUGNN	2007	CAGGGCCUGGCCGGCCGGGNN	30

1391-1409	GUAUUGAGACAUAUUACUGNN	2008	CAGUAAUAUGUCUCAAUACNN	30

139-157	CCGGCCGGCCAGGCCCUGCNN	2009	GCAGGGCCUGGCCGGCCGGNN	30

140-158	CGGCCGGCCAGGCCCUGCCNN	2010	GGCAGGGCCUGGCCGGCCGNN	30

141-159	GGCCGGCCAGGCCCUGCCGNN	2011	CGGCAGGGCCUGGCCGGCCNN	30

1414-1432	UAAGAAAUAUUACUAUAAUNN	2012	AUUAUAGUAAUAUUUCUUANN	30

1415-1433	AAGAAAUAUUACUAUAAUUNN	2013	AAUUAUAGUAAUAUUUCUUNN	30

1416-1434	AGAAAUAUUACUAUAAUUGNN	2014	CAAUUAUAGUAAUAUUUCUNN	30

1417-1435	GAAAUAUUACUAUAAUUGANN	2015	UCAAUUAUAGUAAUAUUUCNN	30

1418-1436	AAAUAUUACUAUAAUUGAGNN	2016	CUCAAUUAUAGUAAUAUUUNN	30

1419-1437	AAUAUUACUAUAAUUGAGANN	2017	UCUCAAUUAUAGUAAUAUUNN	30

1420-1438	AUAUUACUAUAAUUGAGAANN	2018	UUCUCAAUUAUAGUAAUAUNN	30

1421-1439	UAUUACUAUAAUUGAGAACNN	2019	GUUCUCAAUUAUAGUAAUANN	30

142-160	GCCGGCCAGGCCCUGCCGCNN	2020	GCGGCAGGGCCUGGCCGGCNN	30

1422-1440	AUUACUAUAAUUGAGAACUNN	2021	AGUUCUCAAUUAUAGUAAUNN	30

1423-1441	UUACUAUAAUUGAGAACUANN	2022	UAGUUCUCAAUUAUAGUAANN	30

1424-1442	UACUAUAAUUGAGAACUACNN	2023	GUAGUUCUCAAUUAUAGUANN	30

1425-1443	ACUAUAAUUGAGAACUACANN	2024	UGUAGUUCUCAAUUAUAGUNN	30

1426-1444	CUAUAAUUGAGAACUACAGNN	2025	CUGUAGUUCUCAAUUAUAGNN	30

1427-1445	UAUAAUUGAGAACUACAGCNN	2026	GCUGUAGUUCUCAAUUAUANN	30

1428-1446	AUAAUUGAGAACUACAGCUNN	2027	AGCUGUAGUUCUCAAUUAUNN	30

1429-1447	UAAUUGAGAACUACAGCUUNN	2028	AAGCUGUAGUUCUCAAUUANN	30

1430-1448	AAUUGAGAACUACAGCUUUNN	2029	AAAGCUGUAGUUCUCAAUUNN	30

1431-1449	AUUGAGAACUACAGCUUUUNN	2030	AAAAGCUGUAGUUCUCAAUNN	30

143-161	CCGGCCAGGCCCUGCCGCUNN	2031	AGCGGCAGGGCCUGGCCGGNN	30

1432-1450	UUGAGAACUACAGCUUUUANN	2032	UAAAAGCUGUAGUUCUCAANN	30

1433-1451	UGAGAACUACAGCUUUUAANN	2033	UUAAAAGCUGUAGUUCUCANN	30

1434-1452	GAGAACUACAGCUUUUAAGNN	2034	CUUAAAAGCUGUAGUUCUCNN	30

1435-1453	AGAACUACAGCUUUUAAGANN	2035	UCUUAAAAGCUGUAGUUCUNN	30

1436-1454	GAACUACAGCUUUUAAGAUNN	2036	AUCUUAAAAGCUGUAGUUCNN	30

1437-1455	AACUACAGCUUUUAAGAUUNN	2037	AAUCUUAAAAGCUGUAGUUNN	30

1438-1456	ACUACAGCUUUUAAGAUUGNN	2038	CAAUCUUAAAAGCUGUAGUNN	30

1439-1457	CUACAGCUUUUAAGAUUGUNN	2039	ACAAUCUUAAAAGCUGUAGNN	30

1440-1458	UACAGCUUUUAAGAUUGUANN	2040	UACAAUCUUAAAAGCUGUANN	30

1441-1459	ACAGCUUUUAAGAUUGUACNN	2041	GUACAAUCUUAAAAGCUGUNN	31

144-162	CGGCCAGGCCCUGCCGCUCNN	2042	GAGCGGCAGGGCCUGGCCGNN	31

1442-1460	CAGCUUUUAAGAUUGUACUNN	2043	AGUACAAUCUUAAAAGCUGNN	31

1443-1461	AGCUUUUAAGAUUGUACUUNN	2044	AAGUACAAUCUUAAAAGCUNN	31

1444-1462	GCUUUUAAGAUUGUACUUUNN	2045	AAAGUACAAUCUUAAAAGCNN	31

1445-1463	CUUUUAAGAUUGUACUUUUNN	2046	AAAAGUACAAUCUUAAAAGNN	31

1446-1464	UUUUAAGAUUGUACUUUUANN	2047	UAAAAGUACAAUCUUAAAANN	31

1447-1465	UUUAAGAUUGUACUUUUAUNN	2048	AUAAAAGUACAAUCUUAAANN	31

1448-1466	UUAAGAUUGUACUUUUAUCNN	2049	GAUAAAAGUACAAUCUUAANN	31

1449-1467	UAAGAUUGUACUUUUAUCUNN	2050	AGAUAAAAGUACAAUCUUANN	31

1450-1468	AAGAUUGUACUUUUAUCUUNN	2051	AAGAUAAAAGUACAAUCUUNN	31

1451-1469	AGAUUGUACUUUUAUCUUANN	2052	UAAGAUAAAAGUACAAUCUNN	31

145-163	GGCCAGGCCCUGCCGCUCANN	2053	UGAGCGGCAGGGCCUGGCCNN	31

1452-1470	GAUUGUACUUUUAUCUUAANN	2054	UUAAGAUAAAAGUACAAUCNN	31

1453-1471	AUUGUACUUUUAUCUUAAANN	2055	UUUAAGAUAAAAGUACAAUNN	31

1454-1472	UUGUACUUUUAUCUUAAAANN	2056	UUUUAAGAUAAAAGUACAANN	31

1455-1473	UGUACUUUUAUCUUAAAAGNN	2057	CUUUUAAGAUAAAAGUACANN	31

1456-1474	GUACUUUUAUCUUAAAAGGNN	2058	CCUUUUAAGAUAAAAGUACNN	31

1457-1475	UACUUUUAUCUUAAAAGGGNN	2059	CCCUUUUAAGAUAAAAGUANN	31

1458-1476	ACUUUUAUCUUAAAAGGGUNN	2060	ACCCUUUUAAGAUAAAAGUNN	31

1459-1477	CUUUUAUCUUAAAAGGGUGNN	2061	CACCCUUUUAAGAUAAAAGNN	31

1460-1478	UUUUAUCUUAAAAGGGUGGNN	2062	CCACCCUUUUAAGAUAAAANN	31

1461-1479	UUUAUCUUAAAAGGGUGGUNN	2063	ACCACCCUUUUAAGAUAAANN	31

146-164	GCCAGGCCCUGCCGCUCAUNN	2064	AUGAGCGGCAGGGCCUGGCNN	31

1462-1480	UUAUCUUAAAAGGGUGGUANN	2065	UACCACCCUUUUAAGAUAANN	31

1463-1481	UAUCUUAAAAGGGUGGUAGNN	2066	CUACCACCCUUUUAAGAUANN	31

1464-1482	AUCUUAAAAGGGUGGUAGUNN	2067	ACUACCACCCUUUUAAGAUNN	31

1465-1483	UCUUAAAAGGGUGGUAGUUNN	2068	AACUACCACCCUUUUAAGANN	31

1466-1484	CUUAAAAGGGUGGUAGUUUNN	2069	AAACUACCACCCUUUUAAGNN	31

147-165	CCAGGCCCUGCCGCUCAUGNN	2070	CAUGAGCGGCAGGGCCUGGNN	31

148-166	CAGGCCCUGCCGCUCAUGGNN	2071	CCAUGAGCGGCAGGGCCUGNN	31

1486-1504	CCCUAAAAUACUUAUUAUGNN	2072	CAUAAUAAGUAUUUUAGGGNN	31

1487-1505	CCUAAAAUACUUAUUAUGUNN	2073	ACAUAAUAAGUAUUUUAGGNN	31

1488-1506	CUAAAAUACUUAUUAUGUANN	2074	UACAUAAUAAGUAUUUUAGNN	31

1489-1507	UAAAAUACUUAUUAUGUAANN	2075	UUACAUAAUAAGUAUUUUANN	31

1490-1508	AAAAUACUUAUUAUGUAAGNN	2076	CUUACAUAAUAAGUAUUUUNN	31

1491-1509	AAAUACUUAUUAUGUAAGGNN	2077	CCUUACAUAAUAAGUAUUUNN	31

149-167	AGGCCCUGCCGCUCAUGGUNN	2078	ACCAUGAGCGGCAGGGCCUNN	31

1492-1510	AAUACUUAUUAUGUAAGGGNN	2079	CCCUUACAUAAUAAGUAUUNN	31

1493-1511	AUACUUAUUAUGUAAGGGUNN	2080	ACCCUUACAUAAUAAGUAUNN	31

1494-1512	UACUUAUUAUGUAAGGGUCNN	2081	GACCCUUACAUAAUAAGUANN	31

1495-1513	ACUUAUUAUGUAAGGGUCANN	2082	UGACCCUUACAUAAUAAGUNN	31

1496-1514	CUUAUUAUGUAAGGGUCAUNN	2083	AUGACCCUUACAUAAUAAGNN	31

1497-1515	UUAUUAUGUAAGGGUCAUUNN	2084	AAUGACCCUUACAUAAUAANN	31

1498-1516	UAUUAUGUAAGGGUCAUUANN	2085	UAAUGACCCUUACAUAAUANN	31

1499-1517	AUUAUGUAAGGGUCAUUAGNN	2086	CUAAUGACCCUUACAUAAUNN	31

1500-1518	UUAUGUAAGGGUCAUUAGANN	2087	UCUAAUGACCCUUACAUAANN	31

1501-1519	UAUGUAAGGGUCAUUAGACNN	2088	GUCUAAUGACCCUUACAUANN	31

150-168	GGCCCUGCCGCUCAUGGUGNN	2089	CACCAUGAGCGGCAGGGCCNN	31

1502-1520	AUGUAAGGGUCAUUAGACANN	2090	UGUCUAAUGACCCUUACAUNN	31

1503-1521	UGUAAGGGUCAUUAGACAANN	2091	UUGUCUAAUGACCCUUACANN	31

1504-1522	GUAAGGGUCAUUAGACAAANN	2092	UUUGUCUAAUGACCCUUACNN	31

1505-1523	UAAGGGUCAUUAGACAAAUNN	2093	AUUUGUCUAAUGACCCUUANN	31

1506-1524	AAGGGUCAUUAGACAAAUGNN	2094	CAUUUGUCUAAUGACCCUUNN	31

1507-1525	AGGGUCAUUAGACAAAUGUNN	2095	ACAUUUGUCUAAUGACCCUNN	31

1508-1526	GGGUCAUUAGACAAAUGUCNN	2096	GACAUUUGUCUAAUGACCCNN	31

1509-1527	GGUCAUUAGACAAAUGUCUNN	2097	AGACAUUUGUCUAAUGACCNN	31

1510-1528	GUCAUUAGACAAAUGUCUUNN	2098	AAGACAUUUGUCUAAUGACNN	31

1511-1529	UCAUUAGACAAAUGUCUUGNN	2099	CAAGACAUUUGUCUAAUGANN	31

151-169	GCCCUGCCGCUCAUGGUGCNN	2100	GCACCAUGAGCGGCAGGGCNN	31

1512-1530	CAUUAGACAAAUGUCUUGANN	2101	UCAAGACAUUUGUCUAAUGNN	31

1513-1531	AUUAGACAAAUGUCUUGAANN	2102	UUCAAGACAUUUGUCUAAUNN	31

1514-1532	UUAGACAAAUGUCUUGAAGNN	2103	CUUCAAGACAUUUGUCUAANN	31

1515-1533	UAGACAAAUGUCUUGAAGUNN	2104	ACUUCAAGACAUUUGUCUANN	31

1516-1534	AGACAAAUGUCUUGAAGUANN	2105	UACUUCAAGACAUUUGUCUNN	31

1517-1535	GACAAAUGUCUUGAAGUAGNN	2106	CUACUUCAAGACAUUUGUCNN	31

1518-1536	ACAAAUGUCUUGAAGUAGANN	2107	UCUACUUCAAGACAUUUGUNN	31

152-170	CCCUGCCGCUCAUGGUGCCNN	2108	GGCACCAUGAGCGGCAGGGNN	31

153-171	CCUGCCGCUCAUGGUGCCANN	2109	UGGCACCAUGAGCGGCAGGNN	31

1541-1559	GAAUUUAUGAAUGGUUCUUNN	2110	AAGAACCAUUCAUAAAUUCNN	31

154-172	CUGCCGCUCAUGGUGCCAGNN	2111	CUGGCACCAUGAGCGGCAGNN	31

1542-1560	AAUUUAUGAAUGGUUCUUUNN	2112	AAAGAACCAUUCAUAAAUUNN	31

1543-1561	AUUUAUGAAUGGUUCUUUANN	2113	UAAAGAACCAUUCAUAAAUNN	31

1544-1562	UUUAUGAAUGGUUCUUUAUNN	2114	AUAAAGAACCAUUCAUAAANN	31

1545-1563	UUAUGAAUGGUUCUUUAUCNN	2115	GAUAAAGAACCAUUCAUAANN	31

1546-1564	UAUGAAUGGUUCUUUAUCANN	2116	UGAUAAAGAACCAUUCAUANN	31

1547-1565	AUGAAUGGUUCUUUAUCAUNN	2117	AUGAUAAAGAACCAUUCAUNN	31

1548-1566	UGAAUGGUUCUUUAUCAUUNN	2118	AAUGAUAAAGAACCAUUCANN	31

1549-1567	GAAUGGUUCUUUAUCAUUUNN	2119	AAAUGAUAAAGAACCAUUCNN	31

1550-1568	AAUGGUUCUUUAUCAUUUCNN	2120	GAAAUGAUAAAGAACCAUUNN	31

1551-1569	AUGGUUCUUUAUCAUUUCUNN	2121	AGAAAUGAUAAAGAACCAUNN	31

155-173	UGCCGCUCAUGGUGCCAGCNN	2122	GCUGGCACCAUGAGCGGCANN	31

1552-1570	UGGUUCUUUAUCAUUUCUCNN	2123	GAGAAAUGAUAAAGAACCANN	31

1553-1571	GGUUCUUUAUCAUUUCUCUNN	2124	AGAGAAAUGAUAAAGAACCNN	31

1554-1572	GUUCUUUAUCAUUUCUCUUNN	2125	AAGAGAAAUGAUAAAGAACNN	31

1555-1573	UUCUUUAUCAUUUCUCUUCNN	2126	GAAGAGAAAUGAUAAAGAANN	31

1556-1574	UCUUUAUCAUUUCUCUUCCNN	2127	GGAAGAGAAAUGAUAAAGANN	31

1557-1575	CUUUAUCAUUUCUCUUCCCNN	2128	GGGAAGAGAAAUGAUAAAGNN	31

1558-1576	UUUAUCAUUUCUCUUCCCCNN	2129	GGGGAAGAGAAAUGAUAAANN	31

1559-1577	UUAUCAUUUCUCUUCCCCCNN	2130	GGGGGAAGAGAAAUGAUAANN	31

1560-1578	UAUCAUUUCUCUUCCCCCUNN	2131	AGGGGGAAGAGAAAUGAUANN	31

1561-1579	AUCAUUUCUCUUCCCCCUUNN	2132	AAGGGGGAAGAGAAAUGAUNN	31

156-174	GCCGCUCAUGGUGCCAGCCNN	2133	GGCUGGCACCAUGAGCGGCNN	31

1562-1580	UCAUUUCUCUUCCCCCUUUNN	2134	AAAGGGGGAAGAGAAAUGANN	31

1563-1581	CAUUUCUCUUCCCCCUUUUNN	2135	AAAAGGGGGAAGAGAAAUGNN	31

1564-1582	AUUUCUCUUCCCCCUUUUUNN	2136	AAAAAGGGGGAAGAGAAAUNN	31

1565-1583	UUUCUCUUCCCCCUUUUUGNN	2137	CAAAAAGGGGGAAGAGAAANN	31

1566-1584	UUCUCUUCCCCCUUUUUGGNN	2138	CCAAAAAGGGGGAAGAGAANN	31

1567-1585	UCUCUUCCCCCUUUUUGGCNN	2139	GCCAAAAAGGGGGAAGAGANN	31

1568-1586	CUCUUCCCCCUUUUUGGCANN	2140	UGCCAAAAAGGGGGAAGAGNN	31

1569-1587	UCUUCCCCCUUUUUGGCAUNN	2141	AUGCCAAAAAGGGGGAAGANN	32

1570-1588	CUUCCCCCUUUUUGGCAUCNN	2142	GAUGCCAAAAAGGGGGAAGNN	32

1571-1589	UUCCCCCUUUUUGGCAUCCNN	2143	GGAUGCCAAAAAGGGGGAANN	32

157-175	CCGCUCAUGGUGCCAGCCCNN	2144	GGGCUGGCACCAUGAGCGGNN	32

1572-1590	UCCCCCUUUUUGGCAUCCUNN	2145	AGGAUGCCAAAAAGGGGGANN	32

1573-1591	CCCCCUUUUUGGCAUCCUGNN	2146	CAGGAUGCCAAAAAGGGGGNN	32

1574-1592	CCCCUUUUUGGCAUCCUGGNN	2147	CCAGGAUGCCAAAAAGGGGNN	32

1575-1593	CCCUUUUUGGCAUCCUGGCNN	2148	GCCAGGAUGCCAAAAAGGGNN	32

1576-1594	CCUUUUUGGCAUCCUGGCUNN	2149	AGCCAGGAUGCCAAAAAGGNN	32

1577-1595	CUUUUUGGCAUCCUGGCUUNN	2150	AAGCCAGGAUGCCAAAAAGNN	32

1578-1596	UUUUUGGCAUCCUGGCUUGNN	2151	CAAGCCAGGAUGCCAAAAANN	32

1579-1597	UUUUGGCAUCCUGGCUUGCNN	2152	GCAAGCCAGGAUGCCAAAANN	32

1580-1598	UUUGGCAUCCUGGCUUGCCNN	2153	GGCAAGCCAGGAUGCCAAANN	32

1581-1599	UUGGCAUCCUGGCUUGCCUNN	2154	AGGCAAGCCAGGAUGCCAANN	32

158-176	CGCUCAUGGUGCCAGCCCANN	2155	UGGGCUGGCACCAUGAGCGNN	32

1582-1600	UGGCAUCCUGGCUUGCCUCNN	2156	GAGGCAAGCCAGGAUGCCANN	32

1583-1601	GGCAUCCUGGCUUGCCUCCNN	2157	GGAGGCAAGCCAGGAUGCCNN	32

1584-1602	GCAUCCUGGCUUGCCUCCANN	2158	UGGAGGCAAGCCAGGAUGCNN	32

1585-1603	CAUCCUGGCUUGCCUCCAGNN	2159	CUGGAGGCAAGCCAGGAUGNN	32

1586-1604	AUCCUGGCUUGCCUCCAGUNN	2160	ACUGGAGGCAAGCCAGGAUNN	32

1587-1605	UCCUGGCUUGCCUCCAGUUNN	2161	AACUGGAGGCAAGCCAGGANN	32

1588-1606	CCUGGCUUGCCUCCAGUUUNN	2162	AAACUGGAGGCAAGCCAGGNN	32

1589-1607	CUGGCUUGCCUCCAGUUUUNN	2163	AAAACUGGAGGCAAGCCAGNN	32

1590-1608	UGGCUUGCCUCCAGUUUUANN	2164	UAAAACUGGAGGCAAGCCANN	32

1591-1609	GGCUUGCCUCCAGUUUUAGNN	2165	CUAAAACUGGAGGCAAGCCNN	32

159-177	GCUCAUGGUGCCAGCCCAGNN	2166	CUGGGCUGGCACCAUGAGCNN	32

1592-1610	GCUUGCCUCCAGUUUUAGGNN	2167	CCUAAAACUGGAGGCAAGCNN	32

1593-1611	CUUGCCUCCAGUUUUAGGUNN	2168	ACCUAAAACUGGAGGCAAGNN	32

1594-1612	UUGCCUCCAGUUUUAGGUCNN	2169	GACCUAAAACUGGAGGCAANN	32

1595-1613	UGCCUCCAGUUUUAGGUCCNN	2170	GGACCUAAAACUGGAGGCANN	32

160-178	CUCAUGGUGCCAGCCCAGANN	2171	UCUGGGCUGGCACCAUGAGNN	32

161-179	UCAUGGUGCCAGCCCAGAGNN	2172	CUCUGGGCUGGCACCAUGANN	32

1615-1633	UUAGUUUGCUUCUGUAAGCNN	2173	GCUUACAGAAGCAAACUAANN	32

1616-1634	UAGUUUGCUUCUGUAAGCANN	2174	UGCUUACAGAAGCAAACUANN	32

1617-1635	AGUUUGCUUCUGUAAGCAANN	2175	UUGCUUACAGAAGCAAACUNN	32

162-180	CAUGGUGCCAGCCCAGAGANN	2176	UCUCUGGGCUGGCACCAUGNN	32

163-181	AUGGUGCCAGCCCAGAGAGNN	2177	CUCUCUGGGCUGGCACCAUNN	32

1639-1657	GAACACCUGCUGAGGGGGCNN	2178	GCCCCCUCAGCAGGUGUUCNN	32

1640-1658	AACACCUGCUGAGGGGGCUNN	2179	AGCCCCCUCAGCAGGUGUUNN	32

1641-1659	ACACCUGCUGAGGGGGCUCNN	2180	GAGCCCCCUCAGCAGGUGUNN	32

164-182	UGGUGCCAGCCCAGAGAGGNN	2181	CCUCUCUGGGCUGGCACCANN	32

1642-1660	CACCUGCUGAGGGGGCUCUNN	2182	AGAGCCCCCUCAGCAGGUGNN	32

1643-1661	ACCUGCUGAGGGGGCUCUUNN	2183	AAGAGCCCCCUCAGCAGGUNN	32

1644-1662	CCUGCUGAGGGGGCUCUUUNN	2184	AAAGAGCCCCCUCAGCAGGNN	32

1645-1663	CUGCUGAGGGGGCUCUUUCNN	2185	GAAAGAGCCCCCUCAGCAGNN	32

1646-1664	UGCUGAGGGGGCUCUUUCCNN	2186	GGAAAGAGCCCCCUCAGCANN	32

1647-1665	GCUGAGGGGGCUCUUUCCCNN	2187	GGGAAAGAGCCCCCUCAGCNN	32

1648-1666	CUGAGGGGGCUCUUUCCCUNN	2188	AGGGAAAGAGCCCCCUCAGNN	32

1649-1667	UGAGGGGGCUCUUUCCCUCNN	2189	GAGGGAAAGAGCCCCCUCANN	32

1650-1668	GAGGGGGCUCUUUCCCUCANN	2190	UGAGGGAAAGAGCCCCCUCNN	32

165-183	GGUGCCAGCCCAGAGAGGGNN	2191	CCCUCUCUGGGCUGGCACCNN	32

166-184	GUGCCAGCCCAGAGAGGGGNN	2192	CCCCUCUCUGGGCUGGCACNN	32

1670-1688	GUAUACUUCAAGUAAGAUCNN	2193	GAUCUUACUUGAAGUAUACNN	32

1671-1689	UAUACUUCAAGUAAGAUCANN	2194	UGAUCUUACUUGAAGUAUANN	32

167-185	UGCCAGCCCAGAGAGGGGCNN	2195	GCCCCUCUCUGGGCUGGCANN	32

1672-1690	AUACUUCAAGUAAGAUCAANN	2196	UUGAUCUUACUUGAAGUAUNN	32

1673-1691	UACUUCAAGUAAGAUCAAGNN	2197	CUUGAUCUUACUUGAAGUANN	32

1674-1692	ACUUCAAGUAAGAUCAAGANN	2198	UCUUGAUCUUACUUGAAGUNN	32

1675-1693	CUUCAAGUAAGAUCAAGAANN	2199	UUCUUGAUCUUACUUGAAGNN	32

1676-1694	UUCAAGUAAGAUCAAGAAUNN	2200	AUUCUUGAUCUUACUUGAANN	32

1677-1695	UCAAGUAAGAUCAAGAAUCNN	2201	GAUUCUUGAUCUUACUUGANN	32

1678-1696	CAAGUAAGAUCAAGAAUCUNN	2202	AGAUUCUUGAUCUUACUUGNN	32

1679-1697	AAGUAAGAUCAAGAAUCUUNN	2203	AAGAUUCUUGAUCUUACUUNN	32

1680-1698	AGUAAGAUCAAGAAUCUUUNN	2204	AAAGAUUCUUGAUCUUACUNN	32

1681-1699	GUAAGAUCAAGAAUCUUUUNN	2205	AAAAGAUUCUUGAUCUUACNN	32

1682-1700	UAAGAUCAAGAAUCUUUUGNN	2206	CAAAAGAUUCUUGAUCUUANN	32

1683-1701	AAGAUCAAGAAUCUUUUGUNN	2207	ACAAAAGAUUCUUGAUCUUNN	32

1684-1702	AGAUCAAGAAUCUUUUGUGNN	2208	CACAAAAGAUUCUUGAUCUNN	32

1685-1703	GAUCAAGAAUCUUUUGUGANN	2209	UCACAAAAGAUUCUUGAUCNN	32

1686-1704	AUCAAGAAUCUUUUGUGAANN	2210	UUCACAAAAGAUUCUUGAUNN	32

1687-1705	UCAAGAAUCUUUUGUGAAANN	2211	UUUCACAAAAGAUUCUUGANN	32

1707-1725	UAUAGAAAUUUACUAUGUANN	2212	UACAUAGUAAAUUUCUAUANN	32

1708-1726	AUAGAAAUUUACUAUGUAANN	2213	UUACAUAGUAAAUUUCUAUNN	32

1709-1727	UAGAAAUUUACUAUGUAAANN	2214	UUUACAUAGUAAAUUUCUANN	32

1710-1728	AGAAAUUUACUAUGUAAAUNN	2215	AUUUACAUAGUAAAUUUCUNN	32

1711-1729	GAAAUUUACUAUGUAAAUGNN	2216	CAUUUACAUAGUAAAUUUCNN	32

1712-1730	AAAUUUACUAUGUAAAUGCNN	2217	GCAUUUACAUAGUAAAUUUNN	32

1713-1731	AAUUUACUAUGUAAAUGCUNN	2218	AGCAUUUACAUAGUAAAUUNN	32

1714-1732	AUUUACUAUGUAAAUGCUUNN	2219	AAGCAUUUACAUAGUAAAUNN	32

1715-1733	UUUACUAUGUAAAUGCUUGNN	2220	CAAGCAUUUACAUAGUAAANN	32

1716-1734	UUACUAUGUAAAUGCUUGANN	2221	UCAAGCAUUUACAUAGUAANN	32

1717-1735	UACUAUGUAAAUGCUUGAUNN	2222	AUCAAGCAUUUACAUAGUANN	32

1718-1736	ACUAUGUAAAUGCUUGAUGNN	2223	CAUCAAGCAUUUACAUAGUNN	32

1719-1737	CUAUGUAAAUGCUUGAUGGNN	2224	CCAUCAAGCAUUUACAUAGNN	32

1720-1738	UAUGUAAAUGCUUGAUGGANN	2225	UCCAUCAAGCAUUUACAUANN	32

1721-1739	AUGUAAAUGCUUGAUGGAANN	2226	UUCCAUCAAGCAUUUACAUNN	32

1722-1740	UGUAAAUGCUUGAUGGAAUNN	2227	AUUCCAUCAAGCAUUUACANN	32

1723-1741	GUAAAUGCUUGAUGGAAUUNN	2228	AAUUCCAUCAAGCAUUUACNN	32

1724-1742	UAAAUGCUUGAUGGAAUUUNN	2229	AAAUUCCAUCAAGCAUUUANN	32

1725-1743	AAAUGCUUGAUGGAAUUUUNN	2230	AAAAUUCCAUCAAGCAUUUNN	32

1726-1744	AAUGCUUGAUGGAAUUUUUNN	2231	AAAAAUUCCAUCAAGCAUUNN	32

1727-1745	AUGCUUGAUGGAAUUUUUUNN	2232	AAAAAAUUCCAUCAAGCAUNN	32

1728-1746	UGCUUGAUGGAAUUUUUUCNN	2233	GAAAAAAUUCCAUCAAGCANN	32

1729-1747	GCUUGAUGGAAUUUUUUCCNN	2234	GGAAAAAAUUCCAUCAAGCNN	32

1730-1748	CUUGAUGGAAUUUUUUCCUNN	2235	AGGAAAAAAUUCCAUCAAGNN	32

1731-1749	UUGAUGGAAUUUUUUCCUGNN	2236	CAGGAAAAAAUUCCAUCAANN	32

1732-1750	UGAUGGAAUUUUUUCCUGCNN	2237	GCAGGAAAAAAUUCCAUCANN	32

1733-1751	GAUGGAAUUUUUUCCUGCUNN	2238	AGCAGGAAAAAAUUCCAUCNN	32

1734-1752	AUGGAAUUUUUUCCUGCUANN	2239	UAGCAGGAAAAAAUUCCAUNN	32

1735-1753	UGGAAUUUUUUCCUGCUAGNN	2240	CUAGCAGGAAAAAAUUCCANN	32

1736-1754	GGAAUUUUUUCCUGCUAGUNN	2241	ACUAGCAGGAAAAAAUUCCNN	33

1737-1755	GAAUUUUUUCCUGCUAGUGNN	2242	CACUAGCAGGAAAAAAUUCNN	33

1738-1756	AAUUUUUUCCUGCUAGUGUNN	2243	ACACUAGCAGGAAAAAAUUNN	33

1739-1757	AUUUUUUCCUGCUAGUGUANN	2244	UACACUAGCAGGAAAAAAUNN	33

1740-1758	UUUUUUCCUGCUAGUGUAGNN	2245	CUACACUAGCAGGAAAAAANN	33

1741-1759	UUUUUCCUGCUAGUGUAGCNN	2246	GCUACACUAGCAGGAAAAANN	33

1742-1760	UUUUCCUGCUAGUGUAGCUNN	2247	AGCUACACUAGCAGGAAAANN	33

1743-1761	UUUCCUGCUAGUGUAGCUUNN	2248	AAGCUACACUAGCAGGAAANN	33

1744-1762	UUCCUGCUAGUGUAGCUUCNN	2249	GAAGCUACACUAGCAGGAANN	33

1745-1763	UCCUGCUAGUGUAGCUUCUNN	2250	AGAAGCUACACUAGCAGGANN	33

1746-1764	CCUGCUAGUGUAGCUUCUGNN	2251	CAGAAGCUACACUAGCAGGNN	33

1747-1765	CUGCUAGUGUAGCUUCUGANN	2252	UCAGAAGCUACACUAGCAGNN	33

1748-1766	UGCUAGUGUAGCUUCUGAANN	2253	UUCAGAAGCUACACUAGCANN	33

1749-1767	GCUAGUGUAGCUUCUGAAANN	2254	UUUCAGAAGCUACACUAGCNN	33

1750-1768	CUAGUGUAGCUUCUGAAAGNN	2255	CUUUCAGAAGCUACACUAGNN	33

1751-1769	UAGUGUAGCUUCUGAAAGGNN	2256	CCUUUCAGAAGCUACACUANN	33

1752-1770	AGUGUAGCUUCUGAAAGGUNN	2257	ACCUUUCAGAAGCUACACUNN	33

1753-1771	GUGUAGCUUCUGAAAGGUGNN	2258	CACCUUUCAGAAGCUACACNN	33

1754-1772	UGUAGCUUCUGAAAGGUGCNN	2259	GCACCUUUCAGAAGCUACANN	33

1755-1773	GUAGCUUCUGAAAGGUGCUNN	2260	AGCACCUUUCAGAAGCUACNN	33

1756-1774	UAGCUUCUGAAAGGUGCUUNN	2261	AAGCACCUUUCAGAAGCUANN	33

1757-1775	AGCUUCUGAAAGGUGCUUUNN	2262	AAAGCACCUUUCAGAAGCUNN	33

1758-1776	GCUUCUGAAAGGUGCUUUCNN	2263	GAAAGCACCUUUCAGAAGCNN	33

1777-1795	UCCAUUUAUUUAAAACUACNN	2264	GUAGUUUUAAAUAAAUGGANN	33

1778-1796	CCAUUUAUUUAAAACUACCNN	2265	GGUAGUUUUAAAUAAAUGGNN	33

1779-1797	CAUUUAUUUAAAACUACCCNN	2266	GGGUAGUUUUAAAUAAAUGNN	33

1780-1798	AUUUAUUUAAAACUACCCANN	2267	UGGGUAGUUUUAAAUAAAUNN	33

1781-1799	UUUAUUUAAAACUACCCAUNN	2268	AUGGGUAGUUUUAAAUAAANN	33

1782-1800	UUAUUUAAAACUACCCAUGNN	2269	CAUGGGUAGUUUUAAAUAANN	33

1783-1801	UAUUUAAAACUACCCAUGCNN	2270	GCAUGGGUAGUUUUAAAUANN	33

1784-1802	AUUUAAAACUACCCAUGCANN	2271	UGCAUGGGUAGUUUUAAAUNN	33

1785-1803	UUUAAAACUACCCAUGCAANN	2272	UUGCAUGGGUAGUUUUAAANN	33

1786-1804	UUAAAACUACCCAUGCAAUNN	2273	AUUGCAUGGGUAGUUUUAANN	33

1787-1805	UAAAACUACCCAUGCAAUUNN	2274	AAUUGCAUGGGUAGUUUUANN	33

1788-1806	AAAACUACCCAUGCAAUUANN	2275	UAAUUGCAUGGGUAGUUUUNN	33

1789-1807	AAACUACCCAUGCAAUUAANN	2276	UUAAUUGCAUGGGUAGUUUNN	33

1790-1808	AACUACCCAUGCAAUUAAANN	2277	UUUAAUUGCAUGGGUAGUUNN	33

1791-1809	ACUACCCAUGCAAUUAAAANN	2278	UUUUAAUUGCAUGGGUAGUNN	33

1792-1810	CUACCCAUGCAAUUAAAAGNN	2279	CUUUUAAUUGCAUGGGUAGNN	33

1793-1811	UACCCAUGCAAUUAAAAGGNN	2280	CCUUUUAAUUGCAUGGGUANN	33

1794-1812	ACCCAUGCAAUUAAAAGGUNN	2281	ACCUUUUAAUUGCAUGGGUNN	33

1795-1813	CCCAUGCAAUUAAAAGGUANN	2282	UACCUUUUAAUUGCAUGGGNN	33

1796-1814	CCAUGCAAUUAAAAGGUACNN	2283	GUACCUUUUAAUUGCAUGGNN	33

1797-1815	CAUGCAAUUAAAAGGUACANN	2284	UGUACCUUUUAAUUGCAUGNN	33

1798-1816	AUGCAAUUAAAAGGUACAANN	2285	UUGUACCUUUUAAUUGCAUNN	33

1799-1817	UGCAAUUAAAAGGUACAAUNN	2286	AUUGUACCUUUUAAUUGCANN	33

1800-1818	GCAAUUAAAAGGUACAAUGNN	2287	CAUUGUACCUUUUAAUUGCNN	33

1801-1819	CAAUUAAAAGGUACAAUGCNN	2288	GCAUUGUACCUUUUAAUUGNN	33

1802-1820	AAUUAAAAGGUACAAUGCANN	2289	UGCAUUGUACCUUUUAAUUNN	33

187-205	AGCCCGGAGGCAGCGAGCGNN	2290	CGCTCGCTGCCTCCGGGCTNN	33

188-206	GCCCGGAGGCAGCGAGCGGNN	2291	CCGCTCGCTGCCTCCGGGCNN	33

189-207	CCCGGAGGCAGCGAGCGGGNN	2292	CCCGCTCGCTGCCTCCGGGNN	33

190-208	CCGGAGGCAGCGAGCGGGGNN	2293	CCCCGCTCGCTGCCTCCGGNN	33

191-209	CGGAGGCAGCGAGCGGGGGNN	2294	CCCCCGCTCGCTGCCTCCGNN	33

192-210	GGAGGCAGCGAGCGGGGGGNN	2295	CCCCCCGCTCGCTGCCTCCNN	33

193-211	GAGGCAGCGAGCGGGGGGCNN	2296	GCCCCCCGCTCGCTGCCTCNN	33

194-212	AGGCAGCGAGCGGGGGGCUNN	2297	AGCCCCCCGCUCGCUGCCUNN	33

195-213	GGCAGCGAGCGGGGGGCUGNN	2298	CAGCCCCCCGCUCGCUGCCNN	33

196-214	GCAGCGAGCGGGGGGCUGCNN	2299	GCAGCCCCCCGCUCGCUGCNN	33

197-215	CAGCGAGCGGGGGGCUGCCNN	2300	GGCAGCCCCCCGCUCGCUGNN	33

198-216	AGCGAGCGGGGGGCUGCCCNN	2301	GGGCAGCCCCCCGCUCGCUNN	33

199-217	GCGAGCGGGGGGCUGCCCCNN	2302	GGGGCAGCCCCCCGCUCGCNN	33

200-218	CGAGCGGGGGGCUGCCCCANN	2303	UGGGGCAGCCCCCCGCUCGNN	33

201-219	GAGCGGGGGGCUGCCCCAGNN	2304	CUGGGGCAGCCCCCCGCUCNN	33

202-220	AGCGGGGGGCUGCCCCAGGNN	2305	CCUGGGGCAGCCCCCCGCUNN	33

203-221	GCGGGGGGCUGCCCCAGGCNN	2306	GCCUGGGGCAGCCCCCCGCNN	33

204-222	CGGGGGGCUGCCCCAGGCGNN	2307	CGCCUGGGGCAGCCCCCCGNN	33

205-223	GGGGGGCUGCCCCAGGCGCNN	2308	GCGCCUGGGGCAGCCCCCCNN	33

206-224	GGGGGCUGCCCCAGGCGCGNN	2309	CGCGCCUGGGGCAGCCCCCNN	33

207-225	GGGGCUGCCCCAGGCGCGCNN	2310	GCGCGCCUGGGGCAGCCCCNN	33

208-226	GGGCUGCCCCAGGCGCGCANN	2311	UGCGCGCCUGGGGCAGCCCNN	33

209-227	GGCUGCCCCAGGCGCGCAANN	2312	UUGCGCGCCUGGGGCAGCCNN	33

210-228	GCUGCCCCAGGCGCGCAAGNN	2313	CUUGCGCGCCUGGGGCAGCNN	33

211-229	CUGCCCCAGGCGCGCAAGCNN	2314	GCUUGCGCGCCUGGGGCAGNN	33

212-230	UGCCCCAGGCGCGCAAGCGNN	2315	CGCUUGCGCGCCUGGGGCANN	33

247-265	CUGAGCCCCGAGGAGAAGGNN	2316	CCUUCUCCUCGGGGCUCAGNN	33

248-266	UGAGCCCCGAGGAGAAGGCNN	2317	GCCUUCUCCUCGGGGCUCANN	33

249-267	GAGCCCCGAGGAGAAGGCGNN	2318	CGCCTTCTCCTCGGGGCTCNN	33

250-268	AGCCCCGAGGAGAAGGCGCNN	2319	GCGCCTTCTCCTCGGGGCTNN	33

251-269	GCCCCGAGGAGAAGGCGCUNN	2320	AGCGCCUUCUCCUCGGGGCNN	33

252-270	CCCCGAGGAGAAGGCGCUGNN	2321	CAGCGCCUUCUCCUCGGGGNN	33

253-271	CCCGAGGAGAAGGCGCUGANN	2322	UCAGCGCCUUCUCCUCGGGNN	33

254-272	CCGAGGAGAAGGCGCUGAGNN	2323	CUCAGCGCCUUCUCCUCGGNN	33

255-273	CGAGGAGAAGGCGCUGAGGNN	2324	CCUCAGCGCCUUCUCCUCGNN	33

256-274	GAGGAGAAGGCGCUGAGGANN	2325	UCCUCAGCGCCUUCUCCUCNN	33

257-275	AGGAGAAGGCGCUGAGGAGNN	2326	CUCCUCAGCGCCUUCUCCUNN	33

258-276	GGAGAAGGCGCUGAGGAGGNN	2327	CCUCCUCAGCGCCUUCUCCNN	33

259-277	GAGAAGGCGCUGAGGAGGANN	2328	UCCUCCUCAGCGCCUUCUCNN	33

260-278	AGAAGGCGCUGAGGAGGAANN	2329	UUCCUCCUCAGCGCCUUCUNN	33

261-279	GAAGGCGCUGAGGAGGAAANN	2330	UUUCCUCCUCAGCGCCUUCNN	33

262-280	AAGGCGCUGAGGAGGAAACNN	2331	GUUUCCUCCUCAGCGCCUUNN	33

263-281	AGGCGCUGAGGAGGAAACUNN	2332	AGUUUCCUCCUCAGCGCCUNN	33

264-282	GGCGCUGAGGAGGAAACUGNN	2333	CAGUUUCCUCCUCAGCGCCNN	33

265-283	GCGCUGAGGAGGAAACUGANN	2334	UCAGUUUCCUCCUCAGCGCNN	33

266-284	CGCUGAGGAGGAAACUGAANN	2335	UUCAGUUUCCUCCUCAGCGNN	33

267-285	GCUGAGGAGGAAACUGAAANN	2336	UUUCAGUUUCCUCCUCAGCNN	33

268-286	CUGAGGAGGAAACUGAAAANN	2337	UUUUCAGUUUCCUCCUCAGNN	33

269-287	UGAGGAGGAAACUGAAAAANN	2338	UUUUUCAGUUUCCUCCUCANN	33

270-288	GAGGAGGAAACUGAAAAACNN	2339	GUUUUUCAGUUUCCUCCUCNN	33

271-289	AGGAGGAAACUGAAAAACANN	2340	UGUUUUUCAGUUUCCUCCUNN	33

272-290	GGAGGAAACUGAAAAACAGNN	2341	CUGUUUUUCAGUUUCCUCCNN	34

273-291	GAGGAAACUGAAAAACAGANN	2342	UCUGUUUUUCAGUUUCCUCNN	34

274-292	AGGAAACUGAAAAACAGAGNN	2343	CUCUGUUUUUCAGUUUCCUNN	34

275-293	GGAAACUGAAAAACAGAGUNN	2344	ACUCUGUUUUUCAGUUUCCNN	34

276-294	GAAACUGAAAAACAGAGUANN	2345	UACUCUGUUUUUCAGUUUCNN	34

277-295	AAACUGAAAAACAGAGUAGNN	2346	CUACUCUGUUUUUCAGUUUNN	34

278-296	AACUGAAAAACAGAGUAGCNN	2347	GCUACUCUGUUUUUCAGUUNN	34

279-297	ACUGAAAAACAGAGUAGCANN	2348	UGCUACUCUGUUUUUCAGUNN	34

280-298	CUGAAAAACAGAGUAGCAGNN	2349	CUGCUACUCUGUUUUUCAGNN	34

281-299	UGAAAAACAGAGUAGCAGCNN	2350	GCUGCUACUCUGUUUUUCANN	34

282-300	GAAAAACAGAGUAGCAGCUNN	2351	AGCUGCUACUCUGUUUUUCNN	34

283-301	AAAAACAGAGUAGCAGCUCNN	2352	GAGCUGCUACUCUGUUUUUNN	34

284-302	AAAACAGAGUAGCAGCUCANN	2353	UGAGCUGCUACUCUGUUUUNN	34

285-303	AAACAGAGUAGCAGCUCAGNN	2354	CUGAGCUGCUACUCUGUUUNN	34

286-304	AACAGAGUAGCAGCUCAGANN	2355	UCUGAGCUGCUACUCUGUUNN	34

287-305	ACAGAGUAGCAGCUCAGACNN	2356	GUCUGAGCUGCUACUCUGUNN	34

288-306	CAGAGUAGCAGCUCAGACUNN	2357	AGUCUGAGCUGCUACUCUGNN	34

289-307	AGAGUAGCAGCUCAGACUGNN	2358	CAGUCUGAGCUGCUACUCUNN	34

290-308	GAGUAGCAGCUCAGACUGCNN	2359	GCAGUCUGAGCUGCUACUCNN	34

291-309	AGUAGCAGCUCAGACUGCCNN	2360	GGCAGUCUGAGCUGCUACUNN	34

292-310	GUAGCAGCUCAGACUGCCANN	2361	UGGCAGUCUGAGCUGCUACNN	34

293-311	UAGCAGCUCAGACUGCCAGNN	2362	CUGGCAGUCUGAGCUGCUANN	34

294-312	AGCAGCUCAGACUGCCAGANN	2363	UCUGGCAGUCUGAGCUGCUNN	34

295-313	GCAGCUCAGACUGCCAGAGNN	2364	CUCUGGCAGUCUGAGCUGCNN	34

296-314	CAGCUCAGACUGCCAGAGANN	2365	UCUCUGGCAGUCUGAGCUGNN	34

297-315	AGCUCAGACUGCCAGAGAUNN	2366	AUCUCUGGCAGUCUGAGCUNN	34

298-316	GCUCAGACUGCCAGAGAUCNN	2367	GAUCUCUGGCAGUCUGAGCNN	34

299-317	CUCAGACUGCCAGAGAUCGNN	2368	CGAUCUCUGGCAGUCUGAGNN	34

300-318	UCAGACUGCCAGAGAUCGANN	2369	UCGAUCUCUGGCAGUCUGANN	34

301-319	CAGACUGCCAGAGAUCGAANN	2370	UUCGAUCUCUGGCAGUCUGNN	34

302-320	AGACUGCCAGAGAUCGAAANN	2371	UUUCGAUCUCUGGCAGUCUNN	34

303-321	GACUGCCAGAGAUCGAAAGNN	2372	CUUUCGAUCUCUGGCAGUCNN	34

304-322	ACUGCCAGAGAUCGAAAGANN	2373	UCUUUCGAUCUCUGGCAGUNN	34

305-323	CUGCCAGAGAUCGAAAGAANN	2374	UUCUUUCGAUCUCUGGCAGNN	34

325-343	GCUCGAAUGAGUGAGCUGGNN	2375	CCAGCUCACUCAUUCGAGCNN	34

326-344	CUCGAAUGAGUGAGCUGGANN	2376	UCCAGCUCACUCAUUCGAGNN	34

327-345	UCGAAUGAGUGAGCUGGAANN	2377	UUCCAGCUCACUCAUUCGANN	34

328-346	CGAAUGAGUGAGCUGGAACNN	2378	GUUCCAGCUCACUCAUUCGNN	34

329-347	GAAUGAGUGAGCUGGAACANN	2379	UGUUCCAGCUCACUCAUUCNN	34

330-348	AAUGAGUGAGCUGGAACAGNN	2380	CUGUUCCAGCUCACUCAUUNN	34

331-349	AUGAGUGAGCUGGAACAGCNN	2381	GCUGUUCCAGCUCACUCAUNN	34

332-350	UGAGUGAGCUGGAACAGCANN	2382	UGCUGUUCCAGCUCACUCANN	34

333-351	GAGUGAGCUGGAACAGCAANN	2383	UUGCUGUUCCAGCUCACUCNN	34

334-352	AGUGAGCUGGAACAGCAAGNN	2384	CUUGCUGUUCCAGCUCACUNN	34

335-353	GUGAGCUGGAACAGCAAGUNN	2385	ACUUGCUGUUCCAGCUCACNN	34

336-354	UGAGCUGGAACAGCAAGUGNN	2386	CACUUGCUGUUCCAGCUCANN	34

337-355	GAGCUGGAACAGCAAGUGGNN	2387	CCACUUGCUGUUCCAGCUCNN	34

338-356	AGCUGGAACAGCAAGUGGUNN	2388	ACCACUUGCUGUUCCAGCUNN	34

339-357	GCUGGAACAGCAAGUGGUANN	2389	UACCACUUGCUGUUCCAGCNN	34

340-358	CUGGAACAGCAAGUGGUAGNN	2390	CUACCACUUGCUGUUCCAGNN	34

341-359	UGGAACAGCAAGUGGUAGANN	2391	UCUACCACUUGCUGUUCCANN	34

342-360	GGAACAGCAAGUGGUAGAUNN	2392	AUCUACCACUUGCUGUUCCNN	34

343-361	GAACAGCAAGUGGUAGAUUNN	2393	AAUCUACCACUUGCUGUUCNN	34

344-362	AACAGCAAGUGGUAGAUUUNN	2394	AAAUCUACCACUUGCUGUUNN	34

345-363	ACAGCAAGUGGUAGAUUUANN	2395	UAAAUCUACCACUUGCUGUNN	34

346-364	CAGCAAGUGGUAGAUUUAGNN	2396	CUAAAUCUACCACUUGCUGNN	34

347-365	AGCAAGUGGUAGAUUUAGANN	2397	UCUAAAUCUACCACUUGCUNN	34

348-366	GCAAGUGGUAGAUUUAGAANN	2398	UUCUAAAUCUACCACUUGCNN	34

349-367	CAAGUGGUAGAUUUAGAAGNN	2399	CUUCUAAAUCUACCACUUGNN	34

350-368	AAGUGGUAGAUUUAGAAGANN	2400	UCUUCUAAAUCUACCACUUNN	34

351-369	AGUGGUAGAUUUAGAAGAANN	2401	UUCUUCUAAAUCUACCACUNN	34

352-370	GUGGUAGAUUUAGAAGAAGNN	2402	CUUCUUCUAAAUCUACCACNN	34

353-371	UGGUAGAUUUAGAAGAAGANN	2403	UCUUCUUCUAAAUCUACCANN	34

354-372	GGUAGAUUUAGAAGAAGAGNN	2404	CUCUUCUUCUAAAUCUACCNN	34

355-373	GUAGAUUUAGAAGAAGAGANN	2405	UCUCUUCUUCUAAAUCUACNN	34

356-374	UAGAUUUAGAAGAAGAGAANN	2406	UUCUCUUCUUCUAAAUCUANN	34

357-375	AGAUUUAGAAGAAGAGAACNN	2407	GUUCUCUUCUUCUAAAUCUNN	34

358-376	GAUUUAGAAGAAGAGAACCNN	2408	GGUUCUCUUCUUCUAAAUCNN	34

359-377	AUUUAGAAGAAGAGAACCANN	2409	UGGUUCUCUUCUUCUAAAUNN	34

360-378	UUUAGAAGAAGAGAACCAANN	2410	UUGGUUCUCUUCUUCUAAANN	34

361-379	UUAGAAGAAGAGAACCAAANN	2411	UUUGGUUCUCUUCUUCUAANN	34

362-380	UAGAAGAAGAGAACCAAAANN	2412	UUUUGGUUCUCUUCUUCUANN	34

363-381	AGAAGAAGAGAACCAAAAANN	2413	TTTTTGGTTCTCTTCTTCTNN	34

364-382	GAAGAAGAGAACCAAAAACNN	2414	GTTTTTGGTTCTCTTCTTCNN	34

365-383	AAGAAGAGAACCAAAAACUNN	2415	AGUUUUUGGUUCUCUUCUUNN	34

366-384	AGAAGAGAACCAAAAACUUNN	2416	AAGUUUUUGGUUCUCUUCUNN	34

367-385	GAAGAGAACCAAAAACUUUNN	2417	AAAGUUUUUGGUUCUCUUCNN	34

368-386	AAGAGAACCAAAAACUUUUNN	2418	AAAAGUUUUUGGUUCUCUUNN	34

369-387	AGAGAACCAAAAACUUUUGNN	2419	CAAAAGUUUUUGGUUCUCUNN	34

370-388	GAGAACCAAAAACUUUUGCNN	2420	GCAAAAGUUUUUGGUUCUCNN	34

371-389	AGAACCAAAAACUUUUGCUNN	2421	AGCAAAAGUUUUUGGUUCUNN	34

372-390	GAACCAAAAACUUUUGCUANN	2422	UAGCAAAAGUUUUUGGUUCNN	34

373-391	AACCAAAAACUUUUGCUAGNN	2423	CUAGCAAAAGUUUUUGGUUNN	34

374-392	ACCAAAAACUUUUGCUAGANN	2424	UCUAGCAAAAGUUUUUGGUNN	34

375-393	CCAAAAACUUUUGCUAGAANN	2425	UUCUAGCAAAAGUUUUUGGNN	34

376-394	CAAAAACUUUUGCUAGAAANN	2426	UUUCUAGCAAAAGUUUUUGNN	34

377-395	AAAAACUUUUGCUAGAAAANN	2427	UUUUCUAGCAAAAGUUUUUNN	34

378-396	AAAACUUUUGCUAGAAAAUNN	2428	AUUUUCUAGCAAAAGUUUUNN	34

379-397	AAACUUUUGCUAGAAAAUCNN	2429	GAUUUUCUAGCAAAAGUUUNN	34

380-398	AACUUUUGCUAGAAAAUCANN	2430	UGAUUUUCUAGCAAAAGUUNN	34

381-399	ACUUUUGCUAGAAAAUCAGNN	2431	CUGAUUUUCUAGCAAAAGUNN	34

382-400	CUUUUGCUAGAAAAUCAGCNN	2432	GCUGAUUUUCUAGCAAAAGNN	34

383-401	UUUUGCUAGAAAAUCAGCUNN	2433	AGCUGAUUUUCUAGCAAAANN	34

384-402	UUUGCUAGAAAAUCAGCUUNN	2434	AAGCUGAUUUUCUAGCAAANN	34

385-403	UUGCUAGAAAAUCAGCUUUNN	2435	AAAGCUGAUUUUCUAGCAANN	34

386-404	UGCUAGAAAAUCAGCUUUUNN	2436	AAAAGCUGAUUUUCUAGCANN	34

387-405	GCUAGAAAAUCAGCUUUUANN	2437	UAAAAGCUGAUUUUCUAGCNN	34

388-406	CUAGAAAAUCAGCUUUUACNN	2438	GUAAAAGCUGAUUUUCUAGNN	34

389-407	UAGAAAAUCAGCUUUUACGNN	2439	CGUAAAAGCUGAUUUUCUANN	34

390-408	AGAAAAUCAGCUUUUACGANN	2440	UCGUAAAAGCUGAUUUUCUNN	34

391-409	GAAAAUCAGCUUUUACGAGNN	2441	CUCGUAAAAGCUGAUUUUCNN	35

392-410	AAAAUCAGCUUUUACGAGANN	2442	UCUCGUAAAAGCUGAUUUUNN	35

393-411	AAAUCAGCUUUUACGAGAGNN	2443	CUCUCGUAAAAGCUGAUUUNN	35

394-412	AAUCAGCUUUUACGAGAGANN	2444	UCUCUCGUAAAAGCUGAUUNN	35

395-413	AUCAGCUUUUACGAGAGAANN	2445	UUCUCUCGUAAAAGCUGAUNN	35

396-414	UCAGCUUUUACGAGAGAAANN	2446	UUUCUCUCGUAAAAGCUGANN	35

397-415	CAGCUUUUACGAGAGAAAANN	2447	UUUUCUCUCGUAAAAGCUGNN	35

398-416	AGCUUUUACGAGAGAAAACNN	2448	GUUUUCUCUCGUAAAAGCUNN	35

399-417	GCUUUUACGAGAGAAAACUNN	2449	AGUUUUCUCUCGUAAAAGCNN	35

400-418	CUUUUACGAGAGAAAACUCNN	2450	GAGUUUUCUCUCGUAAAAGNN	35

401-419	UUUUACGAGAGAAAACUCANN	2451	UGAGUUUUCUCUCGUAAAANN	35

421-439	GGCCUUGUAGUUGAGAACCNN	2452	GGUUCUCAACUACAAGGCCNN	35

422-440	GCCUUGUAGUUGAGAACCANN	2453	UGGUUCUCAACUACAAGGCNN	35

423-441	CCUUGUAGUUGAGAACCAGNN	2454	CUGGUUCUCAACUACAAGGNN	35

424-442	CUUGUAGUUGAGAACCAGGNN	2455	CCUGGUUCUCAACUACAAGNN	35

425-443	UUGUAGUUGAGAACCAGGANN	2456	UCCUGGUUCUCAACUACAANN	35

426-444	UGUAGUUGAGAACCAGGAGNN	2457	CUCCUGGUUCUCAACUACANN	35

427-445	GUAGUUGAGAACCAGGAGUNN	2458	ACUCCUGGUUCUCAACUACNN	35

428-446	UAGUUGAGAACCAGGAGUUNN	2459	AACUCCUGGUUCUCAACUANN	35

429-447	AGUUGAGAACCAGGAGUUANN	2460	UAACUCCUGGUUCUCAACUNN	35

430-448	GUUGAGAACCAGGAGUUAANN	2461	UUAACUCCUGGUUCUCAACNN	35

431-449	UUGAGAACCAGGAGUUAAGNN	2462	CUUAACUCCUGGUUCUCAANN	35

432-450	UGAGAACCAGGAGUUAAGANN	2463	UCUUAACUCCUGGUUCUCANN	35

433-451	GAGAACCAGGAGUUAAGACNN	2464	GUCUUAACUCCUGGUUCUCNN	35

434-452	AGAACCAGGAGUUAAGACANN	2465	UGUCUUAACUCCUGGUUCUNN	35

435-453	GAACCAGGAGUUAAGACAGNN	2466	CUGUCUUAACUCCUGGUUCNN	35

436-454	AACCAGGAGUUAAGACAGCNN	2467	GCUGUCUUAACUCCUGGUUNN	35

437-455	ACCAGGAGUUAAGACAGCGNN	2468	CGCUGUCUUAACUCCUGGUNN	35

438-456	CCAGGAGUUAAGACAGCGCNN	2469	GCGCUGUCUUAACUCCUGGNN	35

44-62	GAGCUAUGGUGGUGGUGGCNN	2470	GCCACCACCACCAUAGCUCNN	35

45-63	AGCUAUGGUGGUGGUGGCANN	2471	UGCCACCACCACCAUAGCUNN	35

458-476	UGGGGAUGGAUGCCCUGGUNN	2472	ACCAGGGCAUCCAUCCCCANN	35

459-477	GGGGAUGGAUGCCCUGGUUNN	2473	AACCAGGGCAUCCAUCCCCNN	35

460-478	GGGAUGGAUGCCCUGGUUGNN	2474	CAACCAGGGCAUCCAUCCCNN	35

461-479	GGAUGGAUGCCCUGGUUGCNN	2475	GCAACCAGGGCAUCCAUCCNN	35

462-480	GAUGGAUGCCCUGGUUGCUNN	2476	AGCAACCAGGGCAUCCAUCNN	35

46-64	GCUAUGGUGGUGGUGGCAGNN	2477	CUGCCACCACCACCAUAGCNN	35

47-65	CUAUGGUGGUGGUGGCAGCNN	2478	GCUGCCACCACCACCAUAGNN	35

482-500	AAGAGGAGGCGGAAGCCAANN	2479	TTGGCTTCCGCCTCCTCTTNN	35

483-501	AGAGGAGGCGGAAGCCAAGNN	2480	CTTGGCTTCCGCCTCCTCTNN	35

484-502	GAGGAGGCGGAAGCCAAGGNN	2481	CCTTGGCTTCCGCCTCCTCNN	35

485-503	AGGAGGCGGAAGCCAAGGGNN	2482	CCCTTGGCTTCCGCCTCCTNN	35

486-504	GGAGGCGGAAGCCAAGGGGNN	2483	CCCCTTGGCTTCCGCCTCCNN	35

48-66	UAUGGUGGUGGUGGCAGCCNN	2484	GGCUGCCACCACCACCAUANN	35

487-505	GAGGCGGAAGCCAAGGGGANN	2485	TCCCCTTGGCTTCCGCCTCNN	35

488-506	AGGCGGAAGCCAAGGGGAANN	2486	TTCCCCTTGGCTTCCGCCTNN	35

489-507	GGCGGAAGCCAAGGGGAAUNN	2487	AUUCCCCUUGGCUUCCGCCNN	35

490-508	GCGGAAGCCAAGGGGAAUGNN	2488	CAUUCCCCUUGGCUUCCGCNN	35

49-67	AUGGUGGUGGUGGCAGCCGNN	2489	CGGCUGCCACCACCACCAUNN	35

50-68	UGGUGGUGGUGGCAGCCGCNN	2490	GCGGCUGCCACCACCACCANN	35

510-528	AGUGAGGCCAGUGGCCGGGNN	2491	CCCGGCCACUGGCCUCACUNN	35

511-529	GUGAGGCCAGUGGCCGGGUNN	2492	ACCCGGCCACUGGCCUCACNN	35

512-530	UGAGGCCAGUGGCCGGGUCNN	2493	GACCCGGCCACUGGCCUCANN	35

513-531	GAGGCCAGUGGCCGGGUCUNN	2494	AGACCCGGCCACUGGCCUCNN	35

514-532	AGGCCAGUGGCCGGGUCUGNN	2495	CAGACCCGGCCACUGGCCUNN	35

515-533	GGCCAGUGGCCGGGUCUGCNN	2496	GCAGACCCGGCCACUGGCCNN	35

516-534	GCCAGUGGCCGGGUCUGCUNN	2497	AGCAGACCCGGCCACUGGCNN	35

517-535	CCAGUGGCCGGGUCUGCUGNN	2498	CAGCAGACCCGGCCACUGGNN	35

518-536	CAGUGGCCGGGUCUGCUGANN	2499	UCAGCAGACCCGGCCACUGNN	35

519-537	AGUGGCCGGGUCUGCUGAGNN	2500	CUCAGCAGACCCGGCCACUNN	35

520-538	GUGGCCGGGUCUGCUGAGUNN	2501	ACUCAGCAGACCCGGCCACNN	35

521-539	UGGCCGGGUCUGCUGAGUCNN	2502	GACUCAGCAGACCCGGCCANN	35

522-540	GGCCGGGUCUGCUGAGUCCNN	2503	GGACUCAGCAGACCCGGCCNN	35

523-541	GCCGGGUCUGCUGAGUCCGNN	2504	CGGACUCAGCAGACCCGGCNN	35

524-542	CCGGGUCUGCUGAGUCCGCNN	2505	GCGGACUCAGCAGACCCGGNN	35

525-543	CGGGUCUGCUGAGUCCGCANN	2506	UGCGGACUCAGCAGACCCGNN	35

526-544	GGGUCUGCUGAGUCCGCAGNN	2507	CUGCGGACUCAGCAGACCCNN	35

574-592	GUGCAGGCCCAGUUGUCACNN	2508	GUGACAACUGGGCCUGCACNN	35

575-593	UGCAGGCCCAGUUGUCACCNN	2509	GGUGACAACUGGGCCUGCANN	35

576-594	GCAGGCCCAGUUGUCACCCNN	2510	GGGUGACAACUGGGCCUGCNN	35

577-595	CAGGCCCAGUUGUCACCCCNN	2511	GGGGUGACAACUGGGCCUGNN	35

578-596	AGGCCCAGUUGUCACCCCUNN	2512	AGGGGUGACAACUGGGCCUNN	35

579-597	GGCCCAGUUGUCACCCCUCNN	2513	GAGGGGUGACAACUGGGCCNN	35

580-598	GCCCAGUUGUCACCCCUCCNN	2514	GGAGGGGUGACAACUGGGCNN	35

581-599	CCCAGUUGUCACCCCUCCANN	2515	UGGAGGGGUGACAACUGGGNN	35

582-600	CCAGUUGUCACCCCUCCAGNN	2516	CUGGAGGGGUGACAACUGGNN	35

583-601	CAGUUGUCACCCCUCCAGANN	2517	UCUGGAGGGGUGACAACUGNN	35

584-602	AGUUGUCACCCCUCCAGAANN	2518	UUCUGGAGGGGUGACAACUNN	35

585-603	GUUGUCACCCCUCCAGAACNN	2519	GUUCUGGAGGGGUGACAACNN	35

586-604	UUGUCACCCCUCCAGAACANN	2520	UGUUCUGGAGGGGUGACAANN	35

587-605	UGUCACCCCUCCAGAACAUNN	2521	AUGUUCUGGAGGGGUGACANN	35

588-606	GUCACCCCUCCAGAACAUCNN	2522	GAUGUUCUGGAGGGGUGACNN	35

589-607	UCACCCCUCCAGAACAUCUNN	2523	AGAUGUUCUGGAGGGGUGANN	35

590-608	CACCCCUCCAGAACAUCUCNN	2524	GAGAUGUUCUGGAGGGGUGNN	35

591-609	ACCCCUCCAGAACAUCUCCNN	2525	GGAGAUGUUCUGGAGGGGUNN	35

592-610	CCCCUCCAGAACAUCUCCCNN	2526	GGGAGAUGUUCUGGAGGGGNN	35

593-611	CCCUCCAGAACAUCUCCCCNN	2527	GGGGAGAUGUUCUGGAGGGNN	35

594-612	CCUCCAGAACAUCUCCCCANN	2528	UGGGGAGAUGUUCUGGAGGNN	35

595-613	CUCCAGAACAUCUCCCCAUNN	2529	AUGGGGAGAUGUUCUGGAGNN	35

596-614	UCCAGAACAUCUCCCCAUGNN	2530	CAUGGGGAGAUGUUCUGGANN	35

597-615	CCAGAACAUCUCCCCAUGGNN	2531	CCAUGGGGAGAUGUUCUGGNN	35

598-616	CAGAACAUCUCCCCAUGGANN	2532	UCCAUGGGGAGAUGUUCUGNN	35

599-617	AGAACAUCUCCCCAUGGAUNN	2533	AUCCAUGGGGAGAUGUUCUNN	35

600-618	GAACAUCUCCCCAUGGAUUNN	2534	AAUCCAUGGGGAGAUGUUCNN	35

601-619	AACAUCUCCCCAUGGAUUCNN	2535	GAAUCCAUGGGGAGAUGUUNN	35

602-620	ACAUCUCCCCAUGGAUUCUNN	2536	AGAAUCCAUGGGGAGAUGUNN	35

603-621	CAUCUCCCCAUGGAUUCUGNN	2537	CAGAAUCCAUGGGGAGAUGNN	35

604-622	AUCUCCCCAUGGAUUCUGGNN	2538	CCAGAAUCCAUGGGGAGAUNN	35

605-623	UCUCCCCAUGGAUUCUGGCNN	2539	GCCAGAAUCCAUGGGGAGANN	35

606-624	CUCCCCAUGGAUUCUGGCGNN	2540	CGCCAGAAUCCAUGGGGAGNN	35

607-625	UCCCCAUGGAUUCUGGCGGNN	2541	CCGCCAGAAUCCAUGGGGANN	36

608-626	CCCCAUGGAUUCUGGCGGUNN	2542	ACCGCCAGAAUCCAUGGGGNN	36

609-627	CCCAUGGAUUCUGGCGGUANN	2543	UACCGCCAGAAUCCAUGGGNN	36

610-628	CCAUGGAUUCUGGCGGUAUNN	2544	AUACCGCCAGAAUCCAUGGNN	36

611-629	CAUGGAUUCUGGCGGUAUUNN	2545	AAUACCGCCAGAAUCCAUGNN	36

612-630	AUGGAUUCUGGCGGUAUUGNN	2546	CAAUACCGCCAGAAUCCAUNN	36

613-631	UGGAUUCUGGCGGUAUUGANN	2547	UCAAUACCGCCAGAAUCCANN	36

614-632	GGAUUCUGGCGGUAUUGACNN	2548	GUCAAUACCGCCAGAAUCCNN	36

615-633	GAUUCUGGCGGUAUUGACUNN	2549	AGUCAAUACCGCCAGAAUCNN	36

616-634	AUUCUGGCGGUAUUGACUCNN	2550	GAGUCAAUACCGCCAGAAUNN	36

617-635	UUCUGGCGGUAUUGACUCUNN	2551	AGAGUCAAUACCGCCAGAANN	36

618-636	UCUGGCGGUAUUGACUCUUNN	2552	AAGAGUCAAUACCGCCAGANN	36

619-637	CUGGCGGUAUUGACUCUUCNN	2553	GAAGAGUCAAUACCGCCAGNN	36

620-638	UGGCGGUAUUGACUCUUCANN	2554	UGAAGAGUCAAUACCGCCANN	36

621-639	GGCGGUAUUGACUCUUCAGNN	2555	CUGAAGAGUCAAUACCGCCNN	36

622-640	GCGGUAUUGACUCUUCAGANN	2556	UCUGAAGAGUCAAUACCGCNN	36

623-641	CGGUAUUGACUCUUCAGAUNN	2557	AUCUGAAGAGUCAAUACCGNN	36

624-642	GGUAUUGACUCUUCAGAUUNN	2558	AAUCUGAAGAGUCAAUACCNN	36

625-643	GUAUUGACUCUUCAGAUUCNN	2559	GAAUCUGAAGAGUCAAUACNN	36

626-644	UAUUGACUCUUCAGAUUCANN	2560	UGAAUCUGAAGAGUCAAUANN	36

627-645	AUUGACUCUUCAGAUUCAGNN	2561	CUGAAUCUGAAGAGUCAAUNN	36

628-646	UUGACUCUUCAGAUUCAGANN	2562	UCUGAAUCUGAAGAGUCAANN	36

629-647	UGACUCUUCAGAUUCAGAGNN	2563	CUCUGAAUCUGAAGAGUCANN	36

630-648	GACUCUUCAGAUUCAGAGUNN	2564	ACUCUGAAUCUGAAGAGUCNN	36

631-649	ACUCUUCAGAUUCAGAGUCNN	2565	GACUCUGAAUCUGAAGAGUNN	36

632-650	CUCUUCAGAUUCAGAGUCUNN	2566	AGACUCUGAAUCUGAAGAGNN	36

633-651	UCUUCAGAUUCAGAGUCUGNN	2567	CAGACUCUGAAUCUGAAGANN	36

634-652	CUUCAGAUUCAGAGUCUGANN	2568	UCAGACUCUGAAUCUGAAGNN	36

635-653	UUCAGAUUCAGAGUCUGAUNN	2569	AUCAGACUCUGAAUCUGAANN	36

636-654	UCAGAUUCAGAGUCUGAUANN	2570	UAUCAGACUCUGAAUCUGANN	36

637-655	CAGAUUCAGAGUCUGAUAUNN	2571	AUAUCAGACUCUGAAUCUGNN	36

638-656	AGAUUCAGAGUCUGAUAUCNN	2572	GAUAUCAGACUCUGAAUCUNN	36

639-657	GAUUCAGAGUCUGAUAUCCNN	2573	GGAUAUCAGACUCUGAAUCNN	36

640-658	AUUCAGAGUCUGAUAUCCUNN	2574	AGGAUAUCAGACUCUGAAUNN	36

641-659	UUCAGAGUCUGAUAUCCUGNN	2575	CAGGAUAUCAGACUCUGAANN	36

642-660	UCAGAGUCUGAUAUCCUGUNN	2576	ACAGGAUAUCAGACUCUGANN	36

643-661	CAGAGUCUGAUAUCCUGUUNN	2577	AACAGGAUAUCAGACUCUGNN	36

644-662	AGAGUCUGAUAUCCUGUUGNN	2578	CAACAGGAUAUCAGACUCUNN	36

645-663	GAGUCUGAUAUCCUGUUGGNN	2579	CCAACAGGAUAUCAGACUCNN	36

646-664	AGUCUGAUAUCCUGUUGGGNN	2580	CCCAACAGGAUAUCAGACUNN	36

647-665	GUCUGAUAUCCUGUUGGGCNN	2581	GCCCAACAGGAUAUCAGACNN	36

648-666	UCUGAUAUCCUGUUGGGCANN	2582	UGCCCAACAGGAUAUCAGANN	36

649-667	CUGAUAUCCUGUUGGGCAUNN	2583	AUGCCCAACAGGAUAUCAGNN	36

650-668	UGAUAUCCUGUUGGGCAUUNN	2584	AAUGCCCAACAGGAUAUCANN	36

651-669	GAUAUCCUGUUGGGCAUUCNN	2585	GAAUGCCCAACAGGAUAUCNN	36

652-670	AUAUCCUGUUGGGCAUUCUNN	2586	AGAAUGCCCAACAGGAUAUNN	36

653-671	UAUCCUGUUGGGCAUUCUGNN	2587	CAGAAUGCCCAACAGGAUANN	36

654-672	AUCCUGUUGGGCAUUCUGGNN	2588	CCAGAAUGCCCAACAGGAUNN	36

655-673	UCCUGUUGGGCAUUCUGGANN	2589	UCCAGAAUGCCCAACAGGANN	36

656-674	CCUGUUGGGCAUUCUGGACNN	2590	GUCCAGAAUGCCCAACAGGNN	36

657-675	CUGUUGGGCAUUCUGGACANN	2591	UGUCCAGAAUGCCCAACAGNN	36

658-676	UGUUGGGCAUUCUGGACAANN	2592	UUGUCCAGAAUGCCCAACANN	36

659-677	GUUGGGCAUUCUGGACAACNN	2593	GUUGUCCAGAAUGCCCAACNN	36

660-678	UUGGGCAUUCUGGACAACUNN	2594	AGUUGUCCAGAAUGCCCAANN	36

661-679	UGGGCAUUCUGGACAACUUNN	2595	AAGUUGUCCAGAAUGCCCANN	36

662-680	GGGCAUUCUGGACAACUUGNN	2596	CAAGUUGUCCAGAAUGCCCNN	36

663-681	GGCAUUCUGGACAACUUGGNN	2597	CCAAGUUGUCCAGAAUGCCNN	36

664-682	GCAUUCUGGACAACUUGGANN	2598	UCCAAGUUGUCCAGAAUGCNN	36

665-683	CAUUCUGGACAACUUGGACNN	2599	GUCCAAGUUGUCCAGAAUGNN	36

666-684	AUUCUGGACAACUUGGACCNN	2600	GGUCCAAGUUGUCCAGAAUNN	36

667-685	UUCUGGACAACUUGGACCCNN	2601	GGGUCCAAGUUGUCCAGAANN	36

668-686	UCUGGACAACUUGGACCCANN	2602	UGGGUCCAAGUUGUCCAGANN	36

669-687	CUGGACAACUUGGACCCAGNN	2603	CUGGGUCCAAGUUGUCCAGNN	36

670-688	UGGACAACUUGGACCCAGUNN	2604	ACUGGGUCCAAGUUGUCCANN	36

671-689	GGACAACUUGGACCCAGUCNN	2605	GACUGGGUCCAAGUUGUCCNN	36

672-690	GACAACUUGGACCCAGUCANN	2606	UGACUGGGUCCAAGUUGUCNN	36

673-691	ACAACUUGGACCCAGUCAUNN	2607	AUGACUGGGUCCAAGUUGUNN	36

674-692	CAACUUGGACCCAGUCAUGNN	2608	CAUGACUGGGUCCAAGUUGNN	36

675-693	AACUUGGACCCAGUCAUGUNN	2609	ACAUGACUGGGUCCAAGUUNN	36

676-694	ACUUGGACCCAGUCAUGUUNN	2610	AACAUGACUGGGUCCAAGUNN	36

677-695	CUUGGACCCAGUCAUGUUCNN	2611	GAACAUGACUGGGUCCAAGNN	36

678-696	UUGGACCCAGUCAUGUUCUNN	2612	AGAACAUGACUGGGUCCAANN	36

679-697	UGGACCCAGUCAUGUUCUUNN	2613	AAGAACAUGACUGGGUCCANN	36

680-698	GGACCCAGUCAUGUUCUUCNN	2614	GAAGAACAUGACUGGGUCCNN	36

681-699	GACCCAGUCAUGUUCUUCANN	2615	UGAAGAACAUGACUGGGUCNN	36

682-700	ACCCAGUCAUGUUCUUCAANN	2616	UUGAAGAACAUGACUGGGUNN	36

683-701	CCCAGUCAUGUUCUUCAAANN	2617	UUUGAAGAACAUGACUGGGNN	36

684-702	CCAGUCAUGUUCUUCAAAUNN	2618	AUUUGAAGAACAUGACUGGNN	36

685-703	CAGUCAUGUUCUUCAAAUGNN	2619	CAUUUGAAGAACAUGACUGNN	36

686-704	AGUCAUGUUCUUCAAAUGCNN	2620	GCAUUUGAAGAACAUGACUNN	36

687-705	GUCAUGUUCUUCAAAUGCCNN	2621	GGCAUUUGAAGAACAUGACNN	36

688-706	UCAUGUUCUUCAAAUGCCCNN	2622	GGGCAUUUGAAGAACAUGANN	36

689-707	CAUGUUCUUCAAAUGCCCUNN	2623	AGGGCAUUUGAAGAACAUGNN	36

690-708	AUGUUCUUCAAAUGCCCUUNN	2624	AAGGGCAUUUGAAGAACAUNN	36

691-709	UGUUCUUCAAAUGCCCUUCNN	2625	GAAGGGCAUUUGAAGAACANN	36

692-710	GUUCUUCAAAUGCCCUUCCNN	2626	GGAAGGGCAUUUGAAGAACNN	36

693-711	UUCUUCAAAUGCCCUUCCCNN	2627	GGGAAGGGCAUUUGAAGAANN	36

694-712	UCUUCAAAUGCCCUUCCCCNN	2628	GGGGAAGGGCAUUUGAAGANN	36

695-713	CUUCAAAUGCCCUUCCCCANN	2629	UGGGGAAGGGCAUUUGAAGNN	36

696-714	UUCAAAUGCCCUUCCCCAGNN	2630	CUGGGGAAGGGCAUUUGAANN	36

697-715	UCAAAUGCCCUUCCCCAGANN	2631	UCUGGGGAAGGGCAUUUGANN	36

698-716	CAAAUGCCCUUCCCCAGAGNN	2632	CUCUGGGGAAGGGCAUUUGNN	36

718-736	CUGCCAGCCUGGAGGAGCUNN	2633	AGCUCCUCCAGGCUGGCAGNN	36

719-737	UGCCAGCCUGGAGGAGCUCNN	2634	GAGCUCCUCCAGGCUGGCANN	36

720-738	GCCAGCCUGGAGGAGCUCCNN	2635	GGAGCUCCUCCAGGCUGGCNN	36

721-739	CCAGCCUGGAGGAGCUCCCNN	2636	GGGAGCUCCUCCAGGCUGGNN	36

722-740	CAGCCUGGAGGAGCUCCCANN	2637	UGGGAGCUCCUCCAGGCUGNN	36

723-741	AGCCUGGAGGAGCUCCCAGNN	2638	CUGGGAGCUCCUCCAGGCUNN	36

724-742	GCCUGGAGGAGCUCCCAGANN	2639	UCUGGGAGCUCCUCCAGGCNN	36

725-743	CCUGGAGGAGCUCCCAGAGNN	2640	CUCUGGGAGCUCCUCCAGGNN	36

726-744	CUGGAGGAGCUCCCAGAGGNN	2641	CCUCUGGGAGCUCCUCCAGNN	37

727-745	UGGAGGAGCUCCCAGAGGUNN	2642	ACCUCUGGGAGCUCCUCCANN	37

728-746	GGAGGAGCUCCCAGAGGUCNN	2643	GACCUCUGGGAGCUCCUCCNN	37

729-747	GAGGAGCUCCCAGAGGUCUNN	2644	AGACCUCUGGGAGCUCCUCNN	37

730-748	AGGAGCUCCCAGAGGUCUANN	2645	UAGACCUCUGGGAGCUCCUNN	37

731-749	GGAGCUCCCAGAGGUCUACNN	2646	GUAGACCUCUGGGAGCUCCNN	37

732-750	GAGCUCCCAGAGGUCUACCNN	2647	GGUAGACCUCUGGGAGCUCNN	37

733-751	AGCUCCCAGAGGUCUACCCNN	2648	GGGUAGACCUCUGGGAGCUNN	37

734-752	GCUCCCAGAGGUCUACCCANN	2649	UGGGUAGACCUCUGGGAGCNN	37

735-753	CUCCCAGAGGUCUACCCAGNN	2650	CUGGGUAGACCUCUGGGAGNN	37

736-754	UCCCAGAGGUCUACCCAGANN	2651	UCUGGGUAGACCUCUGGGANN	37

737-755	CCCAGAGGUCUACCCAGAANN	2652	UUCUGGGUAGACCUCUGGGNN	37

738-756	CCAGAGGUCUACCCAGAAGNN	2653	CUUCUGGGUAGACCUCUGGNN	37

739-757	CAGAGGUCUACCCAGAAGGNN	2654	CCUUCUGGGUAGACCUCUGNN	37

740-758	AGAGGUCUACCCAGAAGGANN	2655	UCCUUCUGGGUAGACCUCUNN	37

741-759	GAGGUCUACCCAGAAGGACNN	2656	GUCCUUCUGGGUAGACCUCNN	37

742-760	AGGUCUACCCAGAAGGACCNN	2657	GGUCCUUCUGGGUAGACCUNN	37

743-761	GGUCUACCCAGAAGGACCCNN	2658	GGGUCCUUCUGGGUAGACCNN	37

744-762	GUCUACCCAGAAGGACCCANN	2659	UGGGUCCUUCUGGGUAGACNN	37

745-763	UCUACCCAGAAGGACCCAGNN	2660	CUGGGUCCUUCUGGGUAGANN	37

746-764	CUACCCAGAAGGACCCAGUNN	2661	ACUGGGUCCUUCUGGGUAGNN	37

747-765	UACCCAGAAGGACCCAGUUNN	2662	AACUGGGUCCUUCUGGGUANN	37

748-766	ACCCAGAAGGACCCAGUUCNN	2663	GAACUGGGUCCUUCUGGGUNN	37

749-767	CCCAGAAGGACCCAGUUCCNN	2664	GGAACUGGGUCCUUCUGGGNN	37

750-768	CCAGAAGGACCCAGUUCCUNN	2665	AGGAACUGGGUCCUUCUGGNN	37

751-769	CAGAAGGACCCAGUUCCUUNN	2666	AAGGAACUGGGUCCUUCUGNN	37

752-770	AGAAGGACCCAGUUCCUUANN	2667	UAAGGAACUGGGUCCUUCUNN	37

753-771	GAAGGACCCAGUUCCUUACNN	2668	GUAAGGAACUGGGUCCUUCNN	37

754-772	AAGGACCCAGUUCCUUACCNN	2669	GGUAAGGAACUGGGUCCUUNN	37

755-773	AGGACCCAGUUCCUUACCANN	2670	UGGUAAGGAACUGGGUCCUNN	37

756-774	GGACCCAGUUCCUUACCAGNN	2671	CUGGUAAGGAACUGGGUCCNN	37

757-775	GACCCAGUUCCUUACCAGCNN	2672	GCUGGUAAGGAACUGGGUCNN	37

758-776	ACCCAGUUCCUUACCAGCCNN	2673	GGCUGGUAAGGAACUGGGUNN	37

759-777	CCCAGUUCCUUACCAGCCUNN	2674	AGGCUGGUAAGGAACUGGGNN	37

760-778	CCAGUUCCUUACCAGCCUCNN	2675	GAGGCUGGUAAGGAACUGGNN	37

761-779	CAGUUCCUUACCAGCCUCCNN	2676	GGAGGCUGGUAAGGAACUGNN	37

762-780	AGUUCCUUACCAGCCUCCCNN	2677	GGGAGGCUGGUAAGGAACUNN	37

763-781	GUUCCUUACCAGCCUCCCUNN	2678	AGGGAGGCUGGUAAGGAACNN	37

764-782	UUCCUUACCAGCCUCCCUUNN	2679	AAGGGAGGCUGGUAAGGAANN	37

765-783	UCCUUACCAGCCUCCCUUUNN	2680	AAAGGGAGGCUGGUAAGGANN	37

766-784	CCUUACCAGCCUCCCUUUCNN	2681	GAAAGGGAGGCUGGUAAGGNN	37

767-785	CUUACCAGCCUCCCUUUCUNN	2682	AGAAAGGGAGGCUGGUAAGNN	37

768-786	UUACCAGCCUCCCUUUCUCNN	2683	GAGAAAGGGAGGCUGGUAANN	37

769-787	UACCAGCCUCCCUUUCUCUNN	2684	AGAGAAAGGGAGGCUGGUANN	37

770-788	ACCAGCCUCCCUUUCUCUGNN	2685	CAGAGAAAGGGAGGCUGGUNN	37

771-789	CCAGCCUCCCUUUCUCUGUNN	2686	ACAGAGAAAGGGAGGCUGGNN	37

772-790	CAGCCUCCCUUUCUCUGUCNN	2687	GACAGAGAAAGGGAGGCUGNN	37

773-791	AGCCUCCCUUUCUCUGUCANN	2688	UGACAGAGAAAGGGAGGCUNN	37

774-792	GCCUCCCUUUCUCUGUCAGNN	2689	CUGACAGAGAAAGGGAGGCNN	37

775-793	CCUCCCUUUCUCUGUCAGUNN	2690	ACUGACAGAGAAAGGGAGGNN	37

776-794	CUCCCUUUCUCUGUCAGUGNN	2691	CACUGACAGAGAAAGGGAGNN	37

777-795	UCCCUUUCUCUGUCAGUGGNN	2692	CCACUGACAGAGAAAGGGANN	37

778-796	CCCUUUCUCUGUCAGUGGGNN	2693	CCCACUGACAGAGAAAGGGNN	37

779-797	CCUUUCUCUGUCAGUGGGGNN	2694	CCCCACUGACAGAGAAAGGNN	37

780-798	CUUUCUCUGUCAGUGGGGANN	2695	UCCCCACUGACAGAGAAAGNN	37

781-799	UUUCUCUGUCAGUGGGGACNN	2696	GUCCCCACUGACAGAGAAANN	37

782-800	UUCUCUGUCAGUGGGGACGNN	2697	CGUCCCCACUGACAGAGAANN	37

783-801	UCUCUGUCAGUGGGGACGUNN	2698	ACGUCCCCACUGACAGAGANN	37

784-802	CUCUGUCAGUGGGGACGUCNN	2699	GACGUCCCCACUGACAGAGNN	37

785-803	UCUGUCAGUGGGGACGUCANN	2700	UGACGUCCCCACUGACAGANN	37

786-804	CUGUCAGUGGGGACGUCAUNN	2701	AUGACGUCCCCACUGACAGNN	37

787-805	UGUCAGUGGGGACGUCAUCNN	2702	GAUGACGUCCCCACUGACANN	37

788-806	GUCAGUGGGGACGUCAUCANN	2703	UGAUGACGUCCCCACUGACNN	37

789-807	UCAGUGGGGACGUCAUCAGNN	2704	CUGAUGACGUCCCCACUGANN	37

790-808	CAGUGGGGACGUCAUCAGCNN	2705	GCUGAUGACGUCCCCACUGNN	37

791-809	AGUGGGGACGUCAUCAGCCNN	2706	GGCUGAUGACGUCCCCACUNN	37

792-810	GUGGGGACGUCAUCAGCCANN	2707	UGGCUGAUGACGUCCCCACNN	37

793-811	UGGGGACGUCAUCAGCCAANN	2708	UUGGCUGAUGACGUCCCCANN	37

794-812	GGGGACGUCAUCAGCCAAGNN	2709	CUUGGCUGAUGACGUCCCCNN	37

795-813	GGGACGUCAUCAGCCAAGCNN	2710	GCUUGGCUGAUGACGUCCCNN	37

796-814	GGACGUCAUCAGCCAAGCUNN	2711	AGCUUGGCUGAUGACGUCCNN	37

797-815	GACGUCAUCAGCCAAGCUGNN	2712	CAGCUUGGCUGAUGACGUCNN	37

798-816	ACGUCAUCAGCCAAGCUGGNN	2713	CCAGCUUGGCUGAUGACGUNN	37

799-817	CGUCAUCAGCCAAGCUGGANN	2714	UCCAGCUUGGCUGAUGACGNN	37

800-818	GUCAUCAGCCAAGCUGGAANN	2715	UUCCAGCUUGGCUGAUGACNN	37

801-819	UCAUCAGCCAAGCUGGAAGNN	2716	CUUCCAGCUUGGCUGAUGANN	37

802-820	CAUCAGCCAAGCUGGAAGCNN	2717	GCUUCCAGCUUGGCUGAUGNN	37

803-821	AUCAGCCAAGCUGGAAGCCNN	2718	GGCUUCCAGCUUGGCUGAUNN	37

804-822	UCAGCCAAGCUGGAAGCCANN	2719	UGGCUUCCAGCUUGGCUGANN	37

805-823	CAGCCAAGCUGGAAGCCAUNN	2720	AUGGCUUCCAGCUUGGCUGNN	37

806-824	AGCCAAGCUGGAAGCCAUUNN	2721	AAUGGCUUCCAGCUUGGCUNN	37

807-825	GCCAAGCUGGAAGCCAUUANN	2722	UAAUGGCUUCCAGCUUGGCNN	37

808-826	CCAAGCUGGAAGCCAUUAANN	2723	UUAAUGGCUUCCAGCUUGGNN	37

809-827	CAAGCUGGAAGCCAUUAAUNN	2724	AUUAAUGGCUUCCAGCUUGNN	37

810-828	AAGCUGGAAGCCAUUAAUGNN	2725	CAUUAAUGGCUUCCAGCUUNN	37

811-829	AGCUGGAAGCCAUUAAUGANN	2726	UCAUUAAUGGCUUCCAGCUNN	37

812-830	GCUGGAAGCCAUUAAUGAANN	2727	UUCAUUAAUGGCUUCCAGCNN	37

813-831	CUGGAAGCCAUUAAUGAACNN	2728	GUUCAUUAAUGGCUUCCAGNN	37

814-832	UGGAAGCCAUUAAUGAACUNN	2729	AGUUCAUUAAUGGCUUCCANN	37

815-833	GGAAGCCAUUAAUGAACUANN	2730	UAGUUCAUUAAUGGCUUCCNN	37

816-834	GAAGCCAUUAAUGAACUAANN	2731	UUAGUUCAUUAAUGGCUUCNN	37

817-835	AAGCCAUUAAUGAACUAAUNN	2732	AUUAGUUCAUUAAUGGCUUNN	37

818-836	AGCCAUUAAUGAACUAAUUNN	2733	AAUUAGUUCAUUAAUGGCUNN	37

819-837	GCCAUUAAUGAACUAAUUCNN	2734	GAAUUAGUUCAUUAAUGGCNN	37

820-838	CCAUUAAUGAACUAAUUCGNN	2735	CGAAUUAGUUCAUUAAUGGNN	37

821-839	CAUUAAUGAACUAAUUCGUNN	2736	ACGAAUUAGUUCAUUAAUGNN	37

822-840	AUUAAUGAACUAAUUCGUUNN	2737	AACGAAUUAGUUCAUUAAUNN	37

823-841	UUAAUGAACUAAUUCGUUUNN	2738	AAACGAAUUAGUUCAUUAANN	37

824-842	UAAUGAACUAAUUCGUUUUNN	2739	AAAACGAAUUAGUUCAUUANN	37

825-843	AAUGAACUAAUUCGUUUUGNN	2740	CAAAACGAAUUAGUUCAUUNN	37

826-844	AUGAACUAAUUCGUUUUGANN	2741	UCAAAACGAAUUAGUUCAUNN	38

827-845	UGAACUAAUUCGUUUUGACNN	2742	GUCAAAACGAAUUAGUUCANN	38

828-846	GAACUAAUUCGUUUUGACCNN	2743	GGUCAAAACGAAUUAGUUCNN	38

829-847	AACUAAUUCGUUUUGACCANN	2744	UGGUCAAAACGAAUUAGUUNN	38

830-848	ACUAAUUCGUUUUGACCACNN	2745	GUGGUCAAAACGAAUUAGUNN	38

831-849	CUAAUUCGUUUUGACCACANN	2746	UGUGGUCAAAACGAAUUAGNN	38

832-850	UAAUUCGUUUUGACCACAUNN	2747	AUGUGGUCAAAACGAAUUANN	38

833-851	AAUUCGUUUUGACCACAUANN	2748	UAUGUGGUCAAAACGAAUUNN	38

834-852	AUUCGUUUUGACCACAUAUNN	2749	AUAUGUGGUCAAAACGAAUNN	38

835-853	UUCGUUUUGACCACAUAUANN	2750	UAUAUGUGGUCAAAACGAANN	38

836-854	UCGUUUUGACCACAUAUAUNN	2751	AUAUAUGUGGUCAAAACGANN	38

837-855	CGUUUUGACCACAUAUAUANN	2752	UAUAUAUGUGGUCAAAACGNN	38

838-856	GUUUUGACCACAUAUAUACNN	2753	GUAUAUAUGUGGUCAAAACNN	38

839-857	UUUUGACCACAUAUAUACCNN	2754	GGUAUAUAUGUGGUCAAAANN	38

840-858	UUUGACCACAUAUAUACCANN	2755	UGGUAUAUAUGUGGUCAAANN	38

841-859	UUGACCACAUAUAUACCAANN	2756	UUGGUAUAUAUGUGGUCAANN	38

842-860	UGACCACAUAUAUACCAAGNN	2757	CUUGGUAUAUAUGUGGUCANN	38

843-861	GACCACAUAUAUACCAAGCNN	2758	GCUUGGUAUAUAUGUGGUCNN	38

844-862	ACCACAUAUAUACCAAGCCNN	2759	GGCUUGGUAUAUAUGUGGUNN	38

845-863	CCACAUAUAUACCAAGCCCNN	2760	GGGCUUGGUAUAUAUGUGGNN	38

846-864	CACAUAUAUACCAAGCCCCNN	2761	GGGGCUUGGUAUAUAUGUGNN	38

847-865	ACAUAUAUACCAAGCCCCUNN	2762	AGGGGCUUGGUAUAUAUGUNN	38

867-885	GUCUUAGAGAUACCCUCUGNN	2763	CAGAGGGUAUCUCUAAGACNN	38

868-886	UCUUAGAGAUACCCUCUGANN	2764	UCAGAGGGUAUCUCUAAGANN	38

869-887	CUUAGAGAUACCCUCUGAGNN	2765	CUCAGAGGGUAUCUCUAAGNN	38

870-888	UUAGAGAUACCCUCUGAGANN	2766	UCUCAGAGGGUAUCUCUAANN	38

871-889	UAGAGAUACCCUCUGAGACNN	2767	GUCUCAGAGGGUAUCUCUANN	38

872-890	AGAGAUACCCUCUGAGACANN	2768	UGUCUCAGAGGGUAUCUCUNN	38

873-891	GAGAUACCCUCUGAGACAGNN	2769	CUGUCUCAGAGGGUAUCUCNN	38

874-892	AGAUACCCUCUGAGACAGANN	2770	UCUGUCUCAGAGGGUAUCUNN	38

875-893	GAUACCCUCUGAGACAGAGNN	2771	CUCUGUCUCAGAGGGUAUCNN	38

876-894	AUACCCUCUGAGACAGAGANN	2772	UCUCUGUCUCAGAGGGUAUNN	38

877-895	UACCCUCUGAGACAGAGAGNN	2773	CUCUCUGUCUCAGAGGGUANN	38

878-896	ACCCUCUGAGACAGAGAGCNN	2774	GCUCUCUGUCUCAGAGGGUNN	38

879-897	CCCUCUGAGACAGAGAGCCNN	2775	GGCUCUCUGUCUCAGAGGGNN	38

880-898	CCUCUGAGACAGAGAGCCANN	2776	UGGCUCUCUGUCUCAGAGGNN	38

881-899	CUCUGAGACAGAGAGCCAANN	2777	UUGGCUCUCUGUCUCAGAGNN	38

882-900	UCUGAGACAGAGAGCCAAGNN	2778	CUUGGCUCUCUGUCUCAGANN	38

883-901	CUGAGACAGAGAGCCAAGCNN	2779	GCUUGGCUCUCUGUCUCAGNN	38

884-902	UGAGACAGAGAGCCAAGCUNN	2780	AGCUUGGCUCUCUGUCUCANN	38

885-903	GAGACAGAGAGCCAAGCUANN	2781	UAGCUUGGCUCUCUGUCUCNN	38

886-904	AGACAGAGAGCCAAGCUAANN	2782	UUAGCUUGGCUCUCUGUCUNN	38

887-905	GACAGAGAGCCAAGCUAAUNN	2783	AUUAGCUUGGCUCUCUGUCNN	38

888-906	ACAGAGAGCCAAGCUAAUGNN	2784	CAUUAGCUUGGCUCUCUGUNN	38

889-907	CAGAGAGCCAAGCUAAUGUNN	2785	ACAUUAGCUUGGCUCUCUGNN	38

890-908	AGAGAGCCAAGCUAAUGUGNN	2786	CACAUUAGCUUGGCUCUCUNN	38

891-909	GAGAGCCAAGCUAAUGUGGNN	2787	CCACAUUAGCUUGGCUCUCNN	38

892-910	AGAGCCAAGCUAAUGUGGUNN	2788	ACCACAUUAGCUUGGCUCUNN	38

893-911	GAGCCAAGCUAAUGUGGUANN	2789	UACCACAUUAGCUUGGCUCNN	38

894-912	AGCCAAGCUAAUGUGGUAGNN	2790	CUACCACAUUAGCUUGGCUNN	38

895-913	GCCAAGCUAAUGUGGUAGUNN	2791	ACUACCACAUUAGCUUGGCNN	38

896-914	CCAAGCUAAUGUGGUAGUGNN	2792	CACUACCACAUUAGCUUGGNN	38

897-915	CAAGCUAAUGUGGUAGUGANN	2793	UCACUACCACAUUAGCUUGNN	38

898-916	AAGCUAAUGUGGUAGUGAANN	2794	UUCACUACCACAUUAGCUUNN	38

899-917	AGCUAAUGUGGUAGUGAAANN	2795	UUUCACUACCACAUUAGCUNN	38

900-918	GCUAAUGUGGUAGUGAAAANN	2796	UUUUCACUACCACAUUAGCNN	38

901-919	CUAAUGUGGUAGUGAAAAUNN	2797	AUUUUCACUACCACAUUAGNN	38

902-920	UAAUGUGGUAGUGAAAAUCNN	2798	GAUUUUCACUACCACAUUANN	38

903-921	AAUGUGGUAGUGAAAAUCGNN	2799	CGAUUUUCACUACCACAUUNN	38

904-922	AUGUGGUAGUGAAAAUCGANN	2800	UCGAUUUUCACUACCACAUNN	38

905-923	UGUGGUAGUGAAAAUCGAGNN	2801	CUCGAUUUUCACUACCACANN	38

906-924	GUGGUAGUGAAAAUCGAGGNN	2802	CCUCGAUUUUCACUACCACNN	38

907-925	UGGUAGUGAAAAUCGAGGANN	2803	UCCUCGAUUUUCACUACCANN	38

908-926	GGUAGUGAAAAUCGAGGAANN	2804	UUCCUCGAUUUUCACUACCNN	38

909-927	GUAGUGAAAAUCGAGGAAGNN	2805	CUUCCUCGAUUUUCACUACNN	38

910-928	UAGUGAAAAUCGAGGAAGCNN	2806	GCUUCCUCGAUUUUCACUANN	38

911-929	AGUGAAAAUCGAGGAAGCANN	2807	UGCUUCCUCGAUUUUCACUNN	38

912-930	GUGAAAAUCGAGGAAGCACNN	2808	GUGCUUCCUCGAUUUUCACNN	38

913-931	UGAAAAUCGAGGAAGCACCNN	2809	GGUGCUUCCUCGAUUUUCANN	38

914-932	GAAAAUCGAGGAAGCACCUNN	2810	AGGUGCUUCCUCGAUUUUCNN	38

915-933	AAAAUCGAGGAAGCACCUCNN	2811	GAGGUGCUUCCUCGAUUUUNN	38

916-934	AAAUCGAGGAAGCACCUCUNN	2812	AGAGGUGCUUCCUCGAUUUNN	38

917-935	AAUCGAGGAAGCACCUCUCNN	2813	GAGAGGUGCUUCCUCGAUUNN	38

918-936	AUCGAGGAAGCACCUCUCANN	2814	UGAGAGGUGCUUCCUCGAUNN	38

919-937	UCGAGGAAGCACCUCUCAGNN	2815	CUGAGAGGUGCUUCCUCGANN	38

920-938	CGAGGAAGCACCUCUCAGCNN	2816	GCUGAGAGGUGCUUCCUCGNN	38

921-939	GAGGAAGCACCUCUCAGCCNN	2817	GGCUGAGAGGUGCUUCCUCNN	38

922-940	AGGAAGCACCUCUCAGCCCNN	2818	GGGCUGAGAGGUGCUUCCUNN	38

923-941	GGAAGCACCUCUCAGCCCCNN	2819	GGGGCUGAGAGGUGCUUCCNN	38

924-942	GAAGCACCUCUCAGCCCCUNN	2820	AGGGGCUGAGAGGUGCUUCNN	38

925-943	AAGCACCUCUCAGCCCCUCNN	2821	GAGGGGCUGAGAGGUGCUUNN	38

926-944	AGCACCUCUCAGCCCCUCANN	2822	UGAGGGGCUGAGAGGUGCUNN	38

927-945	GCACCUCUCAGCCCCUCAGNN	2823	CUGAGGGGCUGAGAGGUGCNN	38

928-946	CACCUCUCAGCCCCUCAGANN	2824	UCUGAGGGGCUGAGAGGUGNN	38

929-947	ACCUCUCAGCCCCUCAGAGNN	2825	CUCUGAGGGGCUGAGAGGUNN	38

930-948	CCUCUCAGCCCCUCAGAGANN	2826	UCUCUGAGGGGCUGAGAGGNN	38

931-949	CUCUCAGCCCCUCAGAGAANN	2827	UUCUCUGAGGGGCUGAGAGNN	38

932-950	UCUCAGCCCCUCAGAGAAUNN	2828	AUUCUCUGAGGGGCUGAGANN	38

933-951	CUCAGCCCCUCAGAGAAUGNN	2829	CAUUCUCUGAGGGGCUGAGNN	38

934-952	UCAGCCCCUCAGAGAAUGANN	2830	UCAUUCUCUGAGGGGCUGANN	38

935-953	CAGCCCCUCAGAGAAUGAUNN	2831	AUCAUUCUCUGAGGGGCUGNN	38

936-954	AGCCCCUCAGAGAAUGAUCNN	2832	GAUCAUUCUCUGAGGGGCUNN	38

937-955	GCCCCUCAGAGAAUGAUCANN	2833	UGAUCAUUCUCUGAGGGGCNN	38

938-956	CCCCUCAGAGAAUGAUCACNN	2834	GUGAUCAUUCUCUGAGGGGNN	38

939-957	CCCUCAGAGAAUGAUCACCNN	2835	GGUGAUCAUUCUCUGAGGGNN	38

940-958	CCUCAGAGAAUGAUCACCCNN	2836	GGGUGAUCAUUCUCUGAGGNN	38

941-959	CUCAGAGAAUGAUCACCCUNN	2837	AGGGUGAUCAUUCUCUGAGNN	38

942-960	UCAGAGAAUGAUCACCCUGNN	2838	CAGGGUGAUCAUUCUCUGANN	38

943-961	CAGAGAAUGAUCACCCUGANN	2839	UCAGGGUGAUCAUUCUCUGNN	38

944-962	AGAGAAUGAUCACCCUGAANN	2840	UUCAGGGUGAUCAUUCUCUNN	38

945-963	GAGAAUGAUCACCCUGAAUNN	2841	AUUCAGGGUGAUCAUUCUCNN	39

946-964	AGAAUGAUCACCCUGAAUUNN	2842	AAUUCAGGGUGAUCAUUCUNN	39

947-965	GAAUGAUCACCCUGAAUUCNN	2843	GAAUUCAGGGUGAUCAUUCNN	39

948-966	AAUGAUCACCCUGAAUUCANN	2844	UGAAUUCAGGGUGAUCAUUNN	39

949-967	AUGAUCACCCUGAAUUCAUNN	2845	AUGAAUUCAGGGUGAUCAUNN	39

950-968	UGAUCACCCUGAAUUCAUUNN	2846	AAUGAAUUCAGGGUGAUCANN	39

951-969	GAUCACCCUGAAUUCAUUGNN	2847	CAAUGAAUUCAGGGUGAUCNN	39

952-970	AUCACCCUGAAUUCAUUGUNN	2848	ACAAUGAAUUCAGGGUGAUNN	39

953-971	UCACCCUGAAUUCAUUGUCNN	2849	GACAAUGAAUUCAGGGUGANN	39

954-972	CACCCUGAAUUCAUUGUCUNN	2850	AGACAAUGAAUUCAGGGUGNN	39

955-973	ACCCUGAAUUCAUUGUCUCNN	2851	GAGACAAUGAAUUCAGGGUNN	39

956-974	CCCUGAAUUCAUUGUCUCANN	2852	UGAGACAAUGAAUUCAGGGNN	39

957-975	CCUGAAUUCAUUGUCUCAGNN	2853	CUGAGACAAUGAAUUCAGGNN	39

958-976	CUGAAUUCAUUGUCUCAGUNN	2854	ACUGAGACAAUGAAUUCAGNN	39

959-977	UGAAUUCAUUGUCUCAGUGNN	2855	CACUGAGACAAUGAAUUCANN	39

960-978	GAAUUCAUUGUCUCAGUGANN	2856	UCACUGAGACAAUGAAUUCNN	39

961-979	AAUUCAUUGUCUCAGUGAANN	2857	UUCACUGAGACAAUGAAUUNN	39

962-980	AUUCAUUGUCUCAGUGAAGNN	2858	CUUCACUGAGACAAUGAAUNN	39

963-981	UUCAUUGUCUCAGUGAAGGNN	2859	CCUUCACUGAGACAAUGAANN	39

964-982	UCAUUGUCUCAGUGAAGGANN	2860	UCCUUCACUGAGACAAUGANN	39

965-983	CAUUGUCUCAGUGAAGGAANN	2861	UUCCUUCACUGAGACAAUGNN	39

966-984	AUUGUCUCAGUGAAGGAAGNN	2862	CUUCCUUCACUGAGACAAUNN	39

967-985	UUGUCUCAGUGAAGGAAGANN	2863	UCUUCCUUCACUGAGACAANN	39

968-986	UGUCUCAGUGAAGGAAGAANN	2864	UUCUUCCUUCACUGAGACANN	39

969-987	GUCUCAGUGAAGGAAGAACNN	2865	GUUCUUCCUUCACUGAGACNN	39

970-988	UCUCAGUGAAGGAAGAACCNN	2866	GGUUCUUCCUUCACUGAGANN	39

971-989	CUCAGUGAAGGAAGAACCUNN	2867	AGGUUCUUCCUUCACUGAGNN	39

972-990	UCAGUGAAGGAAGAACCUGNN	2868	CAGGUUCUUCCUUCACUGANN	39

973-991	CAGUGAAGGAAGAACCUGUNN	2869	ACAGGUUCUUCCUUCACUGNN	39

974-992	AGUGAAGGAAGAACCUGUANN	2870	UACAGGUUCUUCCUUCACUNN	39

975-993	GUGAAGGAAGAACCUGUAGNN	2871	CUACAGGUUCUUCCUUCACNN	39

976-994	UGAAGGAAGAACCUGUAGANN	2872	UCUACAGGUUCUUCCUUCANN	39

977-995	GAAGGAAGAACCUGUAGAANN	2873	UUCUACAGGUUCUUCCUUCNN	39

978-996	AAGGAAGAACCUGUAGAAGNN	2874	CUUCUACAGGUUCUUCCUUNN	39

979-997	AGGAAGAACCUGUAGAAGANN	2875	UCUUCUACAGGUUCUUCCUNN	39

980-998	GGAAGAACCUGUAGAAGAUNN	2876	AUCUUCUACAGGUUCUUCCNN	39

981-999	GAAGAACCUGUAGAAGAUGNN	2877	CAUCUUCUACAGGUUCUUCNN	39

982-1000	AAGAACCUGUAGAAGAUGANN	2878	UCAUCUUCUACAGGUUCUUNN	39

983-1001	AGAACCUGUAGAAGAUGACNN	2879	GUCAUCUUCUACAGGUUCUNN	39

984-1002	GAACCUGUAGAAGAUGACCNN	2880	GGUCAUCUUCUACAGGUUCNN	39

985-1003	AACCUGUAGAAGAUGACCUNN	2881	AGGUCAUCUUCUACAGGUUNN	39

986-1004	ACCUGUAGAAGAUGACCUCNN	2882	GAGGUCAUCUUCUACAGGUNN	39

*Target refers location of target sequence in NM_005080 (human XBP-1 mRNA). Sense and antisense sequences are described with optional dinucleotide (NN) overhangs.

TABLE 13

Sequences of dsRNA targeting both mouse and
rhesus monkeyXBP-1.

		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

Target refers location of target sequence in NM_013842 (Mus musculis* XPB1 mRNA). Sense and antisense sequences are described with optional dinucleotide (NN) overhangs.

Claims

1.-30. (canceled)

31. A dual targeting siRNA agent comprising a first dsRNA targeting a PCSK9 gene and a second dsRNA targeting a second gene, wherein

the first dsRNA and the second dsRNA are linked with a covalent linker; and

the first dsRNA comprises at least 15 contiguous nucleotides of an antisense strand of one of Tables 1, 2, or 4-8, or comprises an antisense strand of one of Tables 1, 2, or 4-8, or comprises a sense strand and an antisense strand of one of Tables 1, 2, or 4-8; and

the second dsRNA comprises at least 15 contiguous nucleotides of an antisense strand of one of Tables 3 or 9-13, or comprises an antisense strand of one of Tables 3 or 9-13, or comprises a sense strand and an antisense strand of one of Tables 3 or 9-13; and

the first dsRNA is not AD-10792 and the second dsRNA is not AD-18038.

32. The dual targeting siRNA agent of claim 31, wherein the first and second dsRNA comprises at least one modified nucleotide.

33. The dual targeting siRNA agent of claim 32, wherein the modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.

34. The dual targeting siRNA agent of claim 32, wherein the modified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

35. The dual targeting siRNA agent of claim 31, wherein each strand of each dsRNA is 19-23 bases in length.

36. The dual targeting siRNA agent of claim 31, wherein the covalent linker is a disulfide linker.

37. The dual targeting siRNA agent of claim 31, wherein the covalent linker links the sense strand of the first dsRNA to the sense strand of the second dsRNA.

38. The dual targeting siRNA agent of claim 31, wherein the covalent linker links the antisense strand of the first dsRNA to the antisense strand of the second dsRNA.

39. The dual targeting siRNA agent of claim 31, further comprising a ligand.

40. The dual targeting siRNA agent of claim 31, wherein administration of the dual targeting siRNA agent to a cell inhibits expression of the PCSK9 gene and the second gene in the cell at a level equivalent to inhibition of expression of both genes obtained by administration of each siRNA individually.

41. The dual targeting siRNA agent of claim 31, wherein administration of the dual targeting siRNA agent to a subject results in a greater reduction of total serum cholesterol than that obtained by administration of each siRNA individually.

42. A pharmaceutical composition comprising the dual targeting siRNA agent of claim 31 and a pharmaceutical carrier.

43. The pharmaceutical composition of claim 42, wherein the pharmaceutical carrier is a lipid formulation.

42. The pharmaceutical composition of claim 42, wherein the pharmaceutical carrier is a lipid formulation described in Table A.

43. A method of inhibiting expression of a PCSK9 gene and a second gene in a cell, the method comprising (a) introducing into the cell the dual targeting siRNA agent of claim 31; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcripts of the PCSK9 gene and the second gene, thereby inhibiting expression of the PCSK9 gene and the second gene in the cell.

44. A method of reducing total serum cholesterol in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 42.

45. An isolated cell comprising the dual targeting siRNA agent of claim 31.