# Patent

## Title
Compositions and methods for inhibiting expression of the PCSK9 gene

## Abstract

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the PCSK9 gene (PCSK9 gene), comprising an antisense strand having a nucleotide sequence which is less that 30 nucleotides in length, generally 19-25 nucleotides in length, and which is substantially complementary to at least a part of the PCSK9 gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier and method for treating diseases caused by PCSK9 gene expression.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 13/472,438, filed May 15, 2012, now U.S. Pat. No. 8,809,292, issued Aug. 19, 2014, which is a continuation of U.S. application Ser. No. 12/554,231, filed Sep. 4, 2009, now U.S. Pat. No. 8,222,222, issued on Jul. 17, 2012, which is a divisional of U.S. application Ser. No. 11/746,864, filed May 10, 2007, now U.S. Pat. No. 7,605,251, issued on Oct. 20, 2009, which claims the benefit of and priority to U.S. Provisional Application No. 60/799,458, filed May 11, 2006; U.S. Provisional Application No. 60/817,203, filed Jun. 27, 2006; U.S. Provisional Application No. 60/840,089, filed Aug. 25, 2006; U.S. Provisional Application No. 60/829,914, filed Oct. 18, 2006; and U.S. Provisional Application No. 60/901,134, filed Feb. 13, 2007. The contents of all of these applications are hereby incorporated by reference in their entirety.


FIELD OF THE INVENTION
This invention relates to double-stranded ribonucleic acid (dsRNA), and its use in mediating RNA interference to inhibit the expression of the PCSK9 gene and the use of the dsRNA to treat pathological processes which can be mediated by down regulating PCSK9, such as 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. These results indicate that PCSK9, either directly or indirectly, reduces LDLR protein levels by a posttranscriptional mechanism
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 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). Clearly the evidence to date indicates that lowering of PCSK9 levels will lower LDLc.
Recently, 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.) discloses 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.
Despite significant advances in the field of RNAi and advances in the treatment of pathological processes which can be mediated by down regulating PCSK9 gene expression, there remains a need for agents that can inhibit PCSK9 gene expression and that can treat diseases associated with PCSK9 gene expression such as hyperlipidemia.
SUMMARY OF THE INVENTION
The invention provides a solution to the problem of treating diseases that can be modulated by down regulating the proprotein convertase subtilisin kexin 9 (PCSK9) by using double-stranded ribonucleic acid (dsRNA) to silence PCSK9 expression.
The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the PCSK9 gene in a cell or mammal using such dsRNA. The invention also provides compositions and methods for treating pathological conditions that can modulated by down regulating the expression of the PCSK9 gene, such as hyperlipidemia. The dsRNA of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an mRNA transcript of the PCSK9 gene.
In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the PCSK9 gene. The dsRNA comprises at least two sequences that are complementary to each other. The dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence. The antisense strand comprises a nucleotide sequence which is substantially complementary to at least part of an mRNA encoding PCSK9, and the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length. The dsRNA, upon contacting with a cell expressing the PCSK9, inhibits the expression of the PCSK9 gene by at least 40%.
For example, the dsRNA molecules of the invention can be comprised of a first sequence of the dsRNA that is selected from the group consisting of the sense sequences of Table 1 and Table 2 the second sequence is selected from the group consisting of the antisense sequences of Tables 1 and Table 2. The dsRNA molecules of the invention can be comprised of naturally occurring nucleotides or can be comprised of at least one modified nucleotide, such as a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative. Alternatively, the modified nucleotide may be chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. Generally, such modified sequence will be based on a first sequence of the dsRNA selected from the group consisting of the sense sequences of Tables 1 and Table 2 and a second sequence selected from the group consisting of the antisense sequences of Tables 1, and Table 2.
In another embodiment, the invention provides a cell comprising one of the dsRNAs of the invention. The cell is generally a mammalian cell, such as a human cell.
In another embodiment, the invention provides a pharmaceutical composition for inhibiting the expression of the PCSK9 gene in an organism, generally a human subject, comprising one or more of the dsRNA of the invention and a pharmaceutically acceptable carrier or delivery vehicle.
In another embodiment, the invention provides a method for inhibiting the expression of the PCSK9 gene in a cell, comprising the following steps:

    
    
        (a) introducing into the cell a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises at least two sequences that are complementary to each other. The dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence. The antisense strand comprises a region of complementarity which is substantially complementary to at least a part of a mRNA encoding PCSK9, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein the dsRNA, upon contact with a cell expressing the PCSK9, inhibits expression of the PCSK9 gene by at least 40%; and
        (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the PCSK9 gene, thereby inhibiting expression of the PCSK9 gene in the cell.
    
    


In another embodiment, the invention provides methods for treating, preventing or managing pathological processes which can be mediated by down regulating PCSK9 gene expression, e.g. hyperlipidemia, comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of one or more of the dsRNAs of the invention.
In another embodiment, the invention provides vectors for inhibiting the expression of the PCSK9 gene in a cell, comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.
In another embodiment, the invention provides a cell comprising a vector for inhibiting the expression of the PCSK9 gene in a cell. The vector comprises a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the structure of the ND-98 lipid.
FIG. 2 shows the results of the in vivo screen of 16 mouse specific (AL-DP-9327 through AL-DP-9342) PCSK9 siRNAs directed against different ORF regions of PCSK9 mRNA (having the first nucleotide corresponding to the ORF position indicated on the graph) in C57/BL6 mice (5 animals/group). The ratio of PCSK9 mRNA to GAPDH mRNA in liver lysates was averaged over each treatment group and compared to a control group treated with PBS or a control group treated with an unrelated siRNA (blood coagulation factor VII).
FIG. 3 shows the results of the in vivo screen of 16 human/mouse/rat crossreactive (AL-DP-9311 through AL-DP-9326) PCSK9 siRNAs directed against different ORF regions of PCSK9 mRNA (having the first nucleotide corresponding to the ORF position indicated on the graph) in C57/BL6 mice (5 animals/group). The ratio of PCSK9 mRNA to GAPDH mRNA in liver lysates was averaged over each treatment group and compared to a control group treated with PBS or a control group treated with an unrelated siRNA (blood coagulation factor VII).
Silencing of PCSK9 mRNA resulted in lowering total serum cholesterol levels.
The most efficacious in terms of knocking down PSCK9 message siRNAs showed the most pronounced cholesterol lowering effect (around 20-30%).
FIG. 4 shows the results of the in vivo screen of 16 mouse specific (AL-DP-9327 through AL-DP-9342) PCSK9 siRNAs in C57/BL6 mice (5 animals/group). Total serum cholesterol levels were averaged over each treatment group and compared to a control group treated with PBS or a control group treated with an unrelated siRNA (blood coagulation factor VII).
FIG. 5 shows the results of the in vivo screen of 16 human/mouse/rat crossreactive (AL-DP-9311 through AL-DP-9326) PCSK9 siRNAs in C57/BL6 mice (5 animals/group). Total serum cholesterol levels were averaged over each treatment group and compared to a control group treated with PBS or a control group treated with an unrelated siRNA (blood coagulation factor VII).
FIG. 6A and FIG. 6B. shows a comparison of the in vitro and in vivo results for silencing PCSK9.
FIG. 7A and FIG. 7B show in vitro results for silencing PCSK9 using monkey primary hepatocytes.
FIG. 8 shows in vivo activity of LNP-01 formulated siRNAs to pcsk-9.
FIG. 9 shows in vivo activity of LNP-01 Formulated chemically modified 9314 and 10792 parent molecules at different times. Clearly modified versions of 10792 display in vivo silencing activity.



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, by using double-stranded ribonucleic acid (dsRNA) to silence the PCSK9 gene thus providing treatment for diseases such as hyperlipidemia.
The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the PCSK9 gene in a cell or mammal using the dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases that can be modulated by down regulating the expression of the PCSK9 gene. dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).
The dsRNA of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an mRNA transcript of the PCSK9 gene. The use of these dsRNAs enables the targeted degradation of an mRNA that is involved in sodium transport. Using cell-based and animal assays, the present inventors have demonstrated that very low dosages of these dsRNA can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of the PCSK9 gene. Thus, the methods and compositions of the invention comprising these dsRNAs are useful for treating pathological processes which can be mediated by down regulating PCSK9, such as in the treatment of hyperlipidemia.
The following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of the target PCSK9 gene, as well as compositions and methods for treating diseases that can be modulated by down regulating the expression of PCSK9, such as hyperlipidemia. The pharmaceutical compositions of the invention comprise a dsRNA having an antisense strand comprising a region of complementarity which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of the PCSK9 gene, together with a pharmaceutically acceptable carrier.
Accordingly, certain aspects of the invention provide pharmaceutical compositions comprising the dsRNA of the invention together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the PCSK9 gene, and methods of using the pharmaceutical compositions to treat diseases that can be modulated by down regulating the expression of PCSK9.
I. DEFINITIONS
For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.
“G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. 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 the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.
As used herein, “PCSK9” refers to the proprotein convertase subtilisin kexin 9 gene or protein (also known as FH3, HCHOLA3, NARC-1, NARC1). mRNA sequences to PCSK9 are provided as human: NM—174936; mouse: NM—153565, and rat: NM—199253.
As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the PCSK9 gene, including mRNA that is a product of RNA processing of a primary transcription product.
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.
This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. 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 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. 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 of the invention.
“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.
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 a dsRNA and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide which is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide which is substantially complementary to a contiguous portion of the mRNA of interest (e.g., encoding PCSK9). 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 a mRNA encoding PCSK9.
The term “double-stranded RNA” or “dsRNA”, as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where separate RNA molecules, such dsRNA are often referred to in the literature as siRNA (“short interfering RNA”). Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”, “short hairpin RNA” or “shRNA”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. In addition, as used in this specification, “dsRNA” may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims.
As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule. For clarity, chemical caps or non-nucleotide chemical moieties conjugated to the 3′ end or 5′ end of an siRNA are not considered in determining whether an siRNA has an overhang or is blunt ended.
The term “antisense strand” refers to the strand of 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 are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.
The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.
“Introducing into a cell”, when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA 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; a dsRNA may also be “introduced into a cell”, wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.
The terms “silence” and “inhibit the expression of”, in as far as they refer to the PCSK9 gene, herein refer to the at least partial suppression of the expression of the PCSK9 gene, as manifested by a reduction of the amount of mRNA transcribed from the PCSK9 gene which may be isolated from a first cell or group of cells in which the PCSK9 gene is transcribed and which has or have been treated such that the expression of the PCSK9 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




  
    
      
        
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Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to PCSK9 gene transcription, e.g. the amount of protein encoded by the PCSK9 gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g apoptosis. In principle, PCSK9 gene silencing may be determined in any cell expressing the 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 dsRNA inhibits the expression of the PCSK9 gene by a certain degree and therefore is encompassed by the instant invention, the assay provided in the Examples below shall serve as such reference.
For example, in certain instances, expression of the PCSK9 gene is suppressed by at least about 20%, 25%, 35%, or 50% by administration of the double-stranded oligonucleotide of the invention. In some embodiment, the PCSK9 gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, the PCSK9 gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention. Tables 1, 2, provides a wide range of values for inhibition of expression obtained in an in vitro assay using various PCSK9 dsRNA molecules at various concentrations.
As used herein in the context of PCSK9 expression, the terms “treat”, “treatment”, and the like, refer to relief from or alleviation of pathological processes which can be mediated by down regulating PCSK9 gene. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes which can be mediated by down regulating the PCSK9 gene), 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 of such condition. For example, in the context of hyperlipidemia, treatment will involve a decrease in serum lipid levels.
As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes which can be mediated by down regulating the PCSK9 gene on or an overt symptom of pathological processes which can be mediated by down regulating the PCSK9 gene. The specific amount that is therapeutically effective can be readily determined by ordinary medical practitioner, and may vary depending on factors known in the art, such as, e.g. the type of pathological processes which can be mediated by down regulating the PCSK9 gene, the patient's history and age, the stage of pathological processes which can be mediated by down regulating PCSK9 gene expression, and the administration of other anti-pathological processes which can be mediated by down regulating PCSK9 gene expression.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% 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 25% reduction in that parameter.
The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof and are described in more detail below. The term specifically excludes cell culture medium.
As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.
II. DOUBLE-STRANDED RIBONUCLEIC ACID (dsRNA)
In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the PCSK9 gene in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of the PCSK9 gene, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein the dsRNA, upon contact with a cell expressing the PCSK9 gene, inhibits the expression of the PCSK9 gene by at least 40%. The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises 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 the PCSK9 gene, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 nucleotides in length. The dsRNA of the invention may further comprise one or more single-stranded nucleotide overhang(s). The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. In a preferred embodiment, the PCSK9 gene is the human PCSK9 gene. In specific embodiments, the antisense strand of the dsRNA comprises a strand selected from the sense sequences of Tables 1 and 2, and a second sequence selected from the group consisting of the antisense sequences of Tables 1 and 2. Alternative antisense agents that target elsewhere in the target sequence provided in Tables 1 and 2, can readily be determined using the target sequence and the flanking PCSK9 sequence.
In further embodiments, the dsRNA comprises at least one nucleotide sequence selected from the groups of sequences provided in Tables 1 and 2. In other embodiments, the dsRNA comprises at least two sequences selected from this group, wherein one of the at least two sequences is complementary to another of the at least two sequences, and one of the at least two sequences is substantially complementary to a sequence of an mRNA generated in the expression of the PCSK9 gene. Generally, the dsRNA comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand in Tables 1 and 2 and the second oligonucleotide is described as the antisense strand in Tables 1 and 2
The skilled person is well aware that dsRNAs comprising a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 1 and 2, the dsRNAs of the invention can comprise at least one strand of a length of minimally 21 nt. It can be reasonably expected that shorter dsRNAs comprising one of the sequences of Tables 1 and 2 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs comprising a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 1 and 2, and differing in their ability to inhibit the expression of the PCSK9 gene in a FACS assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further dsRNAs that cleave within the target sequence provided in Tables 1 and 2 can readily be made using the PCSK9 sequence and the target sequence provided.
In addition, the RNAi agents provided in Tables 1 and 2 identify a site in the PCSK9 mRNA that is susceptible to RNAi based cleavage. As such the present invention further includes RNAi agents that target within the sequence targeted by one of the agents of the present invention. As used herein a second RNAi agent is the to target within the sequence of a first RNAi agent if the second RNAi agent cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first RNAi agent. Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 1 and 2 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in the PCSK9 gene. For example, the last 15 nucleotides of SEQ ID NO:1 (minus the added AA sequences) combined with the next 6 nucleotides from the target PCSK9 gene produces a single strand agent of 21 nucleotides that is based on one of the sequences provided in Tables 1 and 2.
The dsRNA of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA of the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA 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 dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of the PCSK9 gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of the PCSK9 gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of the PCSK9 gene is important, especially if the particular region of complementarity in the PCSK9 gene is known to have polymorphic sequence variation within the population.
In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. 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 may also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids of 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. Chemical modifications may include, but are not limited to 2′ modifications, modifications at other sites of the sugar or base of an oligonucleotide, introduction of non-natural bases into the olibonucleotide chain, covalent attachment to a ligand or chemical moiety, and replacement of internucleotide phosphate linkages with alternate linkages such as thiophosphates. More than one such modification may be employed.
Chemical linking of the two separate dsRNA strands may be achieved by any of a variety of well-known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues. Generally, the chemical groups that can be used to modify the dsRNA include, without limitation, methylene blue; bifunctional groups, generally bis-(2-chloroethyl)amine; N-acetyl-N′-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. In one embodiment, the linker is a hexa-ethylene glycol linker. In this case, the dsRNA are produced by solid phase synthesis and the hexa-ethylene glycol linker is incorporated according to standard methods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996) 35:14665-14670). In a particular embodiment, the 5′-end of the antisense strand and the 3′-end of the sense strand are chemically linked via a hexaethylene glycol linker. In another embodiment, at least one nucleotide of the dsRNA comprises a phosphorothioate or phosphorodithioate groups. The chemical bond at the ends of the dsRNA is generally formed by triple-helix bonds. Tables 1 and 2 provides examples of modified RNAi agents of the invention.
In yet another embodiment, the nucleotides at one or both of the two single strands may be modified to prevent or inhibit the degradation activities of cellular enzymes, such as, for example, without limitation, certain nucleases. Techniques for inhibiting the degradation activity of cellular enzymes against nucleic acids are known in the art including, but not limited to, 2′-amino modifications, 2′-amino sugar modifications, 2′-F sugar modifications, 2′-F modifications, 2′-alkyl sugar modifications, uncharged backbone modifications, morpholino modifications, 2′-O-methyl modifications, and phosphoramidate (see, e.g., Wagner, Nat. Med. (1995) 1:1116-8). Thus, at least one 2′-hydroxyl group of the nucleotides on a dsRNA is replaced by a chemical group, generally by a 2′-amino or a 2′-methyl group. Also, at least one nucleotide may be modified to form a locked nucleotide. Such locked nucleotide contains a methylene bridge that connects the 2′-oxygen of ribose with the 4′-carbon of ribose. Oligonucleotides containing the locked nucleotide are described in Koshkin, A. A., et al., Tetrahedron (1998), 54: 3607-3630) and Obika, S. et al., Tetrahedron Lett. (1998), 39: 5401-5404). Introduction of a locked nucleotide into an oligonucleotide improves the affinity for complementary sequences and increases the melting temperature by several degrees (Braasch, D. A. and D. R. Corey, Chem. Biol. (2001), 8:1-7).
Conjugating a ligand to a dsRNA can enhance its cellular absorption as well as targeting to a particular tissue or uptake by specific types of cells such as liver cells. In certain instances, a hydrophobic ligand is conjugated to the dsRNA to facilitate direct permeation of the cellular membrane and or uptake across the liver cells. Alternatively, the ligand conjugated to the dsRNA is a substrate for receptor-mediated endocytosis. These approaches have been used to facilitate cell permeation of antisense oligonucleotides as well as dsRNA agents. For example, cholesterol has been conjugated to various antisense oligonucleotides resulting in compounds that are substantially more active compared to their non-conjugated analogs. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103. Other lipophilic compounds that have been conjugated to oligonucleotides include 1-pyrene butyric acid, 1,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters the cell by folate-receptor-mediated endocytosis. dsRNA compounds bearing folic acid would be efficiently transported into the cell via the folate-receptor-mediated endocytosis. Li and coworkers report that attachment of folic acid to the 3′-terminus of an oligonucleotide resulted in an 8-fold increase in cellular uptake of the oligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res. 1998, 15, 1540. Other ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents, porphyrin conjugates, delivery peptides and lipids such as cholesterol.
In certain instances, conjugation of a cationic ligand to oligonucleotides results in improved resistance to nucleases. Representative examples of cationic ligands are propylammonium and dimethylpropylammonium. Interestingly, antisense oligonucleotides were reported to retain their high binding affinity to mRNA when the cationic ligand was dispersed throughout the oligonucleotide. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103 and references therein.
The ligand-conjugated dsRNA of the invention may be synthesized by the use of a dsRNA that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the dsRNA. This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto. The methods of the invention facilitate the synthesis of ligand-conjugated dsRNA by the use of, in some preferred embodiments, nucleoside monomers that have been appropriately conjugated with ligands and that may further be attached to a solid-support material. Such ligand-nucleoside conjugates, optionally attached to a solid-support material, are prepared according to some preferred embodiments of the methods of the invention via reaction of a selected serum-binding ligand with a linking moiety located on the 5′ position of a nucleoside or oligonucleotide. In certain instances, an dsRNA bearing an aralkyl ligand attached to the 3′-terminus of the dsRNA is prepared by first covalently attaching a monomer building block to a controlled-pore-glass support via a long-chain aminoalkyl group. Then, nucleotides are bonded via standard solid-phase synthesis techniques to the monomer building-block bound to the solid support. The monomer building block may be a nucleoside or other organic compound that is compatible with solid-phase synthesis.
The dsRNA used in the conjugates of the invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.
Teachings regarding the synthesis of particular modified oligonucleotides may be found in the following: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for the preparation of oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides having modified backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modified oligonucleotides and the preparation thereof through reductive coupling; U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the 3-deazapurine ring system and methods of synthesis thereof; U.S. Pat. No. 5,459,255, drawn to modified nucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302, drawn to processes for preparing oligonucleotides having chiral phosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods and materials for the synthesis of oligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides having alkylthio groups, wherein such groups may be used as linkers to other moieties attached at any of a variety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides having phosphorothioate linkages of high chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for the preparation of 2′-O-alkyl guanosine and related compounds, including 2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated 4′-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods of synthesizing 2′-fluoro-oligonucleotides.
In the ligand-conjugated dsRNA and ligand-molecule bearing sequence-specific linked nucleosides of the invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.
When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. Oligonucleotide conjugates bearing a variety of molecules such as steroids, vitamins, lipids and reporter molecules, has previously been described (see Manoharan et al., PCT Application WO 93/07883). In a preferred embodiment, the oligonucleotides or linked nucleosides of the invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.
The incorporation of a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-allyl, 2′-O-aminoalkyl or 2′-deoxy-2′-fluoro group in nucleosides of an oligonucleotide confers enhanced hybridization properties to the oligonucleotide. Further, oligonucleotides containing phosphorothioate backbones have enhanced nuclease stability. Thus, functionalized, linked nucleosides of the invention can be augmented to include either or both a phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-aminoalkyl, 2′-O-allyl or 2′-deoxy-2′-fluoro group. A summary listing of some of the oligonucleotide modifications known in the art is found at, for example, PCT Publication WO 200370918.
In some embodiments, functionalized nucleoside sequences of the invention possessing an amino group at the 5′-terminus are prepared using a DNA synthesizer, and then reacted with an active ester derivative of a selected ligand. Active ester derivatives are well known to those skilled in the art. Representative active esters include N-hydrosuccinimide esters, tetrafluorophenolic esters, pentafluorophenolic esters and pentachlorophenolic esters. The reaction of the amino group and the active ester produces an oligonucleotide in which the selected ligand is attached to the 5′-position through a linking group. The amino group at the 5′-terminus can be prepared utilizing a 5′-Amino-Modifier C6 reagent. In one embodiment, ligand molecules may be conjugated to oligonucleotides at the 5′-position by the use of a ligand-nucleoside phosphoramidite wherein the ligand is linked to the 5′-hydroxy group directly or indirectly via a linker. Such ligand-nucleoside phosphoramidites are typically used at the end of an automated synthesis procedure to provide a ligand-conjugated oligonucleotide bearing the ligand at the 5′-terminus.
Examples of modified internucleoside linkages or 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 United States Patents relating to the preparation of the above phosphorus-atom-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 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; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is herein incorporated by reference.
Examples of modified internucleoside linkages or backbones that do not include a phosphorus atom therein (i.e., oligonucleosides) have backbones that are formed by short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative United States patents relating to 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; 5,264,562; 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; 5,610,289; 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 certain instances, the oligonucleotide may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to oligonucleotides in order to enhance the activity, cellular distribution or cellular uptake of the oligonucleotide, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (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 oligonucleotide conjugates have been listed above. Typical conjugation protocols involve the synthesis of oligonucleotides 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 oligonucleotide still bound to the solid support or following cleavage of the oligonucleotide in solution phase. Purification of the oligonucleotide conjugate by HPLC typically affords the pure conjugate. The use of a cholesterol conjugate is particularly preferred since such a moiety can increase targeting liver cells cells, a site of PCSK9 expression.
Vector Encoded RNAi Agents
The dsRNA of the invention can also be expressed from recombinant viral vectors intracellularly in vivo. The recombinant viral vectors of the invention comprise sequences encoding the dsRNA of the invention and any suitable promoter for expressing the dsRNA sequences. Suitable promoters include, for example, the U6 or H1 RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art. The recombinant viral vectors of the invention can also comprise inducible or regulatable promoters for expression of the dsRNA in a particular tissue or in a particular intracellular environment. The use of recombinant viral vectors to deliver dsRNA of the invention to cells in vivo is discussed in more detail below.
dsRNA of the invention can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g, lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. 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 of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.
Preferred viral vectors are those derived from AV and AAV. In a particularly preferred embodiment, the dsRNA of the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector comprising, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.
A suitable AV vector for expressing the dsRNA of 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.
Suitable AAV vectors for expressing the dsRNA of 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.
III. PHARMACEUTICAL COMPOSITIONS COMPRISING DSRNA
In one embodiment, the invention provides pharmaceutical compositions comprising a dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition comprising the dsRNA is useful for treating a disease or disorder associated with the expression or activity of the PCSK9 gene, such as pathological processes which can be mediated by down regulating PCSK9 gene expression, such as hyperlipidemia. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for delivery to the liver via parenteral delivery.
The pharmaceutical compositions of the invention are administered in dosages sufficient to inhibit expression of the PCSK9 gene. The present inventors have found that, because of their improved efficiency, compositions comprising the dsRNA of the invention can be administered at surprisingly low dosages. A dosage of 5 mg dsRNA per kilogram body weight of recipient per day is sufficient to inhibit or suppress expression of the PCSK9 gene and may be administered systemically to the patient.
In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 microgram to 1 mg per kilogram body weight per day. The pharmaceutical composition may be administered once daily, or the dsRNA 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 dsRNA 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 dsRNA over a several day period. Sustained release formulations are well known in the art.
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 dsRNAs 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 which can be mediated by down regulating PCSK9 gene expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose.
Any method can be used to administer a dsRNA of the present invention to a mammal. For example, administration can be direct; oral; or parenteral (e.g., by subcutaneous, intraventricular, intramuscular, or intraperitoneal injection, or by intravenous drip). Administration can be rapid (e.g., by injection), or can occur over a period of time (e.g., by slow infusion or administration of slow release formulations).
Typically, when treating a mammal with hyperlipidemia, the dsRNA molecules are administered systemically via parental means. For example, dsRNAs, conjugated or unconjugate or formulated with or without liposomes, can be administered intravenously to a patient. For such, a dsRNA molecule can be formulated into compositions such as sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oil bases. Such solutions also can contain buffers, diluents, and other suitable additives. For parenteral, intrathecal, or intraventricular administration, a dsRNA molecule can be formulated into compositions such as sterile aqueous solutions, which also can contain buffers, diluents, and other suitable additives (e.g., penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers).
In addition, dsRNA molecules can be administered to a mammal as biologic or abiologic means as described in, for example, U.S. Pat. No. 6,271,359. Abiologic delivery can be accomplished by a variety of methods including, without limitation, (1) loading liposomes with a dsRNA acid molecule provided herein and (2) complexing a dsRNA molecule with lipids or liposomes to form nucleic acid-lipid or nucleic acid-liposome complexes. The liposome can be composed of cationic and neutral lipids commonly used to transfect cells in vitro. Cationic lipids can complex (e.g., charge-associate) with negatively charged nucleic acids to form liposomes. Examples of cationic liposomes include, without limitation, lipofectin, lipofectamine, lipofectace, and DOTAP. Procedures for forming liposomes are well known in the art. Liposome compositions can be formed, for example, from phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, or dioleoyl phosphatidylethanolamine. Numerous lipophilic agents are commercially available, including Lipofectin® (Invitrogen/Life Technologies, Carlsbad, Calif.) and EffecteneTM (Qiagen, Valencia, Calif.). In addition, systemic delivery methods can be optimized using commercially available cationic lipids such as DDAB or DOTAP, each of which can be mixed with a neutral lipid such as DOPE or cholesterol. In some cases, liposomes such as those described by Templeton et al. (Nature Biotechnology, 15: 647-652 (1997)) can be used. In other embodiments, polycations such as polyethyleneimine can be used to achieve delivery in vivo and ex vivo (Boletta et al., J. Am Soc. Nephrol. 7: 1728 (1996)). Additional information regarding the use of liposomes to deliver nucleic acids can be found in U.S. Pat. No. 6,271,359, PCT Publication WO 96/40964 and Morrissey, D. et al. 2005. Nat Biotechnol. 23(8):1002-7.
Biologic delivery can be accomplished by a variety of methods including, without limitation, the use of viral vectors. For example, viral vectors (e.g., adenovirus and herpesvirus vectors) can be used to deliver dsRNA molecules to liver cells. Standard molecular biology techniques can be used to introduce one or more of the dsRNAs provided herein into one of the many different viral vectors previously developed to deliver nucleic acid to cells. These resulting viral vectors can be used to deliver the one or more dsRNAs to cells by, for example, infection.
dsRNAs of the present invention can be formulated in a pharmaceutically acceptable carrier or diluent. A “pharmaceutically acceptable carrier” (also referred to herein as an “excipient”) is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties. Typical pharmaceutically acceptable carriers include, by way of example and not limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrates (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).
In addition, dsRNA that target the PCSK9 gene can be formulated into compositions containing the dsRNA admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of nucleic acids. For example, a composition containing one or more dsRNA agents that target the PCSK9 gene can contain other therapeutic agents such as othr lipid lowering agents (e.g., statins).
Methods for Treating Diseases that can be Modulated by Down Regulating the Expression of PCSK9
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 inbalance such as hypercholesterolemia, hypertriglyceridemia and the pathological conditions associated with thiese disorders such as heart and circulatory diseases.
Methods for Inhibiting Expression of the PCSK9 Gene
In yet another aspect, the invention provides a method for inhibiting the expression of the PCSK9 gene in a mammal. The method comprises administering a composition of the invention to the mammal such that expression of the target PCSK9 gene is silenced. Because of their high specificity, the dsRNAs of the invention specifically target RNAs (primary or processed) of the target PCSK9 gene. Compositions and methods for inhibiting the expression of these PCSK9 genes using dsRNAs can be performed as described elsewhere herein.
In one embodiment, the method comprises administering a composition comprising a dsRNA, wherein the dsRNA comprises a nucleotide sequence which is complementary to at least a part of an RNA transcript of the PCSK9 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol) administration. In preferred embodiments, the compositions are administered by intravenous infusion or injection.
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 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
Gene Walking of the PCSK9 Gene
siRNA design was carried out to identify in two separate selections
a) siRNAs targeting PCSK9 human and either mouse or rat mRNA and
b) all human reactive siRNAs with predicted specificity to the target gene PCSK9.
mRNA sequences to human, mouse and rat PCSK9 were used: Human sequence NM—174936.2 was used as reference sequence during the complete siRNA selection procedure.
19 mer stretches conserved in human and mouse, and human and rat PCSK9 mRNA sequences were identified in the first step, resulting in the selection of siRNAs crossreactive to human and mouse, and siRNAs crossreactive to human and rat targets
SiRNAs specifically targeting human PCSK9 were identified in a second selection. All potential 19mer sequences of human PCSK9 were extracted and defined as candidate target sequences. Sequences cross-reactive to human, monkey, and those cross-reactive to mouse, rat, human and monkey are all listed in Tables 1 and 2. Chemically modified versions of those sequences and their activity in both in vitro and in vivo assays are also listed in tables 1 and 2 and examples given in FIGS. 2-8.
In order to rank candidate target sequences and their corresponding siRNAs and select appropriate ones, their predicted potential for interacting with irrelevant targets (off-target potential) was taken as a ranking parameter. siRNAs with low off-target potential were defined as preferable and assumed to be more specific in vivo.
For predicting siRNA-specific off-target potential, the following assumptions were made:
1) positions 2 to 9 (counting 5′ to 3′) of a strand (seed region) may contribute more to off-target potential than rest of sequence (non-seed and cleavage site region)
2) positions 10 and 11 (counting 5′ to 3′) of a strand (cleavage site region) may contribute more to off-target potential than non-seed region
3) positions 1 and 19 of each strand are not relevant for off-target interactions
4) an off-target score can be calculated for each gene and each strand, based on complementarity of siRNA strand sequence to the gene's sequence and position of mismatches
5) number of predicted off-targets as well as highest off-target score must be considered for off-target potential
6) off-target scores are to be considered more relevant for off-target potential than numbers of off-targets
7) assuming potential abortion of sense strand activity by internal modifications introduced, only off-target potential of antisense strand will be relevant
To identify potential off-target genes, 19mer candidate sequences were subjected to a homology search against publically available human mRNA sequences.
The following off-target properties for each 19mer input sequence were extracted for each off-target gene to calculate the off-target score:
Number of mismatches in non-seed region
Number of mismatches in seed region
Number of mismatches in cleavage site region
The off-target score was calculated for considering assumption 1 to 3 as follows:

Off-target score=number of seed mismatches*10+number of cleavage site mismatches*1.2+number of non-seed mismatches*1

The most relevant off-target gene for each siRNA corresponding to the input 19mer sequence was defined as the gene with the lowest off-target score. Accordingly, the lowest off-target score was defined as the relevant off-target score for each siRNA.
dsRNA 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.
siRNA Synthesis
Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2′-O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).
Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, UnterschleiBheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The annealed RNA solution was stored at −20° C. until use.
For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referred to as -Chol-3′), an appropriately modified solid support was used for RNA synthesis. The modified solid support was prepared as follows:
Diethyl-2-azabutane-1,4-dicarboxylate AA




A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until completion of the reaction was ascertained by TLC. After 19 h the solution was partitioned with dichloromethane (3×100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%).
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionic acid ethyl ester AB




Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It was then followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was brought to room temperature and stirred further for 6 h. Completion of the reaction was ascertained by TLC. The reaction mixture was concentrated under vacuum and ethyl acetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer was dried over sodium sulfate and concentrated to give the crude product which was purified by column chromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB.
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC




3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at 0° C. The solution was continued stirring for 1 h. The reaction mixture was concentrated under vacuum, water was added to the residue, and the product was extracted with ethyl acetate. The crude product was purified by conversion into its hydrochloride salt.
3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD




The hydrochloride salt of 3-[(6-Amino-hexanoye-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. The suspension was cooled to 0° C. on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified by flash chromatography (10.3 g, 92%).
1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE  




Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) of diester AD was added slowly with stirring within 20 mins. The temperature was kept below 5° C. during the addition. The stirring was continued for 30 mins at 0° C. and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH2PO4.H2O in 40 mL of water. The resultant mixture was extracted twice with 100 mL of dichloromethane each and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0° C. and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which were combined, dried and evaporated to dryness. The residue was purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).
[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF




Methanol (2 mL) was added dropwise over a period of 1 h to a refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted with ethylacetate (3×40 mL). The combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which was purified by column chromatography (10% MeOH/CHCl3) (89%).
(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG




Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was carried out at room temperature overnight. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated under vacuum and to the residue dichloromethane (50 mL) was added. The organic layer was washed with 1M aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine was removed by evaporating with toluene. The crude product was purified by column chromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl3) (1.75 g, 95%).
Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester AH  




Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step.
Cholesterol Derivatised CPG AI




Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using a wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The achieved loading of the CPG was measured by taking UV measurement (37 mM/g).
The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamide group (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivative group (herein referred to as “5′-Chol-”) was performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step was performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the 5′-end of the nucleic acid oligomer.
Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 1-2.
PCSK9 siRNA Screening in HuH7, HepG2, Hela and Primary Monkey Hepatocytes Discovers Highly Active Sequences
HuH-7 cells were obtained from JCRB Cell Bank (Japanese Collection of Research Bioresources) (Shinjuku, Japan, cat. No.: JCRB0403) Cells were cultured in Dulbecco's MEM (Biochrom AG, Berlin, Germany, cat. No. F0435) supplemented to contain 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany, cat. No. S0115), Penicillin 100 U/ml, Streptomycin 100 μg/ml (Biochrom AG, Berlin, Germany, cat. No. A2213) and 2 mM L-Glutamin (Biochrom AG, Berlin, Germany, cat. No K0282) at 37° C. in an atmosphere with 5% CO2 in a humidified incubator (Heraeus HERAcell, Kendro Laboratory Products, Langenselbold, Germany). HepG2 and Hela cells were obtained from American Type Culture Collection (Rockville, Md., cat. No. HB-8065) and cultured in MEM (Gibco Invitrogen, Karlsruhe, Germany, cat. No. 21090-022) supplemented to contain 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany, cat. No. S0115), Penicillin 100 U/ml, Streptomycin 100 μg/ml (Biochrom AG, Berlin, Germany, cat. No. A2213), 1× Non Essential Amino Acids (Biochrom AG, Berlin, Germany, cat. No. K-0293), and 1 mM Sodium Pyruvate (Biochrom AG, Berlin, Germany, cat. No. L-0473) at 37° C. in an atmosphere with 5% CO2 in a humidified incubator (Heraeus HERAcell, Kendro Laboratory Products, Langenselbold, Germany).
For transfection with siRNA, HuH7, HepG2, or Hela cells were seeded at a density of 2.0×104 cells/well in 96-well plates and transfected directly. Transfection of siRNA (30 nM for single dose screen) was carried out with lipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany, cat. No. 11668-019) as described by the manufacturer.
24 hours after transfection HuH7 and HepG2 cells were lysed and PCSK9 mRNA levels were quantified with the Quantigene Explore Kit (Genosprectra, Dumbarton Circle Fremont, USA, cat. No. QG-000-02) according to the protocol. PCSK9 mRNA levels were normalized to GAP-DH mRNA. For each siRNA eight individual datapoints were collected. siRNA duplexes unrelated to PCSK9 gene were used as control. The activity of a given PCSK9 specific siRNA duplex was expressed as percent PCSK9 mRNA concentration in treated cells relative to PCSK9 mRNA concentration in cells treated with the control siRNA duplex.
Primary cynomolgus monkey hepatocytes (cryopreserved) were obtained from In vitro Technologies, Inc. (Baltimore, Md., USA, cat No M00305) and cultured in InVitroGRO CP Medium (cat No Z99029) at 37° C. in an atmosphere with 5% CO2 in a humidified incubator.
For transfection with siRNA, primary cynomolgus monkey cells were seeded on Collagen coated plates (Fisher Scientific, cat. No. 08-774-5) at a density of 3.5×104 cells/well in 96-well plates and transfected directly. Transfection of siRNA (eight 2-fold dilution series starting from 30 nM) in duplicates was carried out with lipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany, cat. No. 11668-019) as described by the manufacturer.
16 hours after transfection medium was changed to fresh InVitroGRO CP Medium with Torpedo Antibiotic Mix (In vitro Technologies, Inc, cat. No Z99000) added.
24 hours after medium change primary cynomolgus monkey cells were lysed and PCSK9 mRNA levels were quantified with the Quantigene Explore Kit (Genosprectra, Dumbarton Circle Fremont, USA, cat. No. QG-000-02) according to the protocol. PCSK9 mRNA levels were normalized to GAPDH mRNA. Normalized PCSK9/GAPDH ratios were then compared to PCSK9/GAPDH ratio of lipofectamine 2000 only control.
Tables 1-2 (and FIG. 6) summarize the results and provides examples of in vitro screens in different cell lines at different doses. Silencing of PCSK9 transcript was expressed as percentage of remaining transcript at a given dose. Highly active sequences are those with less than 70% transcript remaining post treatment with a given siRNA at a dose less than or equal to 100 nm. Very active sequences are those that have less than 60% of transcript remaining after treatment with a dose. less than or equal to 100 nM. Active sequences are those that have less than 85% transcript remaining after treatment with a high dose (100 nM). Examples of active siRNA's were also screened in vivo in mouse in lipidoid formulations as described below. Active sequences in vitro were also generally active in vivo (See figure FIG. 6 example).
In Vivo Efficacy Screen of PCSK9 siRNAs
Formulation Procedure
The lipidoid LNP-01.4HCl (MW 1487) (FIG. 1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were used to prepare lipid-siRNA nanoparticles. Stock solutions of each in ethanol were prepared: LNP-01, 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide C16, 100 mg/mL. LNP-01, Cholesterol, and PEG-Ceramide C16 stock solutions were then combined in a 42:48:10 molar ratio. Combined lipid solution was mixed rapidly with aqueous siRNA (in sodium acetate pH 5) such that the final ethanol concentration was 35-45% and the final sodium acetate concentration was 100-300 mM. Lipid-siRNA nanoparticles formed spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture was in some cases extruded through a polycarbonate membrane (100 nm cut-off) using a thermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In other cases, the extrusion step was omitted. Ethanol removal and simultaneous buffer exchange was accomplished by either dialysis or tangential flow filtration. Buffer was exchanged to phosphate buffered saline (PBS) pH 7.2.
Characterization of Formulations
Formulations prepared by either the standard or extrusion-free method are characterized in a similar manner. Formulations are first characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles are measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be 20-300 nm, and ideally, 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA is incubated with the RNA-binding dye Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, 0.5% Triton-X100. The total siRNA in the formulation is determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%.
Bolus Dosing
Bolus dosing of formulated siRNAs in C57/BL6 mice (5/group, 8-10 weeks old, Charles River Laboratories, MA) was performed by tail vein injection using a 27 G needle. SiRNAs were formulated in LNP-01 (and then dialyzed against PBS) at 0.5 mg/ml concentration allowing the delivery of the 5 mg/kg dose in 10 μl/g body weight. Mice were kept under an infrared lamp for approximately 3 min prior to dosing to ease injection.
48 hour post dosing mice were sacrificed by CO2-asphyxiation. 0.2 ml blood was collected by retro-orbital bleeding and the liver was harvested and frozen in liquid nitrogen. Serum and livers were stored at −80° C.
Frozen livers were grinded using 6850 Freezer/Mill Cryogenic Grinder (SPEX CentriPrep, Inc) and powders stored at −80° C. until analysis.
PCSK9 mRNA levels were detected using the branched-DNA technology based kit from QuantiGene Reagent System (Genospectra) according to the protocol. 10-20 mg of frozen liver powders was lysed in 600 ul of 0.16 ug/ml Proteinase K (Epicentre, #MPRK092) in Tissue and Cell Lysis Solution (Epicentre, #MTC096H) at 65° C. for 3 hours. Then 10 ul of the lysates were added to 90 ul of Lysis Working Reagent (1 volume of stock Lysis Mixture in two volumes of water) and incubated at 52° C. overnight on Genospectra capture plates with probe sets specific to mouse PCSK9 and mouse GAPDH or cyclophilin B. Nucleic acid sequences for Capture Extender (CE), Label Extender (LE) and blocking (BL) probes were selected from the nucleic acid sequences of PCSK9, GAPDH and cyclophilin B with the help of the QuantiGene ProbeDesigner Software 2.0 (Genospectra, Fremont, Calif., USA, cat. No. QG-002-02). Chemo luminescence was read on a Victor2-Light (Perkin Elmer) as Relative light units. The ratio of PCSK9 mRNA to GAPDH or cyclophilin B mRNA in liver lysates was averaged over each treatment group and compared to a control group treated with PBS or a control group treated with an unrelated siRNA (blood coagulation factor VII).
Total serum cholesterol in mouse serum was measured using the StanBio Cholesterol LiquiColor kit (StanBio Laboratoriy, Boerne, Tex., USA) according to manufacturer's instructions. Measurements were taken on a Victor2 1420 Multilabel Counter (Perkin Elmer) at 495 nm.
EXAMPLES
32 PCSK9 siRNAs formulated in LNP-01 liposomes were tested in vivo in a mouse model. The experiment was performed at 5 mg/kg siRNA dose and at least 10 PCSK9 siRNAs showed more than 40% PCSK9 mRNA knock down compared to a control group treated with PBS, while control group treated with an unrelated siRNA (blood coagulation factor VII) had no effect (FIGS. 2-5). Silencing of PCSK9 transcript also coorelated with a lowering of cholesterol in these animals (FIGS. 4-5). In addition there was a strong coorelation between those molecules that were active in vitro and those active in vivo (FIG. 6). Sequences containing different chemical modifications were also screened in vitro (Tables 1 and 2) and in vivo. As an example, less modified sequences 9314 and 9318, and a more modified versions of that sequence 9314-(10792, 10793, and 10796); 9318-(10794, 10795, 10797) were tested both in vitro (In primary monkey hepatocytes) or in vivo (9314 and 10792) formulated in LNP-01. FIG. 7 (also see Tables 1 and 2) shows that the parent molecules 9314 and 9318 and the modified versions are all active in vitro. FIG. 8 as an example shows that both the parent 9314 and the more highly modified 10792 sequences are active in vivo displaying 50-60% silencing of endogenous PCSK9 in mice. FIG. 9 further exemplifies that activity of other chemically modified versions of the parents 9314 and 10792.
dsRNA Expression Vectors
In another aspect of the invention, PCSK9 specific dsRNA molecules that modulate PCSK9 gene expression activity are 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). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. 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 strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In a preferred 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.
The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. NatI. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. NatI. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. NatI. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. NatI. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. NatI. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.
The promoter driving dsRNA expression in either a DNA plasmid or viral vector of the invention may be a eukaryotic RNA polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter or actin promoter or U1 snRNA promoter) or generally RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g. the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).
In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a 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 transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.
Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA 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.
dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-TKOTM). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single PCSK9 gene or multiple PCSK9 genes over a period of a week or more are also contemplated by the invention. Successful introduction of the vectors of the invention 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 ex vivo cells 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.
The PCSK9 specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise 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.
Those skilled in the art are familiar with methods and compositions in addition to those specifically set out in the instant disclosure which will allow them to practice this invention to the full scope of the claims hereinafter appended.







TABLE 1







sequences












position







in human 







access. # 
Sense strand
SEQ
Antisense-strand
SEQ
Duplex


NM_174936
sequence (5′-3′)1
ID NO:
sequence (5′-3′)1
ID NO:
name





 2-20
AGCGACGUCGAGGCGCUCATT
1
UGAGCGCCUCGACGUCGCUTT
2
AD-15220


 


15-33
CGCUCAUGGUUGCAGGCGGTT
3
CCGCCUGCAACCAUGAGCGTT
4
AD-15275


 


16-34
GCUCAUGGUUGCAGGCGGGTT
5
CCCGCCUGCAACCAUGAGCTT
6
AD-15301


 


30-48
GCGGGCGCCGCCGUUCAGUTT
7
ACUGAACGGCGGCGCCCGCTT
8
AD-15276


 


31-49
CGGGCGCCGCCGUUCAGUUTT
9
AACUGAACGGCGGCGCCCGTT
10
AD-15302


 


32-50
GGGCGCCGCCGUUCAGUUCTT
11
GAACUGAACGGCGGCGCCCTT
12
AD-15303


 


40-58
CCGUUCAGUUCAGGGUCUGTT
13
CAGACCCUGAACUGAACGGTT
14
AD-15221


 


43-61
UUCAGUUCAGGGUCUGAGCTT
15
GCUCAGACCCUGAACUGAATT
16
AD-15413


 


 82-100
GUGAGACUGGCUCGGGCGGTT
17
CCGCCCGAGCCAGUCUCACTT
18
AD-15304


 


100-118
GGCCGGGACGCGUCGUUGCTT
19
GCAACGACGCGUCCCGGCCTT
20
AD-15305


 


101-119
GCCGGGACGCGUCGUUGCATT
21
UGCAACGACGCGUCCCGGCTT
22
AD-15306


 


102-120
CCGGGACGCGUCGUUGCAGTT
23
CUGCAACGACGCGUCCCGGTT
24
AD-15307


 


105-123
GGACGCGUCGUUGCAGCAGTT
25
CUGCUGCAACGACGCGUCCTT
26
AD-15277


 


135-153
UCCCAGCCAGGAUUCCGCGTsT
27
CGCGGAAUCCUGGCUGGGATsT
28
AD-9526 


 


135-153
ucccAGccAGGAuuccGcGTsT
29
CGCGGAAUCCUGGCUGGGATsT
30
AD-9652 


 


136-154
CCCAGCCAGGAUUCCGCGCTsT
31
GCGCGGAAUCCUGGCUGGGTsT
32
AD-9519 


 


136-154
cccAGccAGGAuuccGcGcTsT
33
GCGCGGAAUCCUGGCUGGGTsT
34
AD-9645 


 


138-156
CAGCCAGGAUUCCGCGCGCTsT
35
GCGCGCGGAAUCCUGGCUGTsT
36
AD-9523 


 


138-156
cAGccAGGAuuccGcGcGcTsT
37
GCGCGCGGAAUCCUGGCUGTsT
38
AD-9649 


 


185-203
AGCUCCUGCACAGUCCUCCTsT
39
GGAGGACUGUGCAGGAGCUTsT
40
AD-9569 


 


185-203
AGcuccuGcAcAGuccuccTsT
41
GGAGGACUGUGcAGGAGCUTsT
42
AD-9695 


 


205-223
CACCGCAAGGCUCAAGGCGTT
43
CGCCUUGAGCCUUGCGGUGTT
44
AD-15222


 


208-226
CGCAAGGCUCAAGGCGCCGTT
45
CGGCGCCUUGAGCCUUGCGTT
46
AD-15278


 


210-228
CAAGGCUCAAGGCGCCGCCTT
47
GGCGGCGCCUUGAGCCUUGTT
48
AD-15178


 


232-250
GUGGACCGCGCACGGCCUCTT
49
GAGGCCGUGCGCGGUCCACTT
50
AD-15308


 


233-251
UGGACCGCGCACGGCCUCUTT
51
AGAGGCCGUGCGCGGUCCATT
52
AD-15223


 


234-252
GGACCGCGCACGGCCUCUATT
53
UAGAGGCCGUGCGCGGUCCTT
54
AD-15309


 


235-253
GACCGCGCACGGCCUCUAGTT
55
CUAGAGGCCGUGCGCGGUCTT
56
AD-15279


 


236-254
ACCGCGCACGGCCUCUAGGTT
57
CCUAGAGGCCGUGCGCGGUTT
58
AD-15194


 


237-255
CCGCGCACGGCCUCUAGGUTT
59
ACCUAGAGGCCGUGCGCGGTT
60
AD-15310


 


238-256
CGCGCACGGCCUCUAGGUCTT
61
GACCUAGAGGCCGUGCGCGTT
62
AD-15311


 


239-257
GCGCACGGCCUCUAGGUCUTT
63
AGACCUAGAGGCCGUGCGCTT
64
AD-15392


 


240-258
CGCACGGCCUCUAGGUCUCTT
65
GAGACCUAGAGGCCGUGCGTT
66
AD-15312


 


248-266
CUCUAGGUCUCCUCGCCAGTT
67
CUGGCGAGGAGACCUAGAGTT
68
AD-15313


 


249-267
UCUAGGUCUCCUCGCCAGGTT
69
CCUGGCGAGGAGACCUAGATT
70
AD-15280


 


250-268
CUAGGUCUCCUCGCCAGGATT
71
UCCUGGCGAGGAGACCUAGTT
72
AD-15267


 


252-270
AGGUCUCCUCGCCAGGACATT
73
UGUCCUGGCGAGGAGACCUTT
74
AD-15314


 


258-276
CCUCGCCAGGACAGCAACCTT
75
GGUUGCUGUCCUGGCGAGGTT
76
AD-15315


 


300-318
CGUCAGCUCCAGGCGGUCCTsT
77
GGACCGCCUGGAGCUGACGTsT
78
AD-9624 


 


300-318
cGucAGcuccAGGcGGuccTsT
79
GGACCGCCUGGAGCUGACGTsT
80
AD-9750 


 


301-319
GUCAGCUCCAGGCGGUCCUTsT
81
AGGACCGCCUGGAGCUGACTsT
82
AD-9623 


 


301-319
GucAGcuccAGGcGGuccuTsT
83
AGGACCGCCUGGAGCUGACTsT
84
AD-9749 


 


370-388
GGCGCCCGUGCGCAGGAGGTT
85
CCUCCUGCGCACGGGCGCCTT
86
AD-15384


 


408-426
GGAGCUGGUGCUAGCCUUGTsT
87
CAAGGCUAGCACCAGCUCCTsT
88
AD-9607 


 


408-426
GGAGcuGGuGcuAGccuuGTsT
89
cAAGGCuAGcACcAGCUCCTsT
90
AD-9733 


 


411-429
GCUGGUGCUAGCCUUGCGUTsT
91
ACGCAAGGCUAGCACCAGCTsT
92
AD-9524 


 


411-429
GcuGGuGcuAGccuuGcGuTsT
93
ACGcAAGGCuAGcACcAGCTsT
94
AD-9650 


 


412-430
CUGGUGCUAGCCUUGCGUUTsT
95
AACGCAAGGCUAGCACCAGTsT
96
AD-9520 


 


412-430
CUGGUGCUAGCCUUGCGUUTsT
97
AACGCAAGGCUAGCACCAGTsT
98
AD-9520 


 


412-430
cuGGuGcuAGccuuGcGuuTsT
99
AACGcAAGGCuAGcACcAGTsT
100
AD-9646 


 


416-434
UGCUAGCCUUGCGUUCCGATsT
101
UCGGAACGCAAGGCUAGCATsT
102
AD-9608 


 


416-434
uGcuAGccuuGcGuuccGATsT
103
UCGGAACGcAAGGCuAGcATsT
104
AD-9734 


 


419-437
UAGCCUUGCGUUCCGAGGATsT
105
UCCUCGGAACGCAAGGCUATsT
106
AD-9546 


 


419-437
uAGccuuGcGuuccGAGGATsT
107
UCCUCGGAACGcAAGGCuATsT
108
AD-9672 


 


439-457
GACGGCCUGGCCGAAGCACTT
109
GUGCUUCGGCCAGGCCGUCTT
110
AD-15385


 


447-465
GGCCGAAGCACCCGAGCACTT
111
GUGCUCGGGUGCUUCGGCCTT
112
AD-15393


 


448-466
GCCGAAGCACCCGAGCACGTT
113
CGUGCUCGGGUGCUUCGGCTT
114
AD-15316


 


449-467
CCGAAGCACCCGAGCACGGTT
115
CCGUGCUCGGGUGCUUCGGTT
116
AD-15317


 


458-476
CCGAGCACGGAACCACAGCTT
117
GCUGUGGUUCCGUGCUCGGTT
118
AD-15318


 


484-502
CACCGCUGCGCCAAGGAUCTT
119
GAUCCUUGGCGCAGCGGUGTT
120
AD-15195


 


486-504
CCGCUGCGCCAAGGAUCCGTT
121
CGGAUCCUUGGCGCAGCGGTT
122
AD-15224


 


487-505
CGCUGCGCCAAGGAUCCGUTT
123
ACGGAUCCUUGGCGCAGCGTT
124
AD-15188


 


489-507
CUGCGCCAAGGAUCCGUGGTT
125
CCACGGAUCCUUGGCGCAGTT
126
AD-15225


 


500-518
AUCCGUGGAGGUUGCCUGGTT
127
CCAGGCAACCUCCACGGAUTT
128
AD-15281


 


509-527
GGUUGCCUGGCACCUACGUTT
129
ACGUAGGUGCCAGGCAACCTT
130
AD-15282


 


542-560
AGGAGACCCACCUCUCGCATT
131
UGCGAGAGGUGGGUCUCCUTT
132
AD-15319


 


543-561
GGAGACCCACCUCUCGCAGTT
133
CUGCGAGAGGUGGGUCUCCTT
134
AD-15226


 


544-562
GAGACCCACCUCUCGCAGUTT
135
ACUGCGAGAGGUGGGUCUCTT
136
AD-15271


 


549-567
CCACCUCUCGCAGUCAGAGTT
137
CUCUGACUGCGAGAGGUGGTT
138
AD-15283


 


552-570
CCUCUCGCAGUCAGAGCGCTT
139
GCGCUCUGACUGCGAGAGGTT
140
AD-15284


 


553-571
CUCUCGCAGUCAGAGCGCATT
141
UGCGCUCUGACUGCGAGAGTT
142
AD-15189


 


554-572
UCUCGCAGUCAGAGCGCACTT
143
GUGCGCUCUGACUGCGAGATT
144
AD-15227


 


555-573
CUCGCAGUCAGAGCGCACUTsT
145
AGUGCGCUCUGACUGCGAGTsT
146
AD-9547 


 


555-573
cucGcAGucAGAGcGcAcuTsT
147
AGUGCGCUCUGACUGCGAGTsT
148
AD-9673 


 


558-576
GCAGUCAGAGCGCACUGCCTsT
149
GGCAGUGCGCUCUGACUGCTsT
150
AD-9548 


 


558-576
GcAGucAGAGcGcAcuGccTsT
151
GGcAGUGCGCUCUGACUGCTsT
152
AD-9674 


 


606-624
GGGAUACCUCACCAAGAUCTsT
153
GAUCUUGGUGAGGUAUCCCTsT
154
AD-9529 


 


606-624
GGGAuAccucAccAAGAucTsT
155
GAUCUUGGUGAGGuAUCCCTsT
156
AD-9655 


 


659-677
UGGUGAAGAUGAGUGGCGATsT
157
UCGCCACUCAUCUUCACCATsT
158
AD-9605 


 


659-677
uGGuGAAGAuGAGuGGcGATsT
159
UCGCcACUcAUCUUcACcATsT
160
AD-9731 


 


663-681
GAAGAUGAGUGGCGACCUGTsT
161
CAGGUCGCCACUCAUCUUCTsT
162
AD-9596 


 


663-681
GAAGAuGAGuGGcGAccuGTsT
163
cAGGUCGCcACUcAUCUUCTsT
164
AD-9722 


 


704-722
CCCAUGUCGACUACAUCGATsT
165
UCGAUGUAGUCGACAUGGGTsT
166
AD-9583 


 


704-722
cccAuGucGAcuAcAucGATsT
167
UCGAUGuAGUCGAcAUGGGTsT
168
AD-9709 


 


718-736
AUCGAGGAGGACUCCUCUGTsT
169
CAGAGGAGUCCUCCUCGAUTsT
170
AD-9579 


 


718-736
AucGAGGAGGAcuccucuGTsT
171
cAGAGGAGUCCUCCUCGAUTsT
172
AD-9705 


 


758-776
GGAACCUGGAGCGGAUUACTT
173
GUAAUCCGCUCCAGGUUCCTT
174
AD-15394


 


759-777
GAACCUGGAGCGGAUUACCTT
175
GGUAAUCCGCUCCAGGUUCTT
176
AD-15196


 


760-778
AACCUGGAGCGGAUUACCCTT
177
GGGUAAUCCGCUCCAGGUUTT
178
AD-15197


 


777-795
CCCUCCACGGUACCGGGCGTT
179
CGCCCGGUACCGUGGAGGGTT
180
AD-15198


 


782-800
CACGGUACCGGGCGGAUGATsT
181
UCAUCCGCCCGGUACCGUGTsT
182
AD-9609 


 


782-800
cAcGGuAccGGGcGGAuGATsT
183
UcAUCCGCCCGGuACCGUGTsT
184
AD-9735 


 


783-801
ACGGUACCGGGCGGAUGAATsT
185
UUCAUCCGCCCGGUACCGUTsT
186
AD-9537 


 


783-801
AcGGuAccGGGcGGAuGAATsT
187
UUcAUCCGCCCGGuACCGUTsT
188
AD-9663 


 


784-802
CGGUACCGGGCGGAUGAAUTsT
189
AUUCAUCCGCCCGGUACCGTsT
190
AD-9528 


 


784-802
cGGuAccGGGcGGAuGAAuTsT
191
AUUcAUCCGCCCGGuACCGTsT
192
AD-9654 


 


785-803
GGUACCGGGCGGAUGAAUATsT
193
UAUUCAUCCGCCCGGUACCTsT
194
AD-9515 


 


785-803
GGuAccGGGcGGAuGAAuATsT
195
uAUCcAUCCGCCCGGuACCTsT
196
AD-9641 


 


786-804
GUACCGGGCGGAUGAAUACTsT
197
GUAUUCAUCCGCCCGGUACTsT
198
AD-9514 


 


786-804
GuAccGGGcGGAuGAAuAcTsT
199
GuAUUcAUCCGCCCGGuACTsT
200
AD-9640 


 


788-806
ACCGGGCGGAUGAAUACCATsT
201
UGGUAUUCAUCCGCCCGGUTsT
202
AD-9530 


 


788-806
AccGGGcGGAuGAAuAccATsT
203
UGGuAUUcAUCCGCCCGGUTsT
204
AD-9656 


 


789-807
CCGGGCGGAUGAAUACCAGTsT
205
CUGGUAUUCAUCCGCCCGGTsT
206
AD-9538 


 


789-807
ccGGGcGGAuGAAuAccAGTsT
207
CUGGuAUUcAUCCGCCCGGTsT
208
AD-9664 


 


825-843
CCUGGUGGAGGUGUAUCUCTsT
209
GAGAUACACCUCCACCAGGTsT
210
AD-9598 


 


825-843
ccuGGuGGAGGuGuAucucTsT
211
GAGAuAcACCUCcACcAGGTsT
212
AD-9724 


 


826-844
CUGGUGGAGGUGUAUCUCCTsT
213
GGAGAUACACCUCCACCAGTsT
214
AD-9625 


 


826-844
cuGGuGGAGGuGuAucuccTsT
215
GGAGAuAcACCUCcACcAGTsT
216
AD-9751 


 


827-845
UGGUGGAGGUGUAUCUCCUTsT
217
AGGAGAUACACCUCCACCATsT
218
AD-9556 


 


827-845
uGGuGGAGGuGuAucuccuTsT
219
AGGAGAuAcACCUCcACcATsT
220
AD-9682 


 


828-846
GGUGGAGGUGUAUCUCCUATsT
221
UAGGAGAUACACCUCCACCTsT
222
AD-9539 


 


828-846
GGuGGAGGuGuAucuccuATsT
223
uAGGAGAuAcACCUCcACCTsT
224
AD-9665 


 


831-849
GGAGGUGUAUCUCCUAGACTsT
225
GUCUAGGAGAUACACCUCCTsT
226
AD-9517 


 


831-849
GGAGGuGuAucuccuAGAcTsT
227
GUCuAGGAGAuAcACCUCCTsT
228
AD-9643 


 


833-851
AGGUGUAUCUCCUAGACACTsT
229
GUGUCUAGGAGAUACACCUTsT
230
AD-9610 


 


833-851
AGGuGuAucuccuAGAcAcTsT
231
GUGUCuAGGAGAuAcACCUTsT
232
AD-9736 


 


833-851
AfgGfuGfuAfuCfuCfcUfaGf
233
p-gUfgUfcUfaGfgAfgAfuA
234
AD-14681



aCfaCfTsT

fcAfcCfuTsT




 


833-851
AGGUfGUfAUfCfUfCfCfUfAGAC
235
GUfGUfCfUfAGGAGAUfACf
236
AD-14691



fACfTsT

ACfCfUfTsT




 


833-851
AgGuGuAuCuCcUaGaCaCTsT
237
p-gUfgUfcUfaGfgAfgAfuA
238
AD-14701





fcAfcCfuTsT




 


833-851
AgGuGuAuCuCcUaGaCaCTsT
239
GUfGUfCfUfAGGAGAUfACf
240
AD-14711





ACfCfUfTsT




 


833-851
AfgGfuGfuAfuCfuCfcUfaGfaC
241
GUGUCuaGGagAUACAccuTsT
242
AD-14721



faCffsT






 


833-851
AGGUfGUfAUfCfUfCfCfUfAGA
243
GUGUCuaGGagAUACAccuTsT
244
AD-14731



CfACfTsT






 


833-851
AgGuGuAuCuCcUaGaCaCTsT
245
GUGUCuaGGagAUACAccuTsT
246
AD-14741


 


833-851
GfcAfcCfciffcAfuAfgGfcC
247
p-uCfcAfgGfcCfuAfuGfaGfT 
248
AD-15087



fuGfgAfTsT

gGfuGfcTs




 


833-851
GCfACfCfCfUfCfAUfAGGCf
249
UfCfCfAGGCfCfUfAUfG
250
AD-15097



CfUfGGATsT

AGGGUfGCfTsT




 


833-851
GcAcCcUcAuAgGcCuGgATsT
251
p-uCfcAfgGfcCfuAfuGfaGT 
252
AD-15107





fgGfuGfcTs




 


833-851
GcAcCcUcAuAgGcCuGgATsT
253
UfCfCfAGGCfCfUfAUfG
254
AD-15117





AGGGUfGCfTsT




 


833-851
GfcAfcCfcUfcAfuAfgGfcCf
255
UCCAGgcCUauGAGGGugcTsT
256
AD-15127



uGfgAfTsT






 


833-851
GCfACfCfCfUfCfAUfAGGCfC
257
UCCAGgcCUauGAGGGugcTsT
258
AD-15137



fUfGGATsT






 


833-851
GcAcCcUcAuAgGcCuGgATsT
259
UCCAGgcCUauGAGGGugcTsT
260
AD-15147


 


836-854
UGUAUCUCCUAGACACCAGTsT
261
CUGGUGUCUAGGAGAUACATsT
262
AD-9516 


 


836-854
uGuAucuccuAGAcAccAGTsT
263
CUGGUGUCuAGGAGAuAcATsT
264
AD-9642 


 


840-858
UCUCCUAGACACCAGCAUATsT
265
UAUGCUGGUGUCUAGGAGATsT
266
AD-9562 


 


840-858
ucuccuAGAcAccAGcAuATsT
267
uAUGCUGGUGUCuAGGAGATsT
268
AD-9688 


 


840-858
UfcUffcCfuAfgAfcAfcCfaG
269
p-uAfuGfcUfgGfuGfuCfu
270
AD-14677



fcAfuAfTsT

AfgGfaGfaTsT




 


840-858
UfCfUfCfCfUfAGACfACfCf
271
UfAUfGCfUfGGUfGUfCf
272
AD-14687



AGCfAUfATsT

UfAGGAGATsT




 


840-858
UcUcCuAgAcAcCaGcAuATsT
273
p-uAfuGfcUfgGfuGfuCfuA
274
AD-14697





fgGfaGfaTsT




 


840-858
UcUcCuAgAcAcCaGcAuATsT
275
UfAUfGCfUfGGUfGUfCfU
276
AD-14707





fAGGAGATsT




 


840-858
UfcUffcCfuAafAfcAfcCfa
277
UAUGCugGUguCUAGGagaTsT
278
AD-14717



GfcAfuAfTsT






 


840-858
UfCfUfCfCfUfAGACfACfCfA
279
UAUGCugGUguCUAGGagaTsT
280
AD-14727



GCfAUfATsT






 


840-858
UcUcCuAgAcAcCaGcAuATsT
281
UAUGCugGUguCUAGGagaTsT
282
AD-14737


 


840-858
AfgGfcCfuGfgAfgUfuUfaUf
283
p-cCfgAfaUfaAfaCfuCfcAf
284
AD-15083



uCfgGfTsT

gGfcCfuTsT




 


840-858
AGGCfCfUfGGAGUfUfUfAU
285
CfCfGAAUfAAACfUfCfCfAG
286
AD-15093



fUfCfGGTsT

GCfCfUfTsT




 


840-858
AgGcCuGgAgUuUaUuCgGTsT
287
p-cCfgAfaUfaAfaCfuCfcA
288
AD-15103





fgGfcCfuTsT




 


840-858
AgGcCuGgAgUuUaUuCgGTsT
289
CfCfGAAUfAAACfUfCfCfAG
290
AD-15113





GCfCfUfTsT




 


840-858
AfgGfcCfuGfgAfgUfuUfaU
291
CCGAAuaAAcuCCAGGccuTsT
292
AD-15123



fuCfgGfTsT






 


840-858
AGGCfCfUfGGAGUfUfUfAU
293
CCGAAuaAAcuCCAGGccuTsT
294
AD-15133



fUfCfGGTsT






 


840-858
AgGcCuGgAgUuUaUuCgGTsT
295
CCGAAuaAAcuCCAGGccuTsT
296
AD-15143


 


841-859
CUCCUAGACACCAGCAUACTsT
297
GUAUGCUGGUGUCUAGGAGTsT
298
AD-9521 


 


841-859
cuccuAGAcAccAGcAuAcTsT
299
GuAUGCUGGUGUCuAGGAGTsT
300
AD-9647 


 


842-860
UCCUAGACACCAGCAUACATsT
301
UGUAUGCUGGUGUCUAGGATsT
302
AD-9611 


 


842-860
uccuAGAcAccAGcAuAcATsT
303
UGuAUGCUGGUGUCuAGGATsT
304
AD-9737 


 


843-861
CCUAGACACCAGCAUACAGTsT
305
CUGUAUGCUGGUGUCUAGGTsT
306
AD-9592 


 


843-861
ccuAGAcAccAGcAuAcAGTsT
307
CUGuAUGCUGGUGUCuAGGTsT
308
AD-9718 


 


847-865
GACACCAGCAUACAGAGUGTsT
309
CACUCUGUAUGCUGGUGUCTsT
310
AD-9561 


 


847-865
GAcAccAGcAuAcAGAGuGTsT
311
cACUCUGuAUGCUGGUGUCTsT
312
AD-9687 


 


855-873
CAUACAGAGUGACCACCGGTsT
313
CCGGUGGUCACUCUGUAUGTsT
314
AD-9636 


 


855-873
cAuAcAGAGuGAccAccGGTsT
315
CCGGUGGUcACUCUGuAUGTsT
316
AD-9762 


 


860-878
AGAGUGACCACCGGGAAAUTsT
317
AUUUCCCGGUGGUCACUCUTsT
318
AD-9540 


 


860-878
AGAGuGAccAccGGGAAAuTsT
319
AUUUCCCGGUGGUcACUCUTsT
320
AD-9666 


 


861-879
GAGUGACCACCGGGAAAUCTsT
321
GAUUUCCCGGUGGUCACUCTsT
322
AD-9535 


 


861-879
GAGuGAccAccGGGAAAucTsT
323
GAUUUCCCGGUGGUcACUCTsT
324
AD-9661 


 


863-881
GUGACCACCGGGAAAUCGATsT
325
UCGAUUUCCCGGUGGUCACTsT
326
AD-9559 


 


863-881
GuGAccAccGGGAAAucGATsT
327
UCGAUUUCCCGGUGGUcACTsT
328
AD-9685 


 


865-883
GACCACCGGGAAAUCGAGGTsT
329
CCUCGAUUUCCCGGUGGUCTsT
330
AD-9533 


 


865-883
GAccAccGGGAAAucGAGGTsT
331
CCUCGAUUUCCCGGUGGUCTsT
332
AD-9659 


 


866-884
ACCACCGGGAAAUCGAGGGTsT
333
CCCUCGAUUUCCCGGUGGUTsT
334
AD-9612 


 


866-884
AccAccGGGAAAucGAGGGTsT
335
CCCUCGAUUUCCCGGUGGUTsT
336
AD-9738 


 


867-885
CCACCGGGAAAUCGAGGGCTsT
337
GCCCUCGAUUUCCCGGUGGTsT
338
AD-9557 


 


867-885
ccAccGGGAAAucGAGGGcTsT
339
GCCCUCGAUUUCCCGGUGGTsT
340
AD-9683 


 


875-893
AAAUCGAGGGCAGGGUCAUTsT
341
AUGACCCUGCCCUCGAUUUTsT
342
AD-9531 


 


875-893
AAAucGAGGGcAGGGucAuTsT
343
AUGACCCUGCCCUCGAUUUTsT
344
AD-9657 


 


875-893
AfaAfuCfgAfgGfgCfaGfgG
345
p-aUfgAfcCfcUfgCfcCfu
346
AD-14673



fuCfaUfTsT

CfgAfuUfuTsT




 


875-893
AAAUfCfGAGGGCfAGGGUfCf
347
AUfGACfCfCfUfGCfCfCfUfCf
348
AD-14683



AUfTsT

GAUfUfUfTsT




 


875-893
AaAuCgAgGgCaGgGuCaUTsT
349
p-aUfgAfcCfcUfgCfcCfuC
350
AD-14693





fgAfuUfuTsT




 


875-893
AaAuCgAgGgCaGgGuCaUTsT
351
AUfGACfCfCfUfGCfCfCfUf
352
AD-14703





CfGAUfUfUfTsT




 


875-893
AfaAfuCfgAfgGfgCfaGfgG
353
AUGACccUGccCUCGAuuuTsT
354
AD-14713



fuCfaUfTsT






 


875-893
AAAUfCfGAGGGCfAGGGUfCf
355
AUGACccUGccCUCGAuuuTsT
356
AD-14723



AUfTsT






 


875-893
AaAuCgAgGgCaGgGuCaUTsT
357
AUGACccUGccCUCGAuuuTsT
358
AD-14733


 


875-893
CfgGfcAfcCfcUfcAfuAfgGfc
359
p-cAfgGfcCfuAfuGfaGfgGfu
360
AD-15079



CfuGfTsT

GfcCfgTsT




 


875-893
CfGGCfACfCfCfUfCfAUfAGG
361
CfAGGCfCfUfAUfGAGG
362
AD-15089





GUfGCfCfGTsT





CfCfUfGTsT






 


875-893
CgGcAcCcUcAuAgGcCuGTsT
363
p-cAfgGfcCfuAfuGfaGfg
364
AD-15099





GfuGfcCfgTsT




 


875-893
CgGcAcCcUcAuAgGcCuGTsT
365
CfAGGCfCfUfAUfGAGGGUf
366
AD-15109





GCfCfGTsT




 


875-893
CfgGfcAfcCfcUfcAfuAfgG
367
CAGGCcuAUgaGGGUGccgTsT
368
AD-15119



fcCfuGfTsT






 


875-893
CfGGCfACfCfCfUfCfAUfAG
369
CAGGCcuAUgaGGGUGccgTsT
370
AD-15129



GCfCfUfGTsT






 


875-893
CgGcAcCcUcAuAgGcCuGTsT
371
CAGGCcuAUgaGGGUGccgTsT
372
AD-15139


 


877-895
AUCGAGGGCAGGGUCAUGGTsT
373
CCAUGACCCUGCCCUCGAUTsT
374
AD-9542 


 


877-895
AucGAGGGcAGGGucAuGGTsT
375
CcAUGACCCUGCCCUCGAUTsT
376
AD-9668 


 


878-896
cGAGGGcAGGGucAuGGucTsT
377
GACcAUGACCCUGCCCUCGTsT
378
AD-9739 


 


880-898
GAGGGCAGGGUCAUGGUCATsT
379
UGACCAUGACCCUGCCCUCTsT
380
AD-9637 


 


880-898
GAGGGcAGGGucAuGGucATsT
381
UGACcAUGACCCUGCCCUCTsT
382
AD-9763 


 


882-900
GGGCAGGGUCAUGGUCACCTsT
383
GGUGACCAUGACCCUGCCCTsT
384
AD-9630 


 


882-900
GGGcAGGGucAuGGucAccTsT
385
GGUGACcAUGACCCUGCCCTsT
386
AD-9756 


 


885-903
CAGGGUCAUGGUCACCGACTsT
387
GUCGGUGACCAUGACCCUGTsT
388
AD-9593 


 


885-903
cAGGGucAuGGucAccGAcTsT
389
GUCGGUGACcAUGACCCUGTsT
390
AD-9719 


 


886-904
AGGGUCAUGGUCACCGACUTsT
391
AGUCGGUGACCAUGACCCUTsT
392
AD-9601 


 


886-904
AGGGucAuGGucAccGAcuTsT
393
AGUCGGUGACcAUGACCCUTsT
394
AD-9727 


 


892-910
AUGGUCACCGACUUCGAGATsT
395
UCUCGAAGUCGGUGACCAUTsT
396
AD-9573 


 


892-910
AuGGucAccGAcuucGAGATsT
397
UCUCGAAGUCGGUGACcAUTsT
398
AD-9699 


 


899-917
CCGACUUCGAGAAUGUGCCTT
399
GGCACAUUCUCGAAGUCGGTT
400
AD-15228


 


921-939
GGAGGACGGGACCCGCUUCTT
401
GAAGCGGGUCCCGUCCUCCTT
402
AD-15395


 


 993-1011
CAGCGGCCGGGAUGCCGGCTsT
403
GCCGGCAUCCCGGCCGCUGTsT
404
AD-9602 


 


 993-1011
cAGcGGccGGGAuGccGGcTsT
405
GCCGGcAUCCCGGCCGCUGTsT
406
AD-9728 


 


1020-1038
GGGUGCCAGCAUGCGCAGCTT
407
GCUGCGCAUGCUGGCACCCTT
408
AD-15386


 


1038-1056
CCUGCGCGUGCUCAACUGCTsT
409
GCAGUUGAGCACGCGCAGGTsT
410
AD-9580 


 


1038-1056
ccuGcGcGuGcucAAcuGcTsT
411
GcAGUUGAGcACGCGcAGGTsT
412
AD-9706 


 


1040-1058
UGCGCGUGCUCAACUGCCATsT
413
UGGCAGUUGAGCACGCGCATsT
414
AD-9581 


 


1040-1058
uGcGcGuGcucAAcuGccATsT
415
UGGcAGUUGAGcACGCGcATsT
416
AD-9707 


 


1042-1060
CGCGUGCUCAACUGCCAAGTsT
417
CUUGGCAGUUGAGCACGCGTsT
418
AD-9543 


 


1042-1060
cGcGuGcucAAcuGccAAGTsT
419
CUUGGcAGUUGAGcACGCGTsT
420
AD-9669 


 


1053-1071
CUGCCAAGGGAAGGGCACGTsT
421
CGUGCCCUUCCCUUGGCAGTsT
422
AD-9574 


 


1053-1071
cuGccAAGGGAAGGGcAcGTsT
423
CGUGCCCUUCCCUUGGcAGTsT
424
AD-9700 


 


1057-1075
CAAGGGAAGGGCACGGUUATT
425
UAACCGUGCCCUUCCCUUGTT
426
AD-15320


 


1058-1076
AAGGGAAGGGCACGGUUAGTT
427
CUAACCGUGCCCUUCCCUUTT
428
AD-15321


 


1059-1077
AGGGAAGGGCACGGUUAGCTT
429
GCUAACCGUGCCCUUCCCUTT
430
AD-15199


 


1060-1078
GGGAAGGGCACGGUUAGCGTT
431
CGCUAACCGUGCCCUUCCCTT
432
AD-15167


 


1061-1079
GGAAGGGCACGGUUAGCGGTT
433
CCGCUAACCGUGCCCUUCCTT
434
AD-15164


 


1062-1080
GAAGGGCACGGUUAGCGGCTT
435
GCCGCUAACCGUGCCCUUCTT
436
AD-15166


 


1063-1081
AAGGGCACGGUUAGCGGCATT
437
UGCCGCUAACCGUGCCCUUTT
438
AD-15322


 


1064-1082
AGGGCACGGUUAGCGGCACTT
439
GUGCCGCUAACCGUGCCCUTT
440
AD-15200


 


1068-1086
CACGGUUAGCGGCACCCUCTT
441
GAGGGUGCCGCUAACCGUGTT
442
AD-15213


 


1069-1087
ACGGUUAGCGGCACCCUCATT
443
UGAGGGUGCCGCUAACCGUTT
444
AD-15229


 


1072-1090
GUUAGCGGCACCCUCAUAGTT
445
CUAUGAGGGUGCCGCUAACTT
446
AD-15215


 


1073-1091
UUAGCGGCACCCUCAUAGGTT
447
CCUAUGAGGGUGCCGCUAATT
448
AD-15214


 


1076-1094
GCGGCACCCUCAUAGGCCUTsT
449
AGGCCUAUGAGGGUGCCGCTsT
450
AD-9315 


 


1079-1097
GCACCCUCAUAGGCCUGGATsT
451
UCCAGGCCUAUGAGGGUGCTsT
452
AD-9326 


 


1085-1103
UCAUAGGCCUGGAGUTTUAUTsT
453
AUAAACUCCAGGCCUAUGATsT
454
AD-9318 


 


1090-1108
GGCCUGGAGUTTUAUUCGGATsT
455
UCCGAAUAAACUCCAGGCCTsT
456
AD-9323 


 


1091-1109
GCCUGGAGUUUAUUCGGAATsT
457
UUCCGAAUAAACUCCAGGCTsT
458
AD-9314 


 


1091-1109
GccuGGAGuuuAuucGGAATsT
459
UUCCGAAuAAACUCcAGGCTsT
460
AD-10792


 


1091-1109
GccuGGAGuuuAuucGGAATsT
461
UUCCGAAUAACUCCAGGCTsT
462
AD-10796


 


1093-1111
CUGGAGUTTUAUUCGGAAAATsT
463
UUUUCCGAAUAAACUCCAGTsT
464
AD-9638 


 


1093-1111
cuGGAGuuuAuucGGAAAATsT
465
UUUUCCGAAuAAACUCcAGTsT
466
AD-9764 


 


1095-1113
GGAGUTTUAUUCGGAAAAGCTsT
467
GCUUUUCCGAAUAAACUCCTsT
468
AD-9525 


 


1095-1113
GGAGuuuAuucGGAAAAGcTsT
469
GCUUUUCCGAAuAAACUCCTsT
470
AD-9651 


 


1096-1114
GAGUTTUAUUCGGAAAAGCCTsT
471
GGCUUUUCCGAAUAAACUCTsT
472
AD-9560 


 


1096-1114
GAGuuuAuucGGAAAAGccTsT
473
GGCUUUUCCGAAuAAACUCTsT
474
AD-9686 


 


1100-1118
UTTAUUCGGAAAAGCCAGCUTsT
475
AGCUGGCUUUUCCGAAUAATsT
476
AD-9536 


 


1100-1118
uuAuucGGAAAAGccAGcuTsT
477
AGCUGGCUUUUCCGAAuAATsT
478
AD-9662 


 


1154-1172
CCCUGGCGGGUGGGUACAGTsT
479
CUGUACCCACCCGCCAGGGTsT
480
AD-9584 


 


1154-1172
cccuGGcGGGuGGGuAcAGTsT
481
CUGuACCcACCCGCcAGGGTsT
482
AD-9710 


 


1155-1173
CCUGGCGGGUGGGUACAGCTT
483
GCUGUACCCACCCGCCAGGTT
484
AD-15323


 


1157-1175
UGGCGGGUGGGUACAGCCGTsT
485
CGGCUGUACCCACCCGCCATsT
486
AD-9551 


 


1157-1175
uGGcGGGuGGGuAcAGccGTsT
487
CGGCUGuACCcACCCGCcATsT
488
AD-9677 


 


1158-1176
GGCGGGUGGGUACAGCCGCTT
489
GCGGCUGUACCCACCCGCCTT
490
AD-15230


 


1162-1180
GGUGGGUACAGCCGCGUCCTT
491
GGACGCGGCUGUACCCACCTT
492
AD-15231


 


1164-1182
UGGGUACAGCCGCGUCCUCTT
493
GAGGACGCGGCUGUACCCATT
494
AD-15285


 


1172-1190
GCCGCGUCCUCAACGCCGCTT
495
GCGGCGUUGAGGACGCGGCTT
496
AD-15396


 


1173-1191
CCGCGUCCUCAACGCCGCCTT
497
GGCGGCGUUGAGGACGCGGTT
498
AD-15397


 


1216-1234
GUCGUGCUGGUCACCGCUGTsT
499
CAGCGGUGACCAGCACGACTsT
500
AD-9600 


 


1216-1234
GucGuGcuGGucAccGcuGTsT
501
cAGCGGUGACcAGcACGACTsT
502
AD-9726 


 


1217-1235
UCGUGCUGGUCACCGCUGCTsT
503
GCAGCGGUGACCAGCACGATsT
504
AD-9606 


 


1217-1235
ucGuGcuGGucAccGcuGcTsT
505
GcAGCGGUGACcAGcACGATsT
506
AD-9732 


 


1223-1241
UGGUCACCGCUGCCGGCAATsT
507
UUGCCGGCAGCGGUGACCATsT
508
AD-9633 


 


1223-1241
uGGucAccGcuGccGGcAATsT
509
UUGCCGGcAGCGGUGACcATsT
510
AD-9759 


 


1224-1242
GGUCACCGCUGCCGGCAACTsT
511
GUUGCCGGCAGCGGUGACCTsT
512
AD-9588 


 


1224-1242
GGucAccGcuGccGGcAAcTsT
513
GUUGCCGGcAGCGGUGACCTsT
514
AD-9714 


 


1227-1245
CACCGCUGCCGGCAACUUCTsT
515
GAAGUUGCCGGCAGCGGUGTsT
516
AD-9589 


 


1227-1245
cAccGcuGccGGcAAcuucTsT
517
GAAGUUGCCGGcAGCGGUGTsT
518
AD-9715 


 


1229-1247
CCGCUGCCGGCAACUUCCGTsT
519
CGGAAGUUGCCGGCAGCGGTsT
520
AD-9575 


 


1229-1247
ccGcuGccGGcAAcuuccGTsT
521
CGGAAGUUGCCGGcAGCGGTsT
522
AD-9701 


 


1230-1248
CGCUGCCGGCAACUUCCGGTsT
523
CCGGAAGUUGCCGGCAGCGTsT
524
AD-9563 


 


1230-1248
cGcuGccGGcAAcuuccGGTsT
525
CCGGAAGUUGCCGGcAGCGTsT
526
AD-9689 


 


1231-1249
GCUGCCGGCAACUUCCGGGTsT
527
CCCGGAAGUUGCCGGCAGCTsT
528
AD-9594 


 


1231-1249
GcuGccGGcAAcuuccGGGTsT
529
CCCGGAAGUUGCCGGcAGCTsT
530
AD-9720 


 


1236-1254
CGGCAACUUCCGGGACGAUTsT
531
AUCGUCCCGGAAGUUGCCGTsT
532
AD-9585 


 


1236-1254
cGGcAAcuuccGGGAcGAuTsT
533
AUCGUCCCGGAAGUUGCCGTsT
534
AD-9711 


 


1237-1255
GGCAACUUCCGGGACGAUGTsT
535
CAUCGUCCCGGAAGUUGCCTsT
536
AD-9614 


 


1237-1255
GGcAAcuuccGGGAcGAuGTsT
537
cAUCGUCCCGGAAGUUGCCTsT
538
AD-9740 


 


1243-1261
UUCCGGGACGAUGCCUGCCTsT
539
GGCAGGCAUCGUCCCGGAATsT
540
AD-9615 


 


1243-1261
uuccGGGAcGAuGccuGccTsT
541
GGcAGGcAUCGUCCCGGAATsT
542
AD-9741 


 


1248-1266
GGACGAUGCCUGCCUCUACTsT
543
GUAGAGGCAGGCAUCGUCCTsT
544
AD-9534 


 


1248-1266
GGACGAUGCCUGCCUCUACTsT
545
GUAGAGGCAGGCAUCGUCCTsT
546
AD-9534 


 


1248-1266
GGAcGAuGccuGccucuAcTsT
547
GuAGAGGcAGGcAUCGUCCTsT
548
AD-9660 


 


1279-1297
GCUCCCGAGGUCAUCACAGTT
549
CUGUGAUGACCUCGGGAGCTT
550
AD-15324


 


1280-1298
CUCCCGAGGUCAUCACAGUTT
551
ACUGUGAUGACCUCGGGAGTT
552
AD-15232


 


1281-1299
UCCCGAGGUCAUCACAGUUTT
553
AACUGUGAUGACCUCGGGATT
554
AD-15233


 


1314-1332
CCAAGACCAGCCGGUGACCTT
555
GGUCACCGGCUGGUCUUGGTT
556
AD-15234


 


1315-1333
CAAGACCAGCCGGUGACCCTT
557
GGGUCACCGGCUGGUCUUGTT
558
AD-15286


 


1348-1366
ACCAACUUUGGCCGCUGUGTsT
559
CACAGCGGCCAAAGUUGGUTsT
560
AD-9590 


 


1348-1366
AccAAcuuuGGccGcuGuGTsT
561
cAcAGCGGCcAAAGUUGGUTsT
562
AD-9716 


 


1350-1368
CAACUUUGGCCGCUGUGUGTsT
563
CACACAGCGGCCAAAGUUGTsT
564
AD-9632 


 


1350-1368
cAAcuuuGGccGcuGuGuGTsT
565
cAcAcAGCGGCcAAAGUUGTsT
566
AD-9758 


 


1360-1378
CGCUGUGUGGACCUCUUUGTsT
567
CAAAGAGGUCCACACAGCGTsT
568
AD-9567 


 


1360-1378
cGcuGuGuGGAccucuuuGTsT
569
cAAAGAGGUCcAcAcAGCGTsT
570
AD-9693 


 


1390-1408
GACAUCAUUGGUGCCUCCATsT
571
UGGAGGCACCAAUGAUGUCTsT
572
AD-9586 


 


1390-1408
GAcAucAuuGGuGccuccATsT
573
UGGAGGcACcAAUGAUGUCTsT
574
AD-9712 


 


1394-1412
UCAUUGGUGCCUCCAGCGATsT
575
UCGCUGGAGGCACCAAUGATsT
576
AD-9564 


 


1394-1412
ucAuuGGuGccuccAGcGATsT
577
UCGCUGGAGGcACcAAUGATsT
578
AD-9690 


 


1417-1435
AGCACCUGCUUUGUGUCACTsT
579
GUGACACAAAGCAGGUGCUTsT
580
AD-9616 


 


1417-1435
AGcAccuGcuuuGuGucAcTsT
581
GUGAcAcAAAGcAGGUGCUTsT
582
AD-9742 


 


1433-1451
CACAGAGUGGGACAUCACATT
583
UGUGAUGUCCCACUCUGUGTT
584
AD-15398


 


1486-1504
AUGCUGUCUGCCGAGCCGGTsT
585
CCGGCUCGGCAGACAGCAUTsT
586
AD-9617 


 


1486-1504
AuGcuGucuGccGAGccGGTsT
587
CCGGCUCGGcAGAcAGcAUTsT
588
AD-9743 


 


1491-1509
GUCUGCCGAGCCGGAGCUCTsT
589
GAGCUCCGGCUCGGCAGACTsT
590
AD-9635 


 


1491-1509
GucuGccGAGccGGAGcucTsT
591
GAGCUCCGGCUCGGcAGACTsT
592
AD-9761 


 


1521-1539
GUUGAGGCAGAGACUGAUCTsT
593
GAUCAGUCUCUGCCUCAACTsT
594
AD-9568 


 


1521-1539
GuuGAGGcAGAGAcuGAucTsT
595
GAUcAGUCUCUGCCUcAACTsT
596
AD-9694 


 


1527-1545
GCAGAGACUGAUCCACUUCTsT
597
GAAGUGGAUCAGUCUCUGCTsT
598
AD-9576 


 


1527-1545
GcAGAGAcuGAuccAcuucTsT
599
GAAGUGGAUcAGUCUCUGCTsT
600
AD-9702 


 


1529-1547
AGAGACUGAUCCACUUCUCTsT
601
GAGAAGUGGAUCAGUCUCUTsT
602
AD-9627 


 


1529-1547
AGAGAcuGAuccAcuucucTsT
603
GAGAAGUGGAUcAGUCUCUTsT
604
AD-9753 


 


1543-1561
UUCUCUGCCAAAGAUGUCATsT
605
UGACAUCUUUGGCAGAGAATsT
606
AD-9628 


 


1543-1561
uucucuGccAAAGAuGucATsT
607
UGAcAUCUUUGGcAGAGAATsT
608
AD-9754 


 


1545-1563
CUCUGCCAAAGAUGUCAUCTsT
609
GAUGACAUCUUUGGCAGAGTsT
610
AD-9631 


 


1545-1563
cucuGccAAAGAuGucAucTsT
611
GAUGAcAUCUUUGGcAGAGTsT
612
AD-9757 


 


1580-1598
CUGAGGACCAGCGGGUACUTsT
613
AGUACCCGCUGGUCCUCAGTsT
614
AD-9595 


 


1580-1598
cuGAGGAccAGcGGGuAcuTsT
615
AGuACCCGCUGGUCCUcAGTsT
616
AD-9721 


 


1581-1599
UGAGGACCAGCGGGUACUGTsT
617
CAGUACCCGCUGGUCCUCATsT
618
AD-9544 


 


1581-1599
uGAGGAccAGcGGGuAcuGTsT
619
cAGuACCCGCUGGUCCUcATsT
620
AD-9670 


 


1666-1684
ACUGUAUGGUCAGCACACUTT
621
AGUGUGCUGACCAUACAGUTT
622
AD-15235


 


1668-1686
UGUAUGGUCAGCACACUCGTT
623
CGAGUGUGCUGACCAUACATT
624
AD-15236


 


1669-1687
GUAUGGUCAGCACACUCGGTT
625
CCGAGUGUGCUGACCAUACTT
626
AD-15168


 


1697-1715
GGAUGGCCACAGCCGUCGCTT
627
GCGACGGCUGUGGCCAUCCTT
628
AD-15174


 


1698-1716
GAUGGCCACAGCCGUCGCCTT
629
GGCGACGGCUGUGGCCAUCTT
630
AD-15325


 


1806-1824
CAAGCUGGUCUGCCGGGCCTT
631
GGCCCGGCAGACCAGCUUGTT
632
AD-15326


 


1815-1833
CUGCCGGGCCCACAACGCUTsT
633
AGCGUUGUGGGCCCGGCAGTsT
634
AD-9570 


 


1815-1833
cuGccGGGcccAcAAcGcuTsT
635
AGCGUUGUGGGCCCGGcAGTsT
636
AD-9696 


 


1816-1834
UGCCGGGCCCACAACGCUUTsT
637
AAGCGUUGUGGGCCCGGCATsT
638
AD-9566 


 


1816-1834
uGccGGGcccAcAAcGcuuTsT
639
AAGCGUUGUGGGCCCGGcATsT
640
AD-9692 


 


1818-1836
CCGGGCCCACAACGCUUUUTsT
641
AAAAGCGUUGUGGGCCCGGTsT
642
AD-9532 


 


1818-1836
ccGGGcccAcAAcGcuuuuTsT
643
AAAAGCGUUGUGGGCCCGGTsT
644
AD-9658 


 


1820-1838
GGGCCCACAACGCUUUUGGTsT
645
CCAAAAGCGUUGUGGGCCCTsT
646
AD-9549 


 


1820-1838
GGGcccAcAAcGcuuuuGGTsT
647
CcAAAAGCGUUGUGGGCCCTsT
648
AD-9675 


 


1840-1858
GGUGAGGGUGUCUACGCCATsT
649
UGGCGUAGACACCCUCACCTsT
650
AD-9541 


 


1840-1858
GGuGAGGGuGucuAcGccATsT
651
UGGCGuAGAcACCCUcACCTsT
652
AD-9667 


 


1843-1861
GAGGGUGUCUACGCCAUUGTsT
653
CAAUGGCGUAGACACCCUCTsT
654
AD-9550 


 


1843-1861
GAGGGuGucuAcGccAuuGTsT
655
cAAUGGCGuAGAcACCCUCTsT
656
AD-9676 


 


1861-1879
GCCAGGUGCUGCCUGCUACTsT
657
GUAGCAGGCAGCACCUGGCTsT
658
AD-9571 


 


1861-1879
GccAGGuGcuGccuGcuAcTsT
659
GuAGcAGGcAGcACCUGGCTsT
660
AD-9697 


 


1862-1880
CCAGGUGCUGCCUGCUACCTsT
661
GGUAGCAGGCAGCACCUGGTsT
662
AD-9572 


 


1862-1880
ccAGGuGcuGccuGcuAccTsT
663
GGuAGcAGGcAGcACCUGGTsT
664
AD-9698 


 


2008-2026
ACCCACAAGCCGCCUGUGCTT
665
GCACAGGCGGCUUGUGGGUTT
666
AD-15327


 


2023-2041
GUGCUGAGGCCACGAGGUCTsT
667
GACCUCGUGGCCUCAGCACTsT
668
AD-9639 


 


2023-2041
GuGcuGAGGccAcGAGGucTsT
669
GACCUCGUGGCCUcAGcACTsT
670
AD-9765 


 


2024-2042
UGCUGAGGCCACGAGGUCATsT
671
UGACCUCGUGGCCUCAGCATsT
672
AD-9518 


 


2024-2042
UGCUGAGGCCACGAGGUCATsT
673
UGACCUCGUGGCCUCAGCATsT
674
AD-9518 


 


2024-2042
uGcuGAGGccAcGAGGucATsT
675
UGACCUCGUGGCCUcAGcATsT
676
AD-9644 


 


2024-2042
UfgCfuGfaGfgCfcAfcGfaG
677
p-uGfaCfcUfcGfuGfgCfc
678
AD-14672



fgUfcAfTsT

UfcAfgCfaTsT




 


2024-2042
UfGCfUfGAGGCfCfACfGA
679
UfGACfCfUfCfGUfGGCfCfUf
680
AD-14682



GGUfCfATsT

CfAGCfATsT




 


2024-2042
UgCuGaGgCcAcGaGgUcATsT
681
p-uGfaCfcUfcGfuGfgCfcUf
682
AD-14692





cAfgCfaTsT




 


2024-2042
UgCuGaGgCcAcGaGgUcATsT
683
UfGACfCfUfCfGUfGGCfCfUf
684
AD-14702





CfAGCfATsT




 


2024-2042
UfgCfuGfaGfgCfcAfcGfaG
685
UGACCucGUggCCUCAgcaTsT
686
AD-14712



fgUfcAfTsT






 


2024-2042
UfGCfUfGAGGCfCfACfG
687
UGACCucGUggCCUCAgcaTsT
688
AD-14722



AGGUfCfATsT






 


2024-2042
UgCuGaGgCcAcGaGgUcATsT
689
UGACCucGUggCCUCAgcaTsT
690
AD-14732


 


2024-2042
GfuGfgUfcAfgCfgGfcCfgGf
691
p-cAfuCfcCfgGfcCfgCfuGf
692
AD-15078



gAfuGfTsT

aCfcAfcTsT




 


2024-2042
GUfGGUfCfAGCfGGCfCfGGG
693
CfAUfCfCfCfGGCfCfGCfUfGAC
694
AD-15088



AUfGTsT

fCfACfTsT




 


2024-2042
GuGgUcAgCgGcCgGgAuGTsT
695
p-cAfuCfcCfgGfcCfgCfuG
696
AD-15098





faCfcAfcTsT




 


2024-2042
GuGgUcAgCgGcCgGgAuGTsT
697
CfAUfCfCfCfGGCfCfGCfUfG
698
AD-15108





ACfCfACfTsT




 


2024-2042
GfuGfgUfcAfgCfgGfcCf
699
CAUCCcgGCcgCUGACcacTsT
700
AD-15118



gGfgAfuGfTsT






 


2024-2042
GUfGGUfCfAGCfGGCfCfG
701
CAUCCcgGCcgCUGACcacTsT
702
AD-15128



GGAUfGTsT






 


2024-2042
GuGgUcAgCgGcCgGgAuGTsT
703
CAUCCcgGCcgCUGACcacTsT
704
AD-15138


 


2030-2048
GGCCACGAGGUCAGCCCAATT
705
UUGGGCUGACCUCGUGGCCTT
706
AD-15237


 


2035-2053
CGAGGUCAGCCCAACCAGUTT
707
ACUGGUUGGGCUGACCUCGTT
708
AD-15287


 


2039-2057
GUCAGCCCAACCAGUGCGUTT
709
ACGCACUGGUUGGGCUGACTT
710
AD-15238


 


2041-2059
CAGCCCAACCAGUGCGUGGTT
711
CCACGCACUGGUUGGGCUGTT
712
AD-15328


 


2062-2080
CACAGGGAGGCCAGCAUCCTT
713
GGAUGCUGGCCUCCCUGUGTT
714
AD-15399


 


2072-2090
CCAGCAUCCACGCUUCCUGTsT
715
CAGGAAGCGUGGAUGCUGGTsT
716
AD-9582


 


2072-2090
ccAGcAuccAcGcuuccuGTsT
717
cAGGAAGCGUGGAUGCUGGTsT
718
AD-9708


 


2118-2136
AGUCAAGGAGCAUGGAAUCTsT
719
GAUUCCAUGCUCCUUGACUTsT
720
AD-9545


 


2118-2136
AGucAAGGAGcAuGGAAucTsT
721
GAUUCcAUGCUCCUUGACUTsT
722
AD-9671


 


2118-2136
AfgUfcAfaGfgAfgCfaUfgG
723
p-gAfuUfcCfaUfgCfuCfcUf
724
AD-14674



faAfuCfTsT

uGfaCfuTsT




 


2118-2136
AGUfCfAAGGAGCfAUfGG
725
GAUfUfCfCfAUfGCfUfCfCfUf
726
AD-14684



AAUfCfTsT

UfGACfUfTsT




 


2118-2136
AgUcAaGgAgCaUgGaAuCTsT
727
p-gAfuUfcCfaUfgCfuCfcUfu
728
AD-14694





GfaCfuTsT




 


2118-2136
AgUcAaGgAgCaUgGaAuCTsT
729
GAUfUfCfCfAUfGCfUfCfCf
730
AD-14704





UfUfGACfUfTsT




 


2118-2136
AfgUfcAfaGfgAfgCfaUfgG
731
GAUUCcaUGcuCCUUGacuTsT
732
AD-14714



faAfuCfTsT






 


2118-2136
AGUfCfAAGGAGCfAUfGGAA
733
GAUUCcaUGcuCCUUGacuTsT
734
AD-14724



UfCfTsT






 


2118-2136
AgUcAaGgAgCaUgGaAuCTsT
735
GAUUCcaUGcuCCUUGacuTsT
736
AD-14734


 


2118-2136
GfcGfgCfaCfcCfuCfaUfaG
737
p-aGfgCfcUfaUfgAfgGfgUf
738
AD-15080



fgCfcUfTsT

gCfcGfcTsT




 


2118-2136
GCfGGCfACfCfCfUfCfAUfA
739
AGGCfCfUfAUfGAGGGUfGC
740
AD-15090



GGCfCfUfTsT

fCfGCfTsT




 


2118-2136
GcGgCaCcCuCaUaGgCcUTsT
741
p-aGfgCfcUfaUfgAfgGfgUf
742
AD-15100





gCfcGfcTsT




 


2118-2136
GcGgCaCcCuCaUaGgCcUTsT
743
AGGCfCfUfAUfGAGGGUfGCf
744
AD-15110





CfGCfTsT




 


2118-2136
GfcGfgCfaCfcCfuCfaUfaG
745
AGGCCuaUGagGGUGCcgcTsT
746
AD-15120



fgCfcUfTsT






 


2118-2136
GCfGGCfACfCfCfUfCfAUfA
747
AGGCCuaUGagGGUGCcgcTsT
748
AD-15130



GGCfCfUfTsT






 


2118-2136
GcGgCaCcCuCaUaGgCcUTsT
749
AGGCCuaUGagGGUGCcgcTsT
750
AD-15140


 


2122-2140
AAGGAGCAUGGAAUCCCGGTsT
751
CCGGGAUUCCAUGCUCCUUTsT
752
AD-9522 


 


2122-2140
AAGGAGcAuGGAAucccGGTsT
753
CCGGGAUUCcAUGCUCCUUTsT
754
AD-9648 


 


2123-2141
AGGAGCAUGGAAUCCCGGCTsT
755
GCCGGGAUUCCAUGCUCCUTsT
756
AD-9552 


 


2123-2141
AGGAGcAuGGAAucccGGcTsT
757
GCCGGGAUUCcAUGCUCCUTsT
758
AD-9678 


 


2125-2143
GAGCAUGGAAUCCCGGCCCTsT
759
GGGCCGGGAUUCCAUGCUCTsT
760
AD-9618 


 


2125-2143
GAGcAuGGAAucccGGcccTsT
761
GGGCCGGGAUUCcAUGCUCTsT
762
AD-9744 


 


2230-2248
GCCUACGCCGUAGACAACATT
763
UGUUGUCUACGGCGUAGGCTT
764
AD-15239


 


2231-2249
CCUACGCCGUAGACAACACTT
765
GUGUUGUCUACGGCGUAGGTT
766
AD-15212


 


2232-2250
CUACGCCGUAGACAACACGTT
767
CGUGUUGUCUACGGCGUAGTT
768
AD-15240


 


2233-2251
UACGCCGUAGACAACACGUTT
769
ACGUGUUGUCUACGGCGUATT
770
AD-15177


 


2235-2253
CGCCGUAGACAACACGUGUTT
771
ACACGUGUUGUCUACGGCGTT
772
AD-15179


 


2236-2254
GCCGUAGACAACACGUGUGTT
773
CACACGUGUUGUCUACGGCTT
774
AD-15180


 


2237-2255
CCGUAGACAACACGUGUGUTT
775
ACACACGUGUUGUCUACGGTT
776
AD-15241


 


2238-2256
CGUAGACAACACGUGUGUATT
777
UACACACGUGUUGUCUACGTT
778
AD-15268


 


2240-2258
UAGACAACACGUGUGUAGUTT
779
ACUACACACGUGUUGUCUATT
780
AD-15242


 


2241-2259
AGACAACACGUGUGUAGUCTT
781
GACUACACACGUGUUGUCUTT
782
AD-15216


 


2242-2260
GACAACACGUGUGUAGUCATT
783
UGACUACACACGUGUUGUCTT
784
AD-15176


 


2243-2261
ACAACACGUGUGUAGUCAGTT
785
CUGACUACACACGUGUUGUTT
786
AD-15181


 


2244-2262
CAACACGUGUGUAGUCAGGTT
787
CCUGACUACACACGUGUUGTT
788
AD-15243


 


2247-2265
CACGUGUGUAGUCAGGAGCTT
789
GCUCCUGACUACACACGUGTT
790
AD-15182


 


2248-2266
ACGUGUGUAGUCAGGAGCCTT
791
GGCUCCUGACUACACACGUTT
792
AD-15244


 


2249-2267
CGUGUGUAGUCAGGAGCCGTT
793
CGGCUCCUGACUACACACGTT
794
AD-15387


 


2251-2269
UGUGUAGUCAGGAGCCGGGTT
795
CCCGGCUCCUGACUACACATT
796
AD-15245


 


2257-2275
GUCAGGAGCCGGGACGUCATsT
797
UGACGUCCCGGCUCCUGACTsT
798
AD-9555 


 


2257-2275
GucAGGAGccGGGAcGucATsT
799
UGACGUCCCGGCUCCUGACTsT
800
AD-9681 


 


2258-2276
UCAGGAGCCGGGACGUCAGTsT
801
CUGACGUCCCGGCUCCUGATsT
802
AD-9619 


 


2258-2276
ucAGGAGccGGGAcGucAGTsT
803
CUGACGUCCCGGCUCCUGATsT
804
AD-9745 


 


2259-2277
CAGGAGCCGGGACGUCAGCTsT
805
GCUGACGUCCCGGCUCCUGTsT
806
AD-9620 


 


2259-2277
cAGGAGccGGGAcGucAGcTsT
807
GCUGACGUCCCGGCUCCUGTsT
808
AD-9746 


 


2263-2281
AGCCGGGACGUCAGCACUATT
809
UAGUGCUGACGUCCCGGCUTT
810
AD-15288


 


2265-2283
CCGGGACGUCAGCACUACATT
811
UGUAGUGCUGACGUCCCGGTT
812
AD-15246


 


2303-2321
CCGUGACAGCCGUUGCCAUTT
813
AUGGCAACGGCUGUCACGGTT
814
AD-15289


 


2317-2335
GCCAUCUGCUGCCGGAGCCTsT
815
GGCUCCGGCAGCAGAUGGCTsT
816
AD-9324 


 


2375-2393
CCCAUCCCAGGAUGGGUGUTT
817
ACACCCAUCCUGGGAUGGGTT
818
AD-15329


 


2377-2395
CAUCCCAGGAUGGGUGUCUTT
819
AGACACCCAUCCUGGGAUGTT
820
AD-15330


 


2420-2438
AGCUUUAAAAUGGUUCCGATT
821
UCGGAACCAUUUUAAAGCUTT
822
AD-15169


 


2421-2439
GCUUUAAAAUGGUUCCGACTT
823
GUCGGAACCAUUUUAAAGCTT
824
AD-15201


 


2422-2440
CUUUAAAAUGGUUCCGACUTT
825
AGUCGGAACCAUUUUAAAGTT
826
AD-15331


 


2423-2441
UUUAAAAUGGUUCCGACUUTT
827
AAGUCGGAACCAUUUUAAATT
828
AD-15190


 


2424-2442
UUAAAAUGGUUCCGACUUGTT
829
CAAGUCGGAACCAUUUUAATT
830
AD-15247


 


2425-2443
UAAAAUGGUUCCGACUUGUTT
831
ACAAGUCGGAACCAUUUUATT
832
AD-15248


 


2426-2444
AAAAUGGUUCCGACUUGUCTT
833
GACAAGUCGGAACCAUUUUTT
834
AD-15175


 


2427-2445
AAAUGGUUCCGACUUGUCCTT
835
GGACAAGUCGGAACCAUUUTT
836
AD-15249


 


2428-2446
AAUGGUUCCGACUUGUCCCTT
837
GGGACAAGUCGGAACCAUUTT
838
AD-15250


 


2431-2449
GGUUCCGACUUGUCCCUCUTT
839
AGAGGGACAAGUCGGAACCTT
840
AD-15400


 


2457-2475
CUCCAUGGCCUGGCACGAGTT
841
CUCGUGCCAGGCCAUGGAGTT
842
AD-15332


 


2459-2477
CCAUGGCCUGGCACGAGGGTT
843
CCCUCGUGCCAGGCCAUGGTT
844
AD-15388


 


2545-2563
GAACUCACUCACUCUGGGUTT
845
ACCCAGAGUGAGUGAGUUCTT
846
AD-15333


 


2549-2567
UCACUCACUCUGGGUGCCUTT
847
AGGCACCCAGAGUGAGUGATT
848
AD-15334


 


2616-2634
UUUCACCAUUCAAACAGGUTT
849
ACCUGUUUGAAUGGUGAAATT
850
AD-15335


 


2622-2640
CAUUCAAACAGGUCGAGCUTT
851
AGCUCGACCUGUUUGAAUGTT
852
AD-15183


 


2623-2641
AUUCAAACAGGUCGAGCUGTT
853
CAGCUCGACCUGUUUGAAUTT
854
AD-15202


 


2624-2642
UUCAAACAGGUCGAGCUGUTT
855
ACAGCUCGACCUGUUUGAATT
856
AD-15203


 


2625-2643
UCAAACAGGUCGAGCUGUGTT
857
CACAGCUCGACCUGUUUGATT
858
AD-15272


 


2626-2644
CAAACAGGUCGAGCUGUGCTT
859
GCACAGCUCGACCUGUUUGTT
860
AD-15217


 


2627-2645
AAACAGGUCGAGCUGUGCUTT
861
AGCACAGCUCGACCUGUUUTT
862
AD-15290


 


2628-2646
AACAGGUCGAGCUGUGCUCTT
863
GAGCACAGCUCGACCUGUUTT
864
AD-15218


 


2630-2648
CAGGUCGAGCUGUGCUCGGTT
865
CCGAGCACAGCUCGACCUGTT
866
AD-15389


 


2631-2649
AGGUCGAGCUGUGCUCGGGTT
867
CCCGAGCACAGCUCGACCUTT
868
AD-15336


 


2633-2651
GUCGAGCUGUGCUCGGGUGTT
869
CACCCGAGCACAGCUCGACTT
870
AD-15337


 


2634-2652
UCGAGCUGUGCUCGGGUGCTT
871
GCACCCGAGCACAGCUCGATT
872
AD-15191


 


2657-2675
AGCUGCUCCCAAUGUGCCGTT
873
CGGCACAUUGGGAGCAGCUTT
874
AD-15390


 


2658-2676
GCUGCUCCCAAUGUGCCGATT
875
UCGGCACAUUGGGAGCAGCTT
876
AD-15338


 


2660-2678
UGCUCCCAAUGUGCCGAUGTT
877
CAUCGGCACAUUGGGAGCATT
878
AD-15204


 


2663-2681
UCCCAAUGUGCCGAUGUCCTT
879
GGACAUCGGCACAUUGGGATT
880
AD-15251


 


2665-2683
CCAAUGUGCCGAUGUCCGUTT
881
ACGGACAUCGGCACAUUGGTT
882
AD-15205


 


2666-2684
CAAUGUGCCGAUGUCCGUGTT
883
CACGGACAUCGGCACAUUGTT
884
AD-15171


 


2667-2685
AAUGUGCCGAUGUCCGUGGTT
885
CCACGGACAUCGGCACAUUTT
886
AD-15252


 


2673-2691
CCGAUGUCCGUGGGCAGAATT
887
UUCUGCCCACGGACAUCGGTT
888
AD-15339


 


2675-2693
GAUGUCCGUGGGCAGAAUGTT
889
CAUUCUGCCCACGGACAUCTT
890
AD-15253


 


2678-2696
GUCCGUGGGCAGAAUGACUTT
891
AGUCAUUCUGCCCACGGACTT
892
AD-15340


 


2679-2697
UCCGUGGGCAGAAUGACUUTT
893
AAGUCAUUCUGCCCACGGATT
894
AD-15291


 


2683-2701
UGGGCAGAAUGACUUUUAUTT
895
AUAAAAGUCAUUCUGCCCATT
896
AD-15341


 


2694-2712
ACUUUUAUUGAGCUCUUGUTT
897
ACAAGAGCUCAAUAAAAGUTT
898
AD-15401


 


2700-2718
AUUGAGCUCUUGUUCCGUGTT
899
CACGGAACAAGAGCUCAAUTT
900
AD-15342


 


2704-2722
AGCUCUUGUUCCGUGCCAGTT
901
CUGGCACGGAACAAGAGCUTT
902
AD-15343


 


2705-2723
GCUCUUGUUCCGUGCCAGGTT
903
CCUGGCACGGAACAAGAGCTT
904
AD-15292


 


2710-2728
UGUUCCGUGCCAGGCAUUCTT
905
GAAUGCCUGGCACGGAACATT
906
AD-15344


 


2711-2729
GUUCCGUGCCAGGCAUUCATT
907
UGAAUGCCUGGCACGGAACTT
908
AD-15254


 


2712-2730
UUCCGUGCCAGGCAUUCAATT
909
UUGAAUGCCUGGCACGGAATT
910
AD-15345


 


2715-2733
CGUGCCAGGCAUUCAAUCCTT
911
GGAUUGAAUGCCUGGCACGTT
912
AD-15206


 


2716-2734
GUGCCAGGCAUUCAAUCCUTT
913
AGGAUUGAAUGCCUGGCACTT
914
AD-15346


 


2728-2746
CAAUCCUCAGGUCUCCACCTT
915
GGUGGAGACCUGAGGAUUGTT
916
AD-15347


 


2743-2761
CACCAAGGAGGCAGGAUUCTsT
917
GAAUCCUGCCUCCUUGGUGTsT
918
AD-9577 


 


2743-2761
cAccAAGGAGGcAGGAuucTsT
919
GAAUCCUGCCUCCUUGGUGTsT
920
AD-9703 


 


2743-2761
CfaCfcAfaGfgAfgGfcAfgG
921
p-gAfaUfcCfuGfcCfuCfcUf
922
AD-14678



faUfuCfTsT

uGfgUfgTsT




 


2743-2761
CfACfCfAAGGAGGCfAGGA
923
GAAUfCfCfUfGCfCfUfCfC
924
AD-14688



UfUfCfTsT

fUfUfGGUfGTsT




 


2743-2761
CaCcAaGgAgGcAgGaUuCTsT
925
p-gAfaUfcCfuGfcCfuCfc
926
AD-14698





UfuGfgUfgTsT




 


2743-2761
CaCcAaGgAgGcAgGaUuCTsT
927
GAAUfCfCfUfGCfCfUfCfCfU
928
AD-14708





fUfGGUfGTsT




 


2743-2761
CfaCfcAfaGfgAfgGfcAfT
929
GAAUCcuGCcuCCUUGgugTsT
930
AD-14718



gGfaUfuCfTs






 


2743-2761
CfACfCfAAGGAGGCfAGGA
931
GAAUCcuGCcuCCUUGgugTsT
932
AD-14728



UfUfCfTsT






 


2743-2761
CaCcAaGgAgGcAgGaUuCTsT
933
GAAUCcuGCcuCCUUGgugTsT
934
AD-14738


 


2743-2761
GfgCfcUfgGfaGfuUfuAfu
935
p-uCfcGfaAfuAfaAfcUfcC
936
AD-15084



UfcGfgAfTsT

faGfgCfcTsT




 


2743-2761
GGCfCfUfGGAGUfUfUfAU
937
UfCfCfGAAUfAAACfUfCfCf
938
AD-15094



fUfCfGGATsT

AGGCfCfTsT




 


2743-2761
GgCcUgGaGuUuAuUcGgATsT
939
p-uCfcGfaAfuAfaAfcUfcC
940
AD-15104





faGfgCfcTsT




 


2743-2761
GgCcUgGaGuUuAuUcGgATsT
941
UfCfCfGAAUfAAACfUfCfCf
942
AD-15114





AGGCfCfTsT




 


2743-2761
GfgCfcUfgGfaGfuUfuAf
943
UCCGAauAAacUCCAGgccTsT
944
AD-15124



uUfcGfgAfTsT






 


2743-2761
GGCfCfUfGGAGUfUfUfA
945
UCCGAauAAacUCCAGgccTsT
946
AD-15134



UfUfCfGGATsT






 


2743-2761
GgCcUgGaGuUuAuUcGgATsT
947
UCCGAauAAacUCCAGgccTsT
948
AD-15144


 


2753-2771
GCAGGAUUCUUCCCAUGGATT
949
UCCAUGGGAAGAAUCCUGCTT
950
AD-15391


 


2794-2812
UGCAGGGACAAACAUCGUUTT
951
AACGAUGUUUGUCCCUGCATT
952
AD-15348


 


2795-2813
GCAGGGACAAACAUCGUUGTT
953
CAACGAUGUUUGUCCCUGCTT
954
AD-15349


 


2797-2815
AGGGACAAACAUCGUUGGGTT
955
CCCAACGAUGUUUGUCCCUTT
956
AD-15170


 


2841-2859
CCCUCAUCUCCAGCUAACUTT
957
AGUUAGCUGGAGAUGAGGGTT
958
AD-15350


 


2845-2863
CAUCUCCAGCUAACUGUGGTT
959
CCACAGUUAGCUGGAGAUGTT
960
AD-15402


 


2878-2896
GCUCCCUGAUUAAUGGAGGTT
961
CCUCCAUUAAUCAGGGAGCTT
962
AD-15293


 


2881-2899
CCCUGAUUAAUGGAGGCUUTT
963
AAGCCUCCAUUAAUCAGGGTT
964
AD-15351


 


2882-2900
CCUGAUUAAUGGAGGCUUATT
965
UAAGCCUCCAUUAAUCAGGTT
966
AD-15403


 


2884-2902
UGAUUAAUGGAGGCUUAGCTT
967
GCUAAGCCUCCAUUAAUCATT
968
AD-15404


 


2885-2903
GAUUAAUGGAGGCUUAGCUTT
969
AGCUAAGCCUCCAUUAAUCTT
970
AD-15207


 


2886-2904
AUUAAUGGAGGCUUAGCUUTT
971
AAGCUAAGCCUCCAUUAAUTT
972
AD-15352


 


2887-2905
UUAAUGGAGGCUUAGCUUUTT
973
AAAGCUAAGCCUCCAUUAATT
974
AD-15255


 


2903-2921
UUUCUGGAUGGCAUCUAGCTsT
975
GCUAGAUGCCAUCCAGAAATsT
976
AD-9603 


 


2903-2921
uuucuGGAuGGcAucuAGcTsT
977
GCuAGAUGCcAUCcAGAAATsT
978
AD-9729 


 


2904-2922
UUCUGGAUGGCAUCUAGCCTsT
979
GGCUAGAUGCCAUCCAGAATsT
980
AD-9599 


 


2904-2922
uucuGGAuGGcAucuAGccTsT
981
GGCuAGAUGCcAUCcAGAATsT
982
AD-9725 


 


2905-2923
UCUGGAUGGCAUCUAGCCATsT
983
UGGCUAGAUGCCAUCCAGATsT
984
AD-9621 


 


2905-2923
ucuGGAuGGcAucuAGccATsT
985
UGGCuAGAUGCcAUCcAGATsT
986
AD-9747 


 


2925-2943
AGGCUGGAGACAGGUGCGCTT
987
GCGCACCUGUCUCCAGCCUTT
988
AD-15405


 


2926-2944
GGCUGGAGACAGGUGCGCCTT
989
GGCGCACCUGUCUCCAGCCTT
990
AD-15353


 


2927-2945
GCUGGAGACAGGUGCGCCCTT
991
GGGCGCACCUGUCUCCAGCTT
992
AD-15354


 


2972-2990
UUCCUGAGCCACCUUUACUTT
993
AGUAAAGGUGGCUCAGGAATT
994
AD-15406


 


2973-2991
UCCUGAGCCACCUUUACUCTT
995
GAGUAAAGGUGGCUCAGGATT
996
AD-15407


 


2974-2992
CCUGAGCCACCUUUACUCUTT
997
AGAGUAAAGGUGGCUCAGGTT
998
AD-15355


 


2976-2994
UGAGCCACCUUUACUCUGCTT
999
GCAGAGUAAAGGUGGCUCATT
1000
AD-15356


 


2978-2996
AGCCACCUUUACUCUGCUCTT
1001
GAGCAGAGUAAAGGUGGCUTT
1002
AD-15357


 


2981-2999
CACCUUUACUCUGCUCUAUTT
1003
AUAGAGCAGAGUAAAGGUGTT
1004
AD-15269


 


2987-3005
UACUCUGCUCUAUGCCAGGTsT
1005
CCUGGCAUAGAGCAGAGUATsT
1006
AD-9565 


 


2987-3005
uAcucuGcucuAuGccAGGTsT
1007
CCUGGcAuAGAGcAGAGuATsT
1008
AD-9691 


 


2998-3016
AUGCCAGGCUGUGCUAGCATT
1009
UGCUAGCACAGCCUGGCAUTT
1010
AD-15358


 


3003-3021
AGGCUGUGCUAGCAACACCTT
1011
GGUGUUGCUAGCACAGCCUTT
1012
AD-15359


 


3006-3024
CUGUGCUAGCAACACCCAATT
1013
UUGGGUGUUGCUAGCACAGTT
1014
AD-15360


 


3010-3028
GCUAGCAACACCCAAAGGUTT
1015
ACCUUUGGGUGUUGCUAGCTT
1016
AD-15219


 


3038-3056
GGAGCCAUCACCUAGGACUTT
1017
AGUCCUAGGUGAUGGCUCCTT
1018
AD-15361


 


3046-3064
CACCUAGGACUGACUCGGCTT
1019
GCCGAGUCAGUCCUAGGUGTT
1020
AD-15273


 


3051-3069
AGGACUGACUCGGCAGUGUTT
1021
ACACUGCCGAGUCAGUCCUTT
1022
AD-15362


 


3052-3070
GGACUGACUCGGCAGUGUGTT
1023
CACACUGCCGAGUCAGUCCTT
1024
AD-15192


 


3074-3092
UGGUGCAUGCACUGUCUCATT
1025
UGAGACAGUGCAUGCACCATT
1026
AD-15256


 


3080-3098
AUGCACUGUCUCAGCCAACTT
1027
GUUGGCUGAGACAGUGCAUTT
1028
AD-15363


 


3085-3103
CUGUCUCAGCCAACCCGCUTT
1029
AGCGGGUUGGCUGAGACAGTT
1030
AD-15364


 


3089-3107
CUCAGCCAACCCGCUCCACTsT
1031
GUGGAGCGGGUUGGCUGAGTsT
1032
AD-9604 


 


3089-3107
cucAGccAAcccGcuccAcTsT
1033
GUGGAGCGGGUUGGCUGAGTsT
1034
AD-9730 


 


3093-3111
GCCAACCCGCUCCACUACCTsT
1035
GGUAGUGGAGCGGGUUGGCTsT
1036
AD-9527 


 


3093-3111
GccAAcccGcuccAcuAccTsT
1037
GGuAGUGGAGCGGGUUGGCTsT
1038
AD-9653 


 


3096-3114
AACCCGCUCCACUACCCGGTT
1039
CCGGGUAGUGGAGCGGGUUTT
1040
AD-15365


 


3099-3117
CCGCUCCACUACCCGGCAGTT
1041
CUGCCGGGUAGUGGAGCGGTT
1042
AD-15294


 


3107-3125
CUACCCGGCAGGGUACACATT
1043
UGUGUACCCUGCCGGGUAGTT
1044
AD-15173


 


3108-3126
UACCCGGCAGGGUACACAUTT
1045
AUGUGUACCCUGCCGGGUATT
1046
AD-15366


 


3109-3127
ACCCGGCAGGGUACACAUUTT
1047
AAUGUGUACCCUGCCGGGUTT
1048
AD-15367


 


3110-3128
CCCGGCAGGGUACACAUUCTT
1049
GAAUGUGUACCCUGCCGGGTT
1050
AD-15257


 


3112-3130
CGGCAGGGUACACAUUCGCTT
1051
GCGAAUGUGUACCCUGCCGTT
1052
AD-15184


 


3114-3132
GCAGGGUACACAUUCGCACTT
1053
GUGCGAAUGUGUACCCUGCTT
1054
AD-15185


 


3115-3133
CAGGGUACACAUUCGCACCTT
1055
GGUGCGAAUGUGUACCCUGTT
1056
AD-15258


 


3116-3134
AGGGUACACAUUCGCACCCTT
1057
GGGUGCGAAUGUGUACCCUTT
1058
AD-15186


 


3196-3214
GGAACUGAGCCAGAAACGCTT
1059
GCGUUUCUGGCUCAGUUCCTT
1060
AD-15274


 


3197-3215
GAACUGAGCCAGAAACGCATT
1061
UGCGUUUCUGGCUCAGUUCTT
1062
AD-15368


 


3198-3216
AACUGAGCCAGAAACGCAGTT
1063
CUGCGUUUCUGGCUCAGUUTT
1064
AD-15369


 


3201-3219
UGAGCCAGAAACGCAGAUUTT
1065
AAUCUGCGUUUCUGGCUCATT
1066
AD-15370


 


3207-3225
AGAAACGCAGAUUGGGCUGTT
1067
CAGCCCAAUCUGCGUUUCUTT
1068
AD-15259


 


3210-3228
AACGCAGAUUGGGCUGGCUTT
1069
AGCCAGCCCAAUCUGCGUUTT
1070
AD-15408


 


3233-3251
AGCCAAGCCUCUUCUUACUTsT
1071
AGUAAGAAGAGGCUUGGCUTsT
1072
AD-9597 


 


3233-3251
AGccAAGccucuucuuAcuTsT
1073
AGuAAGAAGAGGCUUGGCUTsT
1074
AD-9723 


 


3233-3251
AfgCfcAfaGfcCfuCfuUfcU
1075
p-aGfuAfaGfaAfgAfgGfc
1076
AD-14680



fuAfcUfTsT

UfuGfgCfuTsT




 


3233-3251
AGCfCfAAGCfCfUfCfUfUfC
1077
AGUfAAGAAGAGGCfUfUfGGC
1078
AD-14690



fUfUfACfUfTsT

fUfTsT




 


3233-3251
AgCcAaGcCuCuUcUuAcUTsT
1079
p-aGfuAfaGfaAfgAfgGfcUf
1080
AD-14700





uGfgCfuTsT




 


3233-3251
AgCcAaGcCuCuUcUuAcUTsT
1081
AGUfAAGAAGAGGCfUfUfG
1082
AD-14710





GCfUfTsT




 


3233-3251
AfgCfcAfaGfcCfuCfuUfc
1083
AGUAAgaAGagGCUUGgcuTsT
1084
AD-14720



UfuAfcUfTsT






 


3233-3251
AGCfCfAAGCfCfUfCfUfUfC
1085
AGUAAgaAGagGCUUGgcuTsT
1086
AD-14730



fUfUfACfUfTsT






 


3233-3251
AgCcAaGcCuCuUcUuAcUTsT
1087
AGUAAgaAGagGCUUGgcuTsT
1088
AD-14740


 


3233-3251
UfgGfuUfcCfcUfgAfgGfa
1089
p-gCfuGfgUfcCfuCfaGfg
1090
AD-15086



CfcAfgCfTsT

GfaAfcCfaTsT




 


3233-3251
UfGGUfUfCfCfCfUfGAGG
1091
GCfUfGGUfCfCfUfCfAG
1092
AD-15096



ACfCfAGCfTsT

GGAACfCfATsT




 


3233-3251
UgGuUcCcUgAgGaCcAgCTsT
1093
p-gCfuGfgUfcCfuCfaGfgGfa
1094
AD-15106





AfcCfaTsT




 


3233-3251
UgGuUcCcUgAgGaCcAgCTsT
1095
GCfUfGGUfCfCfUfCfAGGGA
1096
AD-15116





ACfCfATsT




 


3233-3251
UfgGfuUfcCfcUfgAfgGfaC
1097
GCUGGucCUcaGGGAAccaTsT
1098
AD-15126



fcAfgCfTsT






 


3233-3251
UfGGUfUfCfCfCfUfGAGGAC
1099
GCUGGucCUcaGGGAAccaTsT
1100
AD-15136



fCfAGCfTsT






 


3233-3251
UgGuUcCcUgAgGaCcAgCTsT
1101
GCUGGucCUcaGGGAAccaTsT
1102
AD-15146


 


3242-3260
UCUUCUUACUUCACCCGGCTT
1103
GCCGGGUGAAGUAAGAAGATT
1104
AD-15260


 


3243-3261
CUUCUUACUUCACCCGGCUTT
1105
AGCCGGGUGAAGUAAGAAGTT
1106
AD-15371


 


3244-3262
UUCUUACUUCACCCGGCUGTT
1107
CAGCCGGGUGAAGUAAGAATT
1108
AD-15372


 


3262-3280
GGGCUCCUCAUUUUUACGGTT
1109
CCGUAAAAAUGAGGAGCCCTT
1110
AD-15172


 


3263-3281
GGCUCCUCAUUUUUACGGGTT
1111
CCCGUAAAAAUGAGGAGCCTT
1112
AD-15295


 


3264-3282
GCUCCUCAUUUUUACGGGUTT
1113
ACCCGUAAAAAUGAGGAGCTT
1114
AD-15373


 


3265-3283
CUCCUCAUUUUUACGGGUATT
1115
UACCCGUAAAAAUGAGGAGTT
1116
AD-15163


 


3266-3284
UCCUCAUUUUUACGGGUAATT
1117
UUACCCGUAAAAAUGAGGATT
1118
AD-15165


 


3267-3285
CCUCAUUUUUACGGGUAACTT
1119
GUUACCCGUAAAAAUGAGGTT
1120
AD-15374


 


3268-3286
CUCAUUUUUACGGGUAACATT
1121
UGUUACCCGUAAAAAUGAGTT
1122
AD-15296


 


3270-3288
CAUUUUUACGGGUAACAGUTT
1123
ACUGUUACCCGUAAAAAUGTT
1124
AD-15261


 


3271-3289
AUUUUUACGGGUAACAGUGTT
1125
CACUGUUACCCGUAAAAAUTT
1126
AD-15375


 


3274-3292
UUUACGGGUAACAGUGAGGTT
1127
CCUCACUGUUACCCGUAAATT
1128
AD-15262


 


3308-3326
CAGACCAGGAAGCUCGGUGTT
1129
CACCGAGCUUCCUGGUCUGTT
1130
AD-15376


 


3310-3328
GACCAGGAAGCUCGGUGAGTT
1131
CUCACCGAGCUUCCUGGUCTT
1132
AD-15377


 


3312-3330
CCAGGAAGCUCGGUGAGUGTT
1133
CACUCACCGAGCUUCCUGGTT
1134
AD-15409


 


3315-3333
GGAAGCUCGGUGAGUGAUGTT
1135
CAUCACUCACCGAGCUUCCTT
1136
AD-15378


 


3324-3342
GUGAGUGAUGGCAGAACGATT
1137
UCGUUCUGCCAUCACUCACTT
1138
AD-15410


 


3326-3344
GAGUGAUGGCAGAACGAUGTT
1139
CAUCGUUCUGCCAUCACUCTT
1140
AD-15379


 


3330-3348
GAUGGCAGAACGAUGCCUGTT
1141
CAGGCAUCGUUCUGCCAUCTT
1142
AD-15187


 


3336-3354
AGAACGAUGCCUGCAGGCATT
1143
UGCCUGCAGGCAUCGUUCUTT
1144
AD-15263


 


3339-3357
ACGAUGCCUGCAGGCAUGGTT
1145
CCAUGCCUGCAGGCAUCGUTT
1146
AD-15264


 


3348-3366
GCAGGCAUGGAACUUUUUCTT
1147
GAAAAAGUUCCAUGCCUGCTT
1148
AD-15297


 


3356-3374
GGAACUUUUUCCGUUAUCATT
1149
UGAUAACGGAAAAAGUUCCTT
1150
AD-15208


 


3357-3375
GAACUUUUUCCGUUAUCACTT
1151
GUGAUAACGGAAAAAGUUCTT
1152
AD-15209


 


3358-3376
AACUUUUUCCGUUAUCACCTT
1153
GGUGAUAACGGAAAAAGUUTT
1154
AD-15193


 


3370-3388
UAUCACCCAGGCCUGAUUCTT
1155
GAAUCAGGCCUGGGUGAUATT
1156
AD-15380


 


3378-3396
AGGCCUGAUUCACUGGCCUTT
1157
AGGCCAGUGAAUCAGGCCUTT
1158
AD-15298


 


3383-3401
UGAUUCACUGGCCUGGCGGTT
1159
CCGCCAGGCCAGUGAAUCATT
1160
AD-15299


 


3385-3403
AUUCACUGGCCUGGCGGAGTT
1161
CUCCGCCAGGCCAGUGAAUTT
1162
AD-15265


 


3406-3424
GCUUCUAAGGCAUGGUCGGTT
1163
CCGACCAUGCCUUAGAAGCTT
1164
AD-15381


 


3407-3425
CUUCUAAGGCAUGGUCGGGTT
1165
CCCGACCAUGCCUUAGAAGTT
1166
AD-15210


 


3429-3447
GAGGGCCAACAACUGUCCCTT
1167
GGGACAGUUGUUGGCCCUCTT
1168
AD-15270


 


3440-3458
ACUGUCCCUCCUUGAGCACTsT
1169
GUGCUCAAGGAGGGACAGUTsT
1170
AD-9591 


 


3440-3458
AcuGucccuccuuGAGcAcTsT
1171
GUGCUcAAGGAGGGAcAGUTsT
1172
AD-9717 


 


3441-3459
CUGUCCCUCCUUGAGCACCTsT
1173
GGUGCUCAAGGAGGGACAGTsT
1174
AD-9622 


 


3441-3459
cuGucccuccuuGAGcAccTsT
1175
GGUGCUcAAGGAGGGAcAGTsT
1176
AD-9748 


 


3480-3498
ACAUUUAUCUUUUGGGUCUTsT
1177
AGACCCAAAAGAUAAAUGUTsT
1178
AD-9587 


 


3480-3498
AcAuuuAucuuuuGGGucuTsT
1179
AGACCcAAAAGAuAAAUGUTsT
1180
AD-9713 


 


3480-3498
AfcAfuUfuAfuCfuUfuUfg
1181
p-aGfaCfcCfaAfaAfgAfuA
1182
AD-14679



GfgUfcUfTsT

faAfuGfuTsT




 


3480-3498
ACfAUfUfUfAUfCfUfUfUf
1183
AGACfCfCfAAAAGAUfAAA
1184
AD-14689



UfGGGUfCfUfTsT

UfGUfTsT




 


3480-3498
AcAuUuAuCuUuUgGgUcUTsT
1185
p-aGfaCfcCfaAfaAfgAfuA
1186
AD-14699





faAfuGfuTsT




 


3480-3498
AcAuUuAuCuUuUgGgUcUTsT
1187
AGACfCfCfAAAAGAUfAAAUf
1188
AD-14709





GUfTsT




 


3480-3498
AfcAfuUfuAfuCfuUfuUfg
1189
AGACCcaAAagAUAAAuguTsT
1190
AD-14719



GfgUfcUfTsT






 


3480-3498
ACfAUfUfUfAUfCfUfUfUfUf
1191
AGACCcaAAagAUAAAuguTsT
1192
AD-14729



GGGUfCfUfTsT






 


3480-3498
AcAuUuAuCuUuUgGgUcUTsT
1193
AGACCcaAAagAUAAAuguTsT
1194
AD-14739


 


3480-3498
GfcCfaUfcUfgCfuGfcCfgG
1195
p-gGfcUfcCfgGfcAfgCfaGfaU
1196
AD-15085



faGfcCfTsT

fgGfcTsT




 


3480-3498
GCfCfAUfCfUfGCfUfGCfC
1197
GGCfUfCfCfGGCfAGCfAGA
1198
AD-15095



fGGAGCfCfTsT

UfGGCfTsT




 


3480-3498
GcCaUcUgCuGcCgGaGcCTsT
1199
p-gGfcUfcCfgGfcAfgCfaGf
1200
AD-15105





aUfgGfcTsT




 


3480-3498
GcCaUcUgCuGcCgGaGcCTsT
1201
GGCfUfCfCfGGCfAGCfAGA
1202
AD-15115





UfGGCfTsT




 


3480-3498
GfcCfaUfcUfgCfuGfcCf
1203
GGCUCauGCagCAGAUggcTsT
1204
AD-15125



gGfaGfcCfTsT






 


3480-3498
GCfCfAUfCfUfGCfUfGCfCf
1205
GGCUCauGCagCAGAUggcTsT
1206
AD-15135



GGAGCfCfTsT






 


3480-3498
GcCaUcUgCuGcCgGaGcCTsT
1207
GGCUCauGCagCAGAUggcTsT
1208
AD-15145


 


3481-3499
CAUUUAUCUUUUGGGUCUGTsT
1209
CAGACCCAAAAGAUAAAUGTsT
1210
AD-9578 


 


3481-3499
cAuuuAucuuuuGGGucuGTsT
1211
cAGACCcAAAAGAuAAAUGTsT
1212
AD-9704 


 


3485-3503
UAUCUUUUGGGUCUGUCCUTsT
1213
AGGACAGACCCAAAAGAUATsT
1214
AD-9558 


 


3485-3503
uAucuuuuGGGucuGuccuTsT
1215
AGGAcAGACCcAAAAGAuATsT
1216
AD-9684 


 


3504-3522
CUCUGUUGCCUUUUUACAGTsT
1217
CUGUAAAAAGGCAACAGAGTsT
1218
AD-9634 


 


3504-3522
cucuGuuGccuuuuuAcAGTsT
1219
CUGuAAAAAGGcAAcAGAGTsT
1220
AD-9760 


 


3512-3530
CCUUUUUACAGCCAACUUUTT
1221
AAAGUUGGCUGUAAAAAGGTT
1222
AD-15411


 


3521-3539
AGCCAACUUUUCUAGACCUTT
1223
AGGUCUAGAAAAGUUGGCUTT
1224
AD-15266


 


3526-3544
ACUUUUCUAGACCUGUUUUTT
1225
AAAACAGGUCUAGAAAAGUTT
1226
AD-15382


 


3530-3548
UUCUAGACCUGUUUUGCUUTsT
1227
AAGCAAAACAGGUCUAGAATsT
1228
AD-9554 


 


3530-3548
uucuAGAccuGuuuuGcuuTsT
1229
AAGcAAAAcAGGUCuAGAATsT
1230
AD-9680 


 


3530-3548
UfuCfuAfgAfcCfuGfuUfuU
1231
p-aAfgCfaAfaAfcAfgGfuCf
1232
AD-14676



fgCfuUfTsT

uAfgAfaTsT




 


3530-3548
UfUfCfUfAGACfCfUfGUfUf
1233
AAGCfAAAACfAGGUfCfUfAG
1234
AD-14686



UfUfGCfUfUfTsT

AATsT




 


3530-3548
UuCuAgAcCuGuUuUgCuUTsT
1235
p-aAfgCfaAfaAfcAfgGfuCfu
1236
AD-14696





AfgAfaTsT




 


3530-3548
UuCuAgAcCuGuUuUgCuUTsT
1237
AAGCfAAAACfAGGUfCfUfAGAATsT
1238
AD-14706


 


3530-3548
UfuCfuAfgAfcCfuGfuUfuUf
1239
AAGcAaaACagGUCUAgaaTsT
1240
AD-14716



fCfuUfTsT






 


3530-3548
UfUfCfUfAGACfCfUfGUfUfU
1241
AAGcAaaACagGUCUAgaaTsT
1242
AD-14726



fUfGCfUfUfTsT






 


3530-3548
UuCuAgAcCuGuUuUgCuUTsT
1243
AAGcAaaACagGUCUAgaaTsT
1244
AD-14736


 


3530-3548
CfaUfaGfgCfcUfgGfaGfuU
1245
p-aAfuAfaAfcUfcCfaGfgCf
1246
AD-15082



fuAfuUfTsT

cUfaUfgTsT




 


3530-3548
CfAUfAGGCfCfUfGGAGUf
1247
AAUfAAACfUfCfCfAGGCfCf
1248
AD-15092



UfUfAUfUfTsT

UfAUfGTsT




 


3530-3548
CaUaGgCcUgGaGuUuAuUTsT
1249
p-aAfuAfaAfcUfcCfaGfgCf
1250
AD-15102





cUfaUfgTsT




 


3530-3548
CaUaGgCcUgGaGuUuAuUTsT
1251
AAUfAAACfUfCfCfAGGCfCf
1252
AD-15112





UfAUfGTsT




 


3530-3548
CfaUfaGfgCfcUfgGfaGfu
1253
AAUAAacUCcaGGCCUaugTsT
1254
AD-15122



UfuAfuUfTsT






 


3530-3548
CfAUfAGGCfCfUfGGAGUfU
1255
AAUAAacUCcaGGCCUaugTsT
1256
AD-15132



fUfAUfUfTsT






 


3530-3548
CaUaGgCcUgGaGuUuAuUTsT
1257
AAUAAacUCcaGGCCUaugTsT
1258
AD-15142


 


3531-3549
UCUAGACCUGUUUUGCUUUTsT
1259
AAAGCAAAACAGGUCUAGATsT
1260
AD-9553 


 


3531-3549
ucuAGAccuGuuuuGcuuuTsT
1261
AAAGcAAAAcAGGUCuAGATsT
1262
AD-9679 


 


3531-3549
UfcUfaGfaCfcUfgUfu
1263
p-aAfaGfcAfaAfaCfaGfgU
1264
AD-14675



UfuGfcUfuUfTsT

fcUfaGfaTsT




 


3531-3549
UfCfUfAGACfCfUfGUfUfUf
1265
AAAGCfAAAACfAGGUfCfUfAG
1266
AD-14685



UfGCfUfUfUfTsT 

ATsT




 


3531-3549
UcUaGaCcUgUuUuGcUuUTsT
1267
p-aAfaGfcAfaAfaCfaGfgUfcU
1268
AD-14695





faGfaTsT




 


3531-3549
UcUaGaCcUgUuUuGcUuUTsT
1269
AAAGCfAAAACfAGGUfCfUfAGATsT
1270
AD-14705


 


3531-3549
UfcUfaGfaCfcUfgUfuU
1271
AAAGCaaAAcaGGUCUagaTsT
1272
AD-14715



fuGfcUfuUfTsT






 


3531-3549
UfCfUfAGACfCfUfGUfUfUf
1273
AAAGCaaAAcaGGUCUagaTsT
1274
AD-14725



UfGCfUfUfUfTsT 






 


3531-3549
UcUaGaCcUgUuUuGcUuUTsT
1275
AAAGCaaAAcaGGUCUagaTsT
1276
AD-14735


 


3531-3549
UfcAfuAfgGfcCfuGfgAf
1277
p-aUfaAfaCfuCfcAfgGfcCf
1278
AD-15081



gUfuUfaUfTsT

uAfuGfaTsT




 


3531-3549
UfCfAUfAGGCfCfUfGGAGU
1279
AUfAAACfUfCfCfAGGCfC
1280
AD-15091



fUfUfAUfTsT

fUfAUfGATsT




 


3531-3549
UcAuAgGcCuGgAgUuUaUTsT
1281
p-aUfaAfaCfuCfcAfgGfcCfu
1282
AD-15101





AfuGfaTsT




 


3531-3549
UcAuAgGcCuGgAgUuUaUTsT
1283
AUfAAACfUfCfCfAGGCfCfU
1284
AD-15111





fAUfGATsT




 


3531-3549
UfcAfuAfgGfcCfuGfgAfg
1285
AUAAAcuCCagGCCUAugaTsT
1286
AD-15121



UfuUfaUfTsT






 


3531-3549
UfCfAUfAGGCfCfUfGGAGU
1287
AUAAAcuCCagGCCUAugaTsT
1288
AD-15131



fUfUfAUfTsT






 


3531-3549
UcAuAgGcCuGgAgUuUaUTsT
1289
AUAAAcuCCagGCCUAugaTsT
1290
AD-15141


 


3557-3575
UGAAGAUAUUUAUUCUGGGTsT
1291
CCCAGAAUAAAUAUCUUCATsT
1292
AD-9626 


 


3557-3575
uGAAGAuAuuuAuucuGGGTsT
1293
CCcAGAAuAAAuAUCUUcATsT
1294
AD-9752 


 


3570-3588
UCUGGGUUUUGUAGCAUUUTsT
1295
AAAUGCUACAAAACCCAGATsT
1296
AD-9629 


 


3570-3588
ucuGGGuuuuGuAGcAuuuTsT
1297
AAAUGCuAcAAAACCcAGATsT
1298
AD-9755 


 


3613-3631
AUAAAAACAAACAAACGUUTT
1299
AACGUUUGUUUGUUUUUAUTT
1300
AD-15412


 


3617-3635
AAACAAACAAACGUUGUCCTT
1301
GGACAACGUUUGUUUGUUUTT
1302
AD-15211


 


3618-3636
AACAAACAAACGUUGUCCUTT
1303
AGGACAACGUUUGUUUGUUTT
1304
AD-15300










data



















IC50 in








Cynomolgous











Mean percent remaining mRNA
IC50 in
monkey



transcript at siRNA concentration/in cell type
HepG2
Hepatocyte













Duplex name
100 nM/HepG2
30 nM/HepG2
3 nM/HepG2
30 nM/Hela
[nM]
[nM]s





AD-15220



35




AD-15275



56




AD-15301



70




AD-15276



42




AD-15302



32




AD-15303



37




AD-15221



30




AD-15413



61




AD-15304



70




AD-15305



36




AD-15306



20




AD-15307



38




AD-15277



50




AD-9526 
74
89






AD-9652 

97






AD-9519 

78






AD-9645 

66






AD-9523 

55






AD-9649 

60






AD-9569 

112






AD-9695 

102






AD-15222



75




AD-15278



78




AD-15178



83




AD-15308



84




AD-15223



67




AD-15309



34




AD-15279



44




AD-15194



63




AD-15310



42




AD-15311



30




AD-15392



18




AD-15312



21




AD-15313



19




AD-15280



81




AD-15267



82




AD-15314



32




AD-15315



74




AD-9624 

94






AD-9750 

96






AD-9623 
43
66






AD-9749 

105






AD-15384



48




AD-9607 

32
28

0.20



AD-9733 

78
73





AD-9524 

23
28

0.07



AD-9650 

91
90





AD-9520 

23
32





AD-9520 

23






AD-9646 

97
108





AD-9608 

37






AD-9734 

91






AD-9546 

32






AD-9672 

57






AD-15385



54




AD-15393



31




AD-15316



37




AD-15317



37




AD-15318



63




AD-15195



45




AD-15224



57




AD-15188



42




AD-15225



51




AD-15281



89




AD-15282



75




AD-15319



61




AD-15226



56




AD-15271



25




AD-15283



25




AD-15284



64




AD-15189



17




AD-15227



62




AD-9547 

31
29

0.20



AD-9673 

56
57





AD-9548 

54
60





AD-9674 

36
57





AD-9529 

60






AD-9655 

140






AD-9605 

27
31

0.27



AD-9731 

31
31

0.32



AD-9596 

37






AD-9722 

76






AD-9583 

42






AD-9709 

104






AD-9579 

113






AD-9705 

81






AD-15394



32




AD-15196



72




AD-15197



85




AD-15198



71




AD-9609 
66
71






AD-9735 

115






AD-9537 

145






AD-9663 

102






AD-9528 

113






AD-9654 

107






AD-9515 

49






AD-9641 

92






AD-9514 

57






AD-9640 

89






AD-9530 

75






AD-9656 

77






AD-9538 
79
80






AD-9664 

53






AD-9598 
69
83






AD-9724 

127






AD-9625 
58
88






AD-9751 

60






AD-9556 

46






AD-9682 

38






AD-9539 
56
63






AD-9665 

83






AD-9517 

36






AD-9643 

40






AD-9610 

36
34

0.04



AD-9736 

22
29

0.04



AD-14681



33




AD-14691



27




AD-14701



32




AD-14711



33




AD-14721



22




AD-14731



21




AD-14741



22




AD-15087



37




AD-15097



51




AD-15107



26




AD-15117



28




AD-15127



33




AD-15137



54




AD-15147



52




AD-9516 

94






AD-9642 

105






AD-9562 

46
51





AD-9688 

26
34

4.20



AD-14677



38




AD-14687



52




AD-14697



35




AD-14707



58




AD-14717



42




AD-14727



50




AD-14737



32




AD-15083



16




AD-15093



24




AD-15103



11




AD-15113



34




AD-15123



19




AD-15133



15




AD-15143



16




AD-9521 

50






AD-9647 

62






AD-9611 

48






AD-9737 

68






AD-9592 
46
55






AD-9718 

78






AD-9561 

64






AD-9687 

84






AD-9636 

42
41

2.10



AD-9762 

9
28

0.40



AD-9540 

45






AD-9666 

81






AD-9535 
48
73






AD-9661 

83






AD-9559 

35






AD-9685 

77






AD-9533 

100






AD-9659 

88






AD-9612 

122






AD-9738 

83






AD-9557 
75
96






AD-9683 

48






AD-9531 

31
32

0.53



AD-9657 

23
29

0.66



AD-14673



81




AD-14683



56




AD-14693



56




AD-14703



68




AD-14713



55




AD-14723



24




AD-14733



34




AD-15079



85




AD-15089



54




AD-15099



70




AD-15109



67




AD-15119



67




AD-15129



57




AD-15139



69




AD-9542 

160






AD-9668 

92






AD-9739 

109






AD-9637 
56
83






AD-9763 

79






AD-9630 

82






AD-9756 

63






AD-9593 

55






AD-9719 

115






AD-9601 

111






AD-9727 

118






AD-9573 

36
42

1.60



AD-9699 

32
36

2.50



AD-15228



26




AD-15395



53




AD-9602 

126






AD-9728 

94






AD-15386



45




AD-9580 

112






AD-9706 

86






AD-9581 

35






AD-9707 

81






AD-9543 

51






AD-9669 

97






AD-9574 

74






AD-9700 








AD-15320



26




AD-15321



34




AD-15199



64




AD-15167



86




AD-15164



41




AD-15166



43




AD-15322



64




AD-15200



46




AD-15213



27




AD-15229



44




AD-15215



49




AD-15214



101




AD-9315 

15
32

0.98



AD-9326 

35
51





AD-9318 

14
37

0.40



AD-9323 

14
33





AD-9314 

11
22

0.04



AD-10792




0.10
0.10


AD-10796




0.1
0.1


AD-9638 

101






AD-9764 

112






AD-9525 

53






AD-9651 

58






AD-9560 

97






AD-9686 

111






AD-9536 

157






AD-9662 

81






AD-9584 
52
68






AD-9710 

111






AD-15323



62




AD-9551 

91






AD-9677 

62






AD-15230



52




AD-15231



25




AD-15285



36




AD-15396



27




AD-15397



56




AD-9600 

112






AD-9726 

95






AD-9606 

107






AD-9732 

105






AD-9633 
56
75






AD-9759 

111






AD-9588 

66






AD-9714 

106






AD-9589 
67
85






AD-9715 

113






AD-9575 

120






AD-9701 

100






AD-9563 

103






AD-9689 

81






AD-9594 
80
95






AD-9720 

92






AD-9585 

83






AD-9711 

122






AD-9614 

100






AD-9740 

198






AD-9615 

116






AD-9741 

130






AD-9534 

32
30





AD-9534 

32






AD-9660 

89
79





AD-15324



46




AD-15232



19




AD-15233



25




AD-15234



59




AD-15286



109




AD-9590 

122






AD-9716 

114






AD-9632 

34






AD-9758 

96






AD-9567 

41






AD-9693 

50






AD-9586 
81
104






AD-9712 

107






AD-9564 

120






AD-9690 

92






AD-9616 
74
84






AD-9742 

127






AD-15398



24




AD-9617 

111






AD-9743 

104






AD-9635 
73
90






AD-9761 

83






AD-9568 

76






AD-9694 

52






AD-9576 

47






AD-9702 

79






AD-9627 

69






AD-9753 

127






AD-9628 

141






AD-9754 

89






AD-9631 

80






AD-9757 

78






AD-9595 

31
32





AD-9721 

87
70





AD-9544 

68






AD-9670 

67






AD-15235



25




AD-15236



73




AD-15168



100




AD-15174



92




AD-15325



81




AD-15326



65




AD-9570 
35
42






AD-9696 

77






AD-9566 

38






AD-9692 

78






AD-9532 

100






AD-9658 

102






AD-9549 

50






AD-9675 

78






AD-9541 

43






AD-9667 

73






AD-9550 

36






AD-9676 

100






AD-9571 

27
32





AD-9697 

74
89





AD-9572 
47
53






AD-9698 

73






AD-15327



82




AD-9639 

30
35





AD-9765 

82
74





AD-9518 

31
35

0.60



AD-9518 

31






AD-9644 

35
37

2.60



AD-14672



26




AD-14682



27




AD-14692



22




AD-14702



19




AD-14712



25




AD-14722



18




AD-14732



32




AD-15078



86




AD-15088



97




AD-15098



74




AD-15108



67




AD-15118



76




AD-15128



86




AD-15138



74




AD-15237



30




AD-15287



30




AD-15238



36




AD-15328



35




AD-15399



47




AD-9582 

37






AD-9708 

81






AD-9545 

31
43





AD-9671 

15
33

2.50



AD-14674



16




AD-14684



26




AD-14694



18




AD-14704



27




AD-14714



20




AD-14724



18




AD-14734



18




AD-15080



29




AD-15090



23




AD-15100



26




AD-15110



23




AD-15120



20




AD-15130



20




AD-15140



19




AD-9522 

59






AD-9648 

78






AD-9552 

80






AD-9678 

76






AD-9618 

90






AD-9744 

91






AD-15239



38




AD-15212



19




AD-15240



43




AD-15177



59




AD-15179



13




AD-15180



15




AD-15241



14




AD-15268



42




AD-15242



21




AD-15216



28




AD-15176



35




AD-15181



35




AD-15243



22




AD-15182



42




AD-15244



31




AD-15387



23




AD-15245



18




AD-9555 

34






AD-9681 

55






AD-9619 
42
61






AD-9745 

56






AD-9620 
44
77






AD-9746 

89






AD-15288



19




AD-15246



16




AD-15289



37




AD-9324 

59
67





AD-15329



103




AD-15330



62




AD-15169



22




AD-15201



6




AD-15331



14




AD-15190



47




AD-15247



61




AD-15248



22




AD-15175



45




AD-15249



51




AD-15250



96




AD-15400



12




AD-15332



22




AD-15388



30




AD-15333



20




AD-15334



96




AD-15335



75




AD-15183



16




AD-15202



41




AD-15203



39




AD-15272



49




AD-15217



16




AD-15290



15




AD-15218



13




AD-15389



13




AD-15336



40




AD-15337



19




AD-15191



33




AD-15390



25




AD-15338



9




AD-15204



33




AD-15251



76




AD-15205



14




AD-15171



16




AD-15252



58




AD-15339



20




AD-15253



15




AD-15340



18




AD-15291



17




AD-15341



11




AD-15401



13




AD-15342



30




AD-15343



21




AD-15292



16




AD-15344



20




AD-15254



18




AD-15345



18




AD-15206



15




AD-15346



16




AD-15347



62




AD-9577 

33
31





AD-9703 

17
26





AD-14678



22




AD-14688



23




AD-14698



23




AD-14708



14




AD-14718



31




AD-14728



25




AD-14738



31




AD-15084



19




AD-15094



11




AD-15104



16




AD-15114



15




AD-15124



11




AD-15134



12




AD-15144



9




AD-15391



7




AD-15348



13




AD-15349



8




AD-15170



40




AD-15350



14




AD-15402



27




AD-15293



27




AD-15351



14




AD-15403



11




AD-15404



38




AD-15207



15




AD-15352



23




AD-15255



31




AD-9603 

123






AD-9729 

56






AD-9599 

139






AD-9725 

38






AD-9621 

77






AD-9747 

63






AD-15405



32




AD-15353



39




AD-15354



49




AD-15406



35




AD-15407



39




AD-15355



18




AD-15356



50




AD-15357



54




AD-15269



23




AD-9565 

74






AD-9691 

49






AD-15358



12




AD-15359



24




AD-15360



13




AD-15219



19




AD-15361



24




AD-15273



36




AD-15362



31




AD-15192



20




AD-15256



19




AD-15363



33




AD-15364



24




AD-9604 
35
49






AD-9730 

85






AD-9527 

45






AD-9653 

86






AD-15365



62




AD-15294



30




AD-15173



12




AD-15366



21




AD-15367



11




AD-15257



18




AD-15184



50




AD-15185



12




AD-15258



73




AD-15186



36




AD-15274



19




AD-15368



7




AD-15369



17




AD-15370



19




AD-15259



38




AD-15408



52




AD-9597 

23
21

0.04



AD-9723 

12
26





AD-14680



15




AD-14690



18




AD-14700



15




AD-14710



15




AD-14720



18




AD-14730



18




AD-14740



17




AD-15086



85




AD-15096



70




AD-15106



71




AD-15116



73




AD-15126



71




AD-15136



56




AD-15146



72




AD-15260



79




AD-15371



24




AD-15372



52




AD-15172



27




AD-15295



22




AD-15373



11




AD-15163



18




AD-15165



13




AD-15374



23




AD-15296



13




AD-15261



20




AD-15375



90




AD-15262



72




AD-15376



14




AD-15377



19




AD-15409



17




AD-15378



18




AD-15410



8




AD-15379



11




AD-15187



36




AD-15263



18




AD-15264



75




AD-15297



21




AD-15208



6




AD-15209



28




AD-15193



131




AD-15380



88




AD-15298



43




AD-15299



99




AD-15265



95




AD-15381



18




AD-15210



40




AD-15270



83




AD-9591  
75
95






AD-9717 

105






AD-9622 

94






AD-9748 

103






AD-9587 

63
49





AD-9713 

22
25





AD-14679



19




AD-14689



24




AD-14699



19




AD-14709



21




AD-14719



24




AD-14729



23




AD-14739



24




AD-15085



74




AD-15095



60




AD-15105



33




AD-15115



30




AD-15125



54




AD-15135



51




AD-15145



49




AD-9578  
49
61






AD-9704 

111






AD-9558 

66






AD-9684 

63






AD-9634 

29
30





AD-9760 

14
27





AD-15411



5




AD-15266



23




AD-15382



12




AD-9554 

23
24





AD-9680 

12
22

0.10
0.10


AD-14676



12




AD-14686



13




AD-14696



12




AD-14706



18




AD-14716



17




AD-14726



16




AD-14736



9




AD-15082



27




AD-15092



28




AD-15102



19




AD-15112



17




AD-15122



56




AD-15132



39




AD-15142



46




AD-9553 

27
22

0.02



AD-9679 

17
21





AD-14675



11




AD-14685



19




AD-14695



12




AD-14705



16




AD-14715



19




AD-14725



19




AD-14735



19




AD-15081



30




AD-15091



16




AD-15101



16




AD-15111



11




AD-15121



19




AD-15131



17




AD-15141



18




AD-9626 

97
68





AD-9752 

28
33





AD-9629 

23
24





AD-9755 

28
29





AD-15412



21




AD-15211



73




AD-15300



41





1U, 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 conncected 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′-phospate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide

















TABLE 2







Duplex
Sense strand
SEQ ID
Antisense-strand
SEQ ID


number
sequence (5′-3′)1
NO:
sequence (5′-3′)1
NO:





AD-10792
GccuGGAGuuuAuucGGAATsT
1305
UUCCGAAuAAACUCcAGGCTsT
1306


 


AD-10793
GccuGGAGuuuAuucGGAATsT
1307
uUcCGAAuAAACUccAGGCTsT
1308


 


AD-10796
GccuGGAGuuuAuucGGAATsT
1309
UUCCGAAUAAACUCCAGGCTsT
1310


 


AD-12038
GccuGGAGuuuAuucGGAATsT
1311
uUCCGAAUAAACUCCAGGCTsT
1312


 


AD-12039
GccuGGAGuuuAuucGGAATsT
1313
UuCCGAAUAAACUCCAGGCTsT
1314


 


AD-12040
GccuGGAGuuuAuucGGAATsT
1315
UUcCGAAUAAACUCCAGGCTsT
1316


 


AD-12041
GccuGGAGuuuAuucGGAATsT
1317
UUCcGAAUAAACUCCAGGCTsT
1318


 


AD-12042
GCCUGGAGUUUAUUCGGAATsT
1319
uUCCGAAUAAACUCCAGGCTsT
1320


 


AD-12043
GCCUGGAGUUUAUUCGGAATsT
1321
UuCCGAAUAAACUCCAGGCTsT
1322


 


AD-12044
GCCUGGAGUUUAUUCGGAATsT
1323
UUcCGAAUAAACUCCAGGCTsT
1324


 


AD-12045
GCCUGGAGUUUAUUCGGAATsT
1325
UUCcGAAUAAACUCCAGGCTsT
1326


 


AD-12046
GccuGGAGuuuAuucGGAA
1327
UUCCGAAUAAACUCCAGGCscsu
1328


 


AD-12047
GccuGGAGuuuAuucGGAAA
1329
UUUCCGAAUAAACUCCAGGCscsu
1330


 


AD-12048
GccuGGAGuuuAuucGGAAAA
1331
UUUUCCGAAUAAACUCCAGGCscsu
1332


 


AD-12049
GccuGGAGuuuAuucGGAAAAG
1333
CUUUUCCGAAUAAACUCCAGGCscsu
1334


 


AD-12050
GccuGGAGuuuAuucGGAATTab
1335
UUCCGAAUAAACUCCAGGCTTab
1336


 


AD-12051
GccuGGAGuuuAuucGGAAATTab
1337
UUUCCGAAuAAACUCCAGGCTTab
1338


 


AD-12052
GccuGGAGuuuAuucGGAAAATTab
1339
UUUUCCGAAUAAACUCCAGGCTTab
1340


 


AD-12053
GccuGGAGuuuAuucGGAAAAGTTab
1341
CUUUUCCGAAUAAACUCCAGGCTTab
1342


 


AD-12054
GCCUGGAGUUUAUUCGGAATsT
1343
UUCCGAAUAAACUCCAGGCscsu
1344


 


AD-12055
GccuGGAGuuuAuucGGAATsT
1345
UUCCGAAUAAACUCCAGGCscsu
1346


 


AD-12056
GcCuGgAgUuUaUuCgGaA
1347
UUCCGAAUAAACUCCAGGCTTab
1348


 


AD-12057
GcCuGgAgUuUaUuCgGaA
1349
UUCCGAAUAAACUCCAGGCTsT
1350


 


AD-12058
GcCuGgAgUuUaUuCgGaA
1351
UUCCGAAuAAACUCcAGGCTsT
1352


 


AD-12059
GcCuGgAgUuUaUuCgGaA
1353
uUcCGAAuAAACUccAGGCTsT
1354


 


AD-12060
GcCuGgAgUuUaUuCgGaA
1355
UUCCGaaUAaaCUCCAggc
1356


 


AD-12061
GcCuGgnAgUuUaUuCgGaATsT
1357
UUCCGaaUAaaCUCCAggcTsT
1358


 


AD-12062
GcCuGgAgUuUaUuCgGaATTab
1359
UUCCGaaUAaaCUCCAggcTTab
1360


 


AD-12063
GcCuGgAgUuUaUuCgGaA
1361
UUCCGaaUAaaCUCCAggcscsu
1362


 


AD-12064
GcCuGgnAgUuUaUuCgGaATsT
1363
UUCCGAAuAAACUCcAGGCTsT
1364


 


AD-12065
GcCuGgAgUuUaUuCgGaATTab
1365
UUCCGAAuAAACUCcAGGCTTab
1366


 


AD-12066
GcCuGgAgUuUaUuCgGaA
1367
UUCCGAAuAAACUCcAGGCscsu
1368


 


AD-12067
GcCuGgnAgUuUaUuCgGaATsT
1369
UUCCGAAUAAACUCCAGGCTsT
1370


 


AD-12068
GcCuGgAgUuUaUuCgGaATTab
1371
UUCCGAAUAAACUCCAGGCTTab
1372


 


AD-12069
GcCuGgAgUuUaUuCgGaA
1373
UUCCGAAUAAACUCCAGGCscsu
1374


 


AD-12338
GfcCfuGfgAfgUfuUfaUfu
1375
P-uUfcCfgAfaUfaAfaC
1376



CfgGfaAf

fuCfcAfgGfc



 


AD-12339
GcCuGgAgUuUaUuCgGaA
1377
P-uUfcCfgAfaUfaAfaCf
1378





uCfcAfgGfc



 


AD-12340
GccuGGAGuuuAuucGGAA
1379
P-uUfcCfgAfaUfaAfa
1380





CfuCfcAfgGfc



 


AD-12341
GfcCfuGfgAfgUfuUfaUf
1381
P-uUfcCfgAfaUfaAfaCf
1382



uCfgGfaAfTsT

uCfcAfgGfcTsT



 


AD-12342
GfcCfuGfgAfgUfuUfaUfuC
1383
UUCCGAAuAAACUCcAGGCTsT
1384



fgGfaAfTsT





 


AD-12343
GfcCfuGfgAfgUfuUfaUf
1385
uUcCGAAuAAACUccAGGCTsT
1386



uCfgGfaAfTsT





 


AD-12344
GfcCfuGfgAfgUfuUfaUfu
1387
UUCCGAAUAAACUCCAGGCTsT
1388



CfgGfaAfTsT





 


AD-12345
GfcCfuGfgAfgUfuUfaUfu
1389
UUCCGAAUAAACUCCAGGCscsu
1390



CfgGfaAfTsT





 


AD-12346
GfcCfuGfgAfgUfuUfaUfu
1391
UUCCGaaUAaaCUCCAggcscsu
1392



CfgGfaAfTsT





 


AD-12347
GCCUGGAGUUUAUUCGGAATsT
1393
P-uUfcCfgAfaUfaAfaCfu
1394





CfcAfgGfcTsT



 


AD-12348
GccuGGAGuuuAuucGGAATsT
1395
P-uUfcCfgAfaUfaAfaCf
1396





uCfcAfgGfcTsT



 


AD-12349
GcCuGgnAgUuUaUuCgGaATsT
1397
P-uUfcCfgAfaUfaAfaCfuC
1398





fcAfgGfcTsT



 


AD-12350
GfcCfuGfgAfgUfuUfaUfu
1399
P-uUfcCfgAfaUfaAfaC
1400



CfgGfaAfTTab

fuCfcAfgGfcTTab



 


AD-12351
GfcCfuGfgAfgUfuUfaUfuCf
1401
P-uUfcCfgAfaUfaAfaCf
1402



gGfaAf

uCfcAfgGfcsCfsu



 


AD-12352
GfcCfuGfgAfgUfuUfaUfu
1403
UUCCGaaUAaaCUCCAggcscsu
1404



CfgGfaAf





 


AD-12354
GfcCfuGfgAfgUfuUf
1405
UUCCGAAUAAACUCCAGGCscsu
1406



aUfuCfgGfaAf





 


AD-12355
GfcCfuGfgAfgUfuUfaU
1407
UUCCGAAuAAACUCcAGGCTsT
1408



fuCfgGfaAf





 


AD-12356
GfcCfuGfgAfgUfuUfaUfu
1409
uUcCGAAuAAACUccAGGCTsT
1410



CfgGfaAf





 


AD-12357
GmocCmouGmogAm02gUmouUmoa
1411
UUCCGaaUAaaCUCCAggc
1412



UmouCmogGmoaA





 


AD-12358
GmocCmouGmogAm02gUmouU
1413
P-uUfcCfgAfaUfaAfaC
1414



moaUmouCmogGmoaA

fuCfcAfgGfc



 


AD-12359
GmocCmouGmogAm02gUmouU
1415
P-uUfcCfgAfaUfaAfaCfuCfc
1416



moaUmouCmogGmoaA

AfgGfcsCfsu



 


AD-12360
GmocCmouGmogAm02gUmouUm
1417
UUCCGAAUAAACUCCAGGCscsu
1418



oaUmouCmogGmoaA





 


AD-12361
GmocCmouGmogAm02gUmouUmo
1419
UUCCGAAuAAACUCcAGGCTsT
1420



aUmouCmogGmoaA





 


AD-12362
GmocCmouGmogAm02gUmouU
1421
uUcCGAAuAAACUccAGGCTsT
1422



moaUmouCmogGmoaA





 


AD-12363
GmocCmouGmogAm02gUmouU
1423
UUCCGaaUAaaCUCCAggcscsu
1424



moaUmouCmogGmoaA





 


AD-12364
GmocCmouGmogAmogUmouUmo
1425
UUCCGaaUAaaCUCCAggcTsT
1426



aUmouCmogGmoaATsT





 


AD-12365
GmocCmouGmogAmogUmouUm
1427
UUCCGAAuAAACUCcAGGCTsT
1428



oaUmouCmogGmoaATsT





 


AD-12366
GmocCmouGmogAmogUmouUm
1429
UUCCGAAUAAACUCCAGGCTsT
1430



oaUmouCmogGmoaATsT





 


AD-12367
GmocmocmouGGAGmoumoum
1431
UUCCGaaUAaaCUCCAggcTsT
1432



ouAmoumoumocGGAATsT





 


AD-12368
GmocmocmouGGAGmoumoum
1433
UUCCGAAuAAACUCcAGGCTsT
1434



ouAmoumoumocGGAATsT





 


AD-12369
GmocmocmouGGAGmoumoumo
1435
UUCCGAAUAAACUCCAGGCTsT
1436



uAmoumoumocGGAATsT





 


AD-12370
GmocmocmouGGAGmoum
1437
P-UfUfCfCfGAAUfAAACfUfC
1438



oumouAmoumoumocGGAATsT

fCfAGGCfTsT



 


AD-12371
GmocmocmouGGAGmoumoumouA
1439
P-UfUfCfCfGAAUfAAACfUfCfC
1440



moumoumocGGAATsT

fAGGCfsCfsUf



 


AD-12372
GmocmocmouGGAGmoumoumou
1441
P-uUfcCfgAfaUfaAfaCfuCfc
1442



AmoumoumocGGAATsT

AfgGfcsCfsu



 


AD-12373
GmocmocmouGGAGmoumoumou
1443
UUCCGAAUAAACUCCAGGCTsT
1444



AmoumoumocGGAATsT





 


AD-12374
GCfCfUfGGAGUfUfUfAUfUfCf
1445
UfUfCfCfGAAUfAAACfUfCf
1446



GGAATsT

CfAGGCfTsT



 


AD-12375
GCfCfUfGGAGUfUfUfAU
1447
UUCCGAAUAAACUCCAGGCTsT
1448



fUfCfGGAATsT





 


AD-12377
GCfCfUfGGAGUfUfUfAUfU
1449
uUcCGAAuAAACUccAGGCTsT
1450



fCfGGAATsT





 


AD-12378
GCfCfUfGGAGUfUfUf
1451
UUCCGaaUAaaCUCCAggcscsu
1452



AUfUfCfGGAATsT





 


AD-12379
GCfCfUfGGAGUfUfUfAUfU
1453
UUCCGAAUAAACUCCAGGCscsu
1454



fCfGGAATsT





 


AD-12380
GCfCfUfGGAGUfUfUfAUf
1455
P-uUfcCfgAfaUfaAfaCfuCf
1456



UfCfGGAATsT

cAfgGfcsCfsu



 


AD-12381
GCfCfUfGGAGUfUfUfAUfU
1457
P-uUfcCfgAfaUfaAfaCfuC
1458



fCfGGAATsT

fcAfgGfcTsT



 


AD-12382
GCfCfUfGGAGUfUfUfAUf
1459
P-UfUfCfCfGAAUfAAACfU
1460



UfCfGGAATsT

fCfCfAGGCfTst



 


AD-12383
GCCUGGAGUUUAUUCGGAATsT
1461
P-UfUfCfCfGAAUfAAACfUf
1462





CfCfAGGCfTst



 


AD-12384
GccuGGAGuuuAuucGGAATsT
1463
P-UfUfCfCfGAAUfAAACfUfCf
1464





CfAGGCfTst



 


AD-12385
GcCuGgnAgUuUaUuCgGaATsT
1465
P-UfUfCfCfGAAUfAAACfUfC
1466





fCfAGGCfTst



 


AD-12386
GfcCfuGfgAfgUfuUfaUf
1467
P-UfUfCfCfGAAUfAAACfUf
1468



uCfgGfaAf

CfCfAGGCfTst



 


AD-12387
GCfCfUfGGAGGUfUfUf
1469
UfUfCfCfGAAUfAAACfUfCfCfA
1470



AUfUfCfGGAA

GGCfsCfsUf



 


AD-12388
GCfCfUfGGAGGUfUfUfAUf
1471
P-uUfcCfgAfaUfaAfaCfu
1472



UfCfGGAA

CfcAfgGfc



 


AD-12389
GCfCfUfGGAGGUfUfUf
1473
P-uUfcCfgAfaUfaAfaCfuCf
1474



AUfUfCfGGAA

cAfgGfcsCfsu



 


AD-12390
GCfCfUfGGAGGUfUfUfAUfU
1475
UUCCGAAUAAACUCCAGGCscsu
1476



fCfGGAA





 


AD-12391
GCfCfUfGGAGGUfUfUfAUf
1477
UUCCGaaUAaaCUCCAggc
1478



UfCfGGAA





 


AD-12392
GCfCfUfGGAGGUfUfUf
1479
UUCCGAAUAAACUCCAGGCTsT
1480



AUfUfCfGGAA





 


AD-12393
GCfCfUfGGAGGUfUfUfAU
1481
UUCCGAAuAAACUCcAGGCTsT
1482



fUfCfGGAA





 


AD-12394
GCfCfUfGGAGGUfUfUfA
1483
uUcCGAAuAAACUccAGGCTsT
1484



UfUfCfGGAA





 


AD-12395
GmocCmouGmogAmogUmouUmo
1485
P-UfUfCfCfGAAUfAAACfUfCf
1486



aUmouCmogGmoaATsT

CfAGGCfsCfsUf



 


AD-12396
GmocCmouGmogAm02gUmou
1487
P-UfUfCfCfGAAUfAAACfUfCfC
1488



UmoaUmouCmogGmoaA

fAGGCfsCfsU



 


AD-12397
GfcCfuGfgAfgUfuUfa
1489
P-UfUfCfCfGAAUfAAACfUfCf
1490



UfuCfgGfaAf

CfAGGCfsCfsU



 


AD-12398
GfcCfuGfgAfgUfuUfaUfuC
1491
P-UfUfCfCfGAAUfAAACfUfCfCfA
1492



fgGfaAfTsT

GGCfsCfsU



 


AD-12399
GcCuGgnAgUuUaUuCgGaATsT
1493
P-UfUfCfCfGAAUfAAACfUfCfCf
1494





AGGCfsCfsU



 


AD-12400
GCCUGGAGUUUAUUCGGAATsT
1495
P-UfUfCfCfGAAUfAAACfUfCf
1496





CfAGGCfsCfsU



 


AD-12401
GccuGGAGuuuAuucGGAATsT
1497
P-UfUfCfCfGAAUfAAACfUfC
1498





fCfAGGCfsCfsU



 


AD-12402
GccuGGAGuuuAuucGGAA
1499
P-UfUfCfCfGAAUfAAACfUfC
1500





fCfAGGCfsCfsU



 


AD-12403
GCfCfUfGGAGGUfUfUf
1501
P-UfUfCfCfGAAUfAAACfUfCf
1502



AUfUfCfGGAA

CfAGGCfsCfsU



 


AD-9314 
GCCUGGAGUUUAUUCGGAATsT
1503
UUCCGAAUAAACUCCAGGCTsT
1504
















Remaining mRNA

Remaining mRNA 

Remaining mRNA 



 in % of

in % of

in % of 



controls at

controls at

controls at


Duplex
siRNA conc.
Duplex
siRNA conc.
Duplex
siRNA conc.


number
of 30 nM
number
of 30 nM
number
of 30 nM





AD-10792
15
AD-12339
14
AD-12377
88


 


AD-10793
32
AD-12340
19
AD-12378
6


 


AD-10796
13
AD-12341
12
AD-12379
6


 


AD-12038
13
AD-12342
13
AD-12380
8


 


AD-12039
29
AD-12343
24
AD-12381
10


 


AD-12040
10
AD-12344
9
AD-12382
7


 


AD-12041
11
AD-12345
12
AD-12383
7


 


AD-12042
12
AD-12346
13
AD-12377
88


 


AD-12043
13
AD-12347
11
AD-12378
6


 


AD-12044
7
AD-12348
8
AD-12379
6


 


AD-12045
8
AD-12349
11
AD-12380
8


 


AD-12046
13
AD-12350
17
AD-12381
10


 


AD-12047
17
AD-12351
11
AD-12382
7


 


AD-12048
43
AD-12352
11
AD-12383
7


 


AD-12049
34
AD-12354
11
AD-12384
8


 


AD-12050
16
AD-12355
9
AD-12385
8


 


AD-12051
31
AD-12356
25
AD-12386
11


 


AD-12052
81
AD-12357
56
AD-12387
13


 


AD-12053
46
AD-12358
29
AD-12388
19


 


AD-12054
8
AD-12359
30
AD-12389
16


 


AD-12055
13
AD-12360
15
AD-12390
17


 


AD-12056
11
AD-12361
20
AD-12391
21


 


AD-12057
8
AD-12362
51
AD-12392
28


 


AD-12058
9
AD-12363
11
AD-12393
17


 


AD-12059
23
AD-12364
25
AD-12394
75


 


AD-12060
10
AD-12365
18
AD-12395
55


 


AD-12061
7
AD-12366
23
AD-12396
59


 


AD-12062
10
AD-12367
42
AD-12397
20


 


AD-12063
19
AD-12368
40
AD-12398
11


 


AD-12064
15
AD-12369
26
AD-12399
13


 


AD-12065
16
AD-12370
68
AD-12400
12


 


AD-12066
20
AD-12371
60
AD-12401
13


 


AD-12067
17
AD-12372
60
AD-12402
14


 


AD-12068
18
AD-12373
55
AD-12403
4


 


AD-12069
13
AD-12374
9
AD-9314 
9


 


AD-12338
15
AD-12375
16





1U, 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 conncected 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′-phospate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the ′3-most nucleotide

## Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of a human proprotein convertase subtilisin kexin 9 (PCSK9) gene in a cell, wherein the dsRNA comprises a sense strand and an antisense strand complementary to at least 15 contiguous nucleotides of nucleotides 1-19 of SEQ ID NO:453 and comprises a duplex structure between 15 and 30 base pairs in length.

2. The dsRNA of claim 1 comprising a duplex structure between 19 and 21 base pairs in length.

3. The dsRNA of claim 1 wherein the sense strand comprises the nucleotide sequence of nucleotides 1-19 of SEQ ID NO:453 and the antisense strand comprises the nucleotide sequence of nucleotides 1-19 of SEQ ID NO:454.

4. The dsRNA of claim 1, the sense strand consisting of SEQ ID NO:453 and the antisense strand consisting of SEQ ID NO:454.

5. The dsRNA of claim 4, comprising at least one modified nucleotide.

6. The dsRNA of claim 1, wherein the dsRNA comprises at least one modified nucleotide.

7. The dsRNA of claim 1, wherein the dsRNA comprises at least one 2′-O-methyl modified nucleotide and at least one nucleotide comprising a 5′-phosphorothioate group.

8. The dsRNA claim 1, wherein the dsRNA comprises at least one modified nucleotide, wherein the modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a 5′-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, and a non-natural base comprising nucleotide.

9. A cell comprising the dsRNA of claim 1.

10. A pharmaceutical composition comprising the dsRNA of claim 1 and a pharmaceutically acceptable carrier.

11. A composition comprising the dsRNA of claim 1 and a lipid formulation.

12. A composition comprising the dsRNA of claim 1 and a lipid formulation, wherein the lipid formulation comprises a cationic lipid comprising ND-98.

13. A vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of the dsRNA of claim 1.

14. A cell comprising the vector of claim 13.

15. The dsRNA of claim 1, wherein contacting a cell in vitro with 30 nM or less of the dsRNA and maintaining the cell for a time sufficient to obtain degradation of a mRNA transcript of a PCSK9 gene, inhibits expression of the PCSK9 gene in the cell.

16. The dsRNA of claim 1, wherein contacting HepG2 cells expressing the PCSK9 gene in vitro with the dsRNA and maintaining the cells for a time sufficient to obtain degradation of a mRNA transcript of a PCSK9 gene, inhibits expression of the PCSK9 gene in the cell by at least 20%.

17. The dsRNA of claim 1, wherein administering the dsRNA to an animal decreases total serum cholesterol in the animal.

18. A method for inhibiting expression of a proprotein convertase subtilisin kexin 9 (PCSK9) gene in a cell comprising contacting the cell with the dsRNA of claim 1 and maintaining the cell for a time sufficient to obtain degradation of a mRNA transcript of a PCSK9 gene, thereby inhibiting expression of the PCSK9 gene in the cell.

19. A method of treating or managing pathological processes which can be mediated by down regulating expression of a proprotein convertase subtilisin kexin 9 (PCSK9) gene comprising administering to a patient in need of such treatment or management a therapeutically effective amount of the dsRNA of claim 1.

20. A method of treating a proprotein convertase subtilisin kexin 9 (PCSK9) gene-associated disorder comprising administering to a patient in need of such treatment, a therapeutically effective amount of the dsRNA of claim 1.

21. A cell comprising the dsRNA of claim 4.

22. A pharmaceutical composition comprising the dsRNA of claim 4 and a pharmaceutically acceptable carrier.

23. A composition comprising the dsRNA of claim 4 and a lipid formulation.

24. A composition comprising the dsRNA of claim 4 and a lipid formulation, wherein the lipid formulation comprises a cationic lipid comprising ND-98.

25. A method for inhibiting expression of a proprotein convertase subtilisin kexin 9 (PCSK9) gene in a cell comprising contacting the cell with the dsRNA of claim 4 and maintaining the cell for a time sufficient to obtain degradation of a mRNA transcript of a PCSK9 gene, thereby inhibiting expression of the PCSK9 gene in the cell.

26. A method of treating or managing pathological processes which can be mediated by down regulating expression of a proprotein convertase subtilisin kexin 9 (PCSK9) gene comprising administering to a patient in need of such treatment or management a therapeutically effective amount of the dsRNA of claim 4.

27. A method of treating a proprotein convertase subtilisin kexin 9 (PCSK9) gene-associated disorder comprising administering to a patient in need of such treatment, a therapeutically effective amount of the dsRNA of claim 4.