Compositions and methods for inhibiting expression of the PCSK9 gene

专利摘要:
The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human PCSK9 gene in a cell, wherein said dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding PCSK9, and wherein said region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said PCSK9, inhibits expression of said PCSK9 gene, wherein (a) said first sequence is the sequence of SEQ ID NO: 1229 and said second sequence is the sequence of SEQ ID NO: 1230; (b) said first sequence is the sequence of SEQ ID NO: 1227 and said second sequence is the sequence of SEQ ID NO: 1228; or (c) said part of said mRNA encoding PCSK9 is the sequence from position 3530 to 3548 of the mRNA sequence of database entry NM_174936.
公开号:EP2584048A1
申请号:EP12178667.7
申请日:2007-05-10
公开日:2013-04-24
发明作者:Pamela Tan；Birgit Bramlage；Maria Frank-Kamenetsky；Kevin Fitzgerald；Akin Akinc；Victor E. Kotelianski
申请人:Alnylam Pharmaceuticals Inc；
IPC主号:C12N15-00

专利说明:
[0001] This application claims priority to U.S. Provisional Application No. 60/799,458, filed May 11, 2006 ; U.S. Provisional Application No. 60/817,203, filed June 27, 2006 ; U.S. Provisional Application No. 60/840,089, filed August 25, 2006 ; U.S. Provisional Application No. 60/829,914, filed October 18, 2006 ; and U.S. Provisional Application No. 60/901,134, filed February 13, 2007 . The contents of all of these provisional applications are hereby incorporated by reference in their entirety. Field of the Invention
[0002] 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
[0003] 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).
[0004] 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.
[0005] 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
[0006] 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 PGSK9 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.
[0007] 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.
[0008] 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
[0009] 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.
[0010] 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.
[0011] 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%.
[0012] 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 said 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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
[0019] 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. 6 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
[0020] 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.
[0021] 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).
[0022] 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.
[0023] 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.
[0024] 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
[0025] 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.
[0026] "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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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 preferred 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.
[0032] "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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] "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.
[0040] 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 mRNA in control cells - mRNA in treated cellsmRNA in control cells • 100 %
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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)
[0048] 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 said dsRNA, upon contact with a cell expressing said PCSK9 gene, inhibits the expression of said 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.
[0049] 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
[0050] 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.
[0051] 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 RNA agent is said 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.
[0052] 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.
[0053] 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.
[0054] 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, NY, 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.
[0055] 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)eystamine: 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.
[0056] 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).
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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, CA). 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.
[0061] Teachings regarding the synthesis of particular modified oligonucleotides may be found in the following U.S. patents: 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. 6,162,241 , and 5,459,155 , drawn to, inter alia, methods of synthesizing 2'-fluoro-oligonucleotides.
[0062] 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.
[0063] 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.
[0064] 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 .
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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 hexylaminocarbonyl-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
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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
[0079] 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, sucxh 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.
[0080] 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.
[0081] 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 arc well known in the art.
[0082] 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.
[0083] 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.
[0084] 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).
[0085] 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).
[0086] 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.RTM. (Invitrogen/Life Technologies, Carlsbad, Calif.) and Effectene.TM. (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.
[0087] 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.
[0088] 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).
[0089] 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).
[0090] Methods for treating diseases that can be modulated by down regulating the expression of PCSK9
[0091] 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
[0092] 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.
[0093] 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.
[0094] 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
[0095] 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.
[0096] 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.
[0097] 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 siRNA crossreactive to human and mouse, and siRNAs crossreactive to human and rat targets
[0098] 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 Figures 2-8.
[0099] 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.
[0100] 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 I 1 (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
[0101] To identify potential off-target genes, 19mer candidate sequences were subjected to a homology search against publically available human mRNA sequences.
[0102] 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
[0103] The off-target score was calculated for considering assumption 1 to 3 as follows: Off - targer score = number of seed mismatches * 10 + number of cleavage site mismatches * 1.2 + number of non - seed mismatches * 1
[0104] 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
[0105] 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
[0106] 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, 500Å, 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, NY, 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).
[0107] 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, Unterschleißheim, 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.
[0108] 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
[0109]
[0110] 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 x 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
[0111]
[0112] 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
[0113]
[0114] 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
[0115]
[0116] The hydrochloride salt of 3-[(6-Amino-hexanoyl)-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
[0117]
[0118] 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
[0119]
[0120] 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 x 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
[0121]
[0122] Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2 x 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 2,3,4,7,8,9,10,11,12,13,14,15,16,7-tetradecahydro-1H cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester AH
[0123]
[0124] 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 X 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
[0125]
[0126] 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 acetontrile/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).
[0127] 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.
[0128] Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 1-2. Table 1-2: Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5'-3'-phosphodiester bonds. Abbreviation^a Nucleotide(s) A, a 2'-deoxy-adenosine-5'-phosphate, adenosine-5'-phosphate C, c 2'-deoxy-cytidine-5'-phosphate, cytidine-5'-phosphate G, g 2'-deoxy-guanosine-5'-phosphate, guanosine-5'-phosphate T, t 2'-deoxy-thymidine-5'-phosphate, thymidine-5'-phosphate U, u 2'-deoxy-uridine-5'-phosphate, uridine-5'-phosphate N, n any 2'-deoxy-nucleotide/nucleotide (G, A, C, or T, g, a, c or u) Am 2'-O-methyladenosine-5'-phosphate Cm 2'-O-methylcytidine-5'-phosphate Gm 2'-O-methylguanosine-5'-phosphate Tm 2 '-O-methyl-thymidine-5'-phosphate Um 2'-O-methyluridine-5'-phosphate Af 2'-fluoro-2'-deoxy-adenosine-5'-phosphate Cf 2'-fluoro-2'-deoxy-cytidine-5'-phosphate Gf 2'-fluoro-2'-deoxy-guanosine-5'-phosphate Tf 2'-fluoro-2'-deoxy-thymidine-5'-phosphate Uf 2'-fluoro-2'-deoxy-uridine-5'-phosphateA, C, G, T, U, a, c, g, t, u underlined: nucleoside-5'-phosphorothioateam, cm, gm, tm, um underlined: 2-6'-methyl-nucleoside-5'-phosphorothioate ^acapital letters represent 2'-deoxyribonucleotides (DNA), lower case letters represent ribonucleotides (RNA) PCSK9 siRNA screening in HuH7, HepG2, Hela and Primary Monkey Hepatocytes Discovers Highly Active Sequences
[0129] HuH-7cells 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 2mM 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), 1x Non Essential Amino Acids (Biochrom AG, Berlin, Germany, cat. No. K-0293), and 1mM 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).
[0130] For transfection with siRNA, HuH7, HepG2, or Hela cells were seeded at a density of 2.0 x 104 cells/well in 96-well plates and transfected directly. Transfection of siRNA (30nM for single dose screen) was carried out with lipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany, cat. No. 11668-019) as described by the manufacturer.
[0131] 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.
[0132] Primary cynomolgus monkey hepatocytes (cryopreserved) were obtained from In vitro Technologies, Inc. (Baltimore, Maryland, USA, cat No M00305) and cultured in In VitroGRO CP Medium (cat No Z99029) at 37°C in an atmosphere with 5% CO₂ in a humidified incubator.
[0133] 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 x 10⁴ cells/well in 96-well plates and transfected directly. Transfection of siRNA (eight 2-fold dilution series starting from 30nM in duplicates was carried out with lipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany, cat. No. 11668-019) as described by the manufacturer.
[0134] 16 hours after transfection medium was changed to fresh InVitroGRO CP Medium with Torpedo Antibiotic Mix (In vitro Technologies, Inc, cat. No Z99000) added.
[0135] 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.
[0136] Tables 1-2 (and Figure 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 100nm. Very active sequences are those that have less than 60% of transcript remaining after treatment with a dose.less than or equal to 100nM. Active sequences are those that have less than 85% transcript remaining after treatment with a high dose (100nM). 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 Figure 6 example). In vivo Efficacy Screen of PCSK9 siRNAs Formulation Procedure
[0137] The lipidoid LNP-01·4HCl (MW 1487) (Figure 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
[0138] 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
[0139] 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 27G needle. SiRNAs were formulated in LNP-01 (and then dialyzed against PBS) at 0.5 mg/ml concentration allowing the delivery of the 5mg/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.
[0140] 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.
[0141] Frozen livers were grinded using 6850 Freezer/Mill Cryogenic Grinder (SPEX CentriPrep, Inc) and powders stored at -80°C until analysis.
[0142] PCSK9 mRNA levels were detected using the branched-DNA technology based kit from QuantiGene Reagent System (Genospectra) according to the protocol. 10-20mg 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 3hours. Then 10 ul of the lysates were added to 90ul 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, CA, 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).
[0143] Total serum cholesterol in mouse serum was measured using the StanBio Cholesterol LiquiColor kit (StanBio Laboratoriy, Boerne, Texas, USA) according to manufacturer's instructions. Measurements were taken on a Victor2 1420 Multilabel Counter (Perkin Elmer) at 495 nm. Examples
[0144] 32 PCSK9 siRNAs formulated in LNP-01 liposomes were tested in vivo in a mouse model. The experiment was performed at 5mg/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 (Figures 2-5). Silencing of PCSK9 transcript also coorelated with a lowering of cholesterol in these animals (Figures 4-5). In addition there was a strong coorelation between those molecules that were active in vitro and those active in vivo (Figure 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. Figure 7 (also see Tables 1 and2) shows that the parent molecules 9314 and 9318 and the modified versions are all active in vitro. Figure 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. Figure 9 furthur exemplifies that activity of other chemically modified versions of the parents 9314 and 10792. dsRNA expression vectors
[0145] 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, US 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).
[0146] 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.
[0147] 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. NatI.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116 ; U.S. Patent 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. NatI. 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.
[0148] 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 U 1 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)).
[0149] 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.
[0150] 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.
[0151] 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-TKO™). 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.
[0152] 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. Patent 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.
[0153] 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.position in human access. # NM_17493 6 Sense strand sequence (5'-3')¹ SEQ ID NO: Antisense-strand sequence (5'-3')¹ SEQ ID NO: Duplex name Mean percent remaining mRNA transcript at siRNA concentration/in cell type IC50 in HepG2 [nM] IC50 in Cynomogous monkey Hepatoeyte [nM]s 100 nM/HepG 2 30 nM/ HepG 2 3nM/HepG 2 30 nM/Hel a 2-20 AGCGACGUCGAGGCGCUCATT 1 UGAGCGCCUCGACGUCGCGCUTT 2 AD-15220 35 15-33 CGCUCAUGUUUGCAGGCGGTT 3 CCGCCUGCAACCAUGAGCGTT 4 AD-15275 56 16-34 GCUCAUGGUUGCACCCCGGTT 5 CCCGCCUCCAACCAUCAGCTT 6 AD-15301 70 30-48 GCGGGCGCCGCCGUUCAGUTT 7 ACUGAACGGCGGCGCCCGCTT 8 AD-15276 42 31-49 CGGGCGCCGCCGUUCAGUUTT 9 AACUCAACGGCGGCCCCCGTT 10 AD-15302 32 32-50 GGGCGCCGCCGUUCAGUUCTT 11 GAACUGAACGGCGGCGCCCTT 12 AD-15303 37 40-58 CCGUUCAGUUCAGGGUCUGTT 13 CAGACCCUGAACUGAACGGTT 14 AD-15221 30 43-61 UUCAGUUCAGGGUCUGAGCTT 15 GCUCAGACCCUGAACUGAATT 16 AD-15413 61 82-100 GUGAGACUGGCUCGGGCGGTT 17 CCGCCCGAGCCAUUCUCACTT 18 AD-15304 70 100-118 GGCCGGGACCCGUCGUUGCTT 19 GCAACGACGCGUCCCGGCCTT 20 AD-15305 36 101-119 GCCGGGACGCGUCGUUGCATT 21 UGCAACGACGCGUCCCGGCTT 22 AD-15306 20 102-120 CCGGGACGCGUCGUUGCAGTT 23 CUGCAACGACGCGUCCCGGTT 24 AD-15307 38 105-123 GGACGCGUCGUUGCAGCAGTT 25 CUGCUGCAACGACGCUUCCTT 26 AD-15277 50 135-153 UCCCAGCCAGGAUUCCGCGTsT 27 CGCGGAAUCCUGGCUGGGATsT 28 AD-9526 74 89 135-153 ucccAGccAGCAuuccGcGTsT 29 CGCGGAAUCCUGGCUGGGATsT 30 AD-9652 97 136-154 CCCAGCCAGGAUUCCGCGCTsT 31 GCGCGGAA UCCUGGCUGGGTsT 32 AD-9519 78 136-154 cccAGccAGGAuuccGcGcTsT 33 GCGCGGAAUCCUGGCUGGGTsT 34 AD-9645 66 138-156 CAGCCAGGAUUCCGCGCGCTsT 35 GCGCGCGGAAUCCUGGCUTsT 36 AD-9523 55 138-156 cAGccAGGAuuccGcGcGcTsT 37 GCGCGCGGAAUCCUGCCUGTsT 38 AD-9649 60 185-20:3 AGCUCCUGCACAGUCCUCCTsT 39 GGAGGACUGUGCAGGAGCUTsT 40 AD-9569 112 185-203 AGcuccuGcAcAGuccuccTsT 41 GGAGGACUGUGcAGGAGCUTsT 42 AD-9695 102 205-223 CACCGCAAGGCUCAAGCCGTT 43 CGCCUUGAGCCUUGCGGUGTT 44 AD-15222 75 208-226 CGCAAGGCUCAAGGCGCCGTT 45 CGGCGCCUUGAGCCUUGCGTT 46 AD-15278 78 210-228 CAAGGCUCAAGGCGCCGCCTT 47 GGCGGCGCCUUGAGCCUUGTT 48 AD-15178 83 232-250 GUGGACCGCGCACGGCCUCTT 49 GAGGCCGUGCGCGGUCCACTT 50 AD-15308 84 233-251 UGGACCGCGCACGGCCUCUTT 51 AGAGGCCGUGCGCGGUCCATT 52 AD-15223 67 234-252 GGACCGCGCACGGCUCUATT 53 UAGAGGCCGUGCGCGGUCCTT 54 AD-15309 34 235-253 GACCGCGCACGGCCUCUAGTT 55 CUAGAGGCCGUGCGCGGUCTT 56 AD-15279 44 236-254 ACCGCGCACGGCCUCUAGGTT 57 CCUAGAGGCCGUCCGCCGUTT 58 AD-15194 63 237-255 CCGCGCACGGCCUCUAGGUTT 59 ACCUAGAGGCCGUGCGCGGTT 60 AD-15310 42 238-256 CGCCCACGCCCUCUAGGUCTT 61 GACCUAGAGGCCGUGCGCGTT 62 AD-15311 30 239-257 GCGCACGGCCUCUAGGUCUTT 63 AGACCUAGAGGCCGUGCCGCTT 64 AD-15392 18 240-258 CGCACGGCCUCUAGGUCUCTT 65 GAGACCUAGAGCCCGUGCGTT 66 AD-15312 21 248-266 CUCUAGGUCUCCUCGCCAGTT 67 CUGCCCAGGACACCUAGAGTT 68 AD-15313 19 249-267 UCUAGGUCUCCUCOCCAGGTT 69 CCUGGCGAGGAGACCUAGATT 70 AD-15280 81 250-268 CUAGGUCUCCUCGCCAGGATT 71 UCCUGGCGAGGAGACCUAGTT 72 AD-15267 82 252-270 AGGUCUCCUCGCCAGGACATT 73 UGUCCUGGCGAGGAGACCUTT 74 AD-15314 32 258-276 CCUCGCCAGGACAGCAACCTT 75 GGUUGCUGUCCUGGCGAGGTT 76 AD-15315 74 300-318 CGUCAGCUCCAGGCGGUCCTsT 77 GGACCGCCUGGAGCUGACGTsT 78 AD-9624 94 300-318 cGucAGcuccAGGcGGuccTsT 79 GGACCGCCUGGAGCUGACGTsT 80 AD-9750 96 301-319 GUCAGCUCCAGGCGGUCCUTsT 81 AGGACCGCCUGGAGCUGACTsT 82 AD-9623 43 66 301-319 GucAGcuccAGGcGGuccuTsT 83 AGGACCGCCUGGAGCUGACTsT 84 AD-9749 105 370-388 GGCGCCCGUGCGCAGGAGGTT 85 CCUCCUUCCCACGGGCGCCTT 86 AD-15384 48 408-426 GGAGCUGGUGCUAGCCUUGTsT 87 CAAGGCUAGCACCAGCUCCTsT 88 AD-9607 32 28 0,20 408-426 GGAGcuGGuGcuAGccuuGTsT 89 cAAGGCuAGcACcAGCUCCTsT 90 AD-9733 78 73 411-429 GCUGGUGCUAGCCUUGCGUTsT 91 ACGCAAGGCUACCACCAGCTsT 92 AD-9524 23 28 0,07 411-429 GcuGGuGcuAGccuuGcGuTsT 93 ACGcAAGGCuAGcAGCTsT 94 AD-9650 91 90 412-430 CUGGUGCUAGCCUUGCGUUTsT 95 AACGCAAGGCUAGCACCAGTsT 96 AD-9520 23 32 412-430 CUGGUGCUAGCCUUGCGUUTsT 97 AACGCAAGGCUAGCACCAGTsT 98 AD-9520 23 412-430 cuGGuGcuAGccuuGcGuuTsT 99 AACGcAAGGCuAGcACcACTsT 100 AD-9646 97 108 416-434 UGCUAGCCUUGCGUUCCGATsT 101 UCGGAACGCAAGGCUAGCATsT 102 AD-9608 37 416-434 uGcuAGccuuGcGuuccGATsT 103 UCGGAACGcAAGGCuAGcATsT 104 AD-9734 91 419-437 UAGCCUUGCGUUCCGAGGATsT 105 UCCUCGGAACGCAAGGCUATsT 106 AD-9546 32 419-437 uAGccuuGcGuuccGAGGATsT 107 UCCUCGCAACGcAAGGCuATsT 108 AD-9672 57 439-457 GACGGCCUGGCCGAAGCACTT 109 GUGCUUCGGCCAGGCCGUCTT 110 AD-15385 54 447-465 GGCCGAAGCACCCGAGCACTT 111 GUGCUCGGGUGCUUCGGCCTT 112 AD-15393 31 448-466 GCCGAAGCACCCGAGCACGTT 113 CGUGCUCGGGUGCUUCGGCTT 114 AD-15316 37 449-467 CCGAAGCACCCGAGCACGGTT 115 CCGUGCUCGGGUGCUUCGGTT 116 AD-15317 37 459-476 CCGAGCACGGAACCACAGCTT 117 GCUGUGGUUCCGUGCUCCCTT 118 AD-15318 63 484-502 CACCGCUGCGCCAAGGAUUCTT 119 GAUCCUUGGCGCAGCGGUGTT 120 AD-15195 45 480-504 CCGCUGCGCCAAGGAUCCGTT 121 CGGAUCCUUGGCGCAGCGGTT 122 AD-15224 57 487-505 CGCUGCGCCAAGGAUCCGUTT 123 ACGGAUCCUUGGCGCAGCGTT 124 AD-15188 42 489-507 CUGCGCCAAGGAUCCGUGGT T 125 CCACGGAUCCUUGGCGCAGTT 126 AD-15225 51 500-518 AUCCGUGGAGGUUGCCUGGTT 127 CCAGGCAACCUCCACGGAUTT 128 AD-15281 89 5119-527 GGUUGCCUGGCACCUACCUTT 129 ACGUAGGUGCCAGGCAACCTT 130 AD-15282 75 542-560 AGGAGACCCACCUCUCGCATT 131 UGCGAGAGGUGGGUCUCCUTT 132 AD-15319 61 543-561 GGAGACCCACCUCUCGCAGTT 133 CUGCGAGAGGUGCGUCUCCTT 134 AD-15226 56 544-562 GAGACCCACCUCUCGCAGUTT 135 ACUGCGAGAGGUGGGUCUCTT 136 AD-15271 25 549-567 CCACCUCUCGCAGUCAGAGTT 137 CUCUGACUGCGAGAGGUGGTT 138 AD-15283 25 552-570 CCUCUCGCAGUCAGAGCGCTT 139 GCGCUCUGACUGCGAGAGGTT 140 AD-15284 64 553-571 CUCUCGCAGUCAGAGCGCATT 141 UGCGCUCUGACUGCGAGAGTT 142 AD-15189 17 554-572 UCUCGCAGUCAGAGCUCACTT 143 CUGCGCUCUGACUGCGAGATT 144 AD-15227 62 555-573 CUCGCAGUCAGACCGCACUTsT 145 ACUGCGCUCUGACUGCGACTsT 146 AD-9547 31 29 0,20 555-573 cucGcuAGucAGAGcGcAcuTsT 147 AGUGCGCUCUGACUGCGAGTsT 148 AD-9673 56 57 558-576 GCAGUCAGAGCGCACUGCCTsT 149 GGCAGUGCGCUCUGACUGCTsT 150 AD-9548 54 60 558-576 GcAGucAGAGcGcAcuGccTsT 151 GGcAGUGCGCUCUGACUGCTsT 152 AD-9774 36 57 606-624 GGGAUACCUCACCAAGAUCTsT 153 GAUCUUGGUGAGGUAUCCCTsT 154 AD-9529 60 606-624 GGGAuAccucAccAAGAucTsT 155 GAUCUUGGUGAGGuAUCCCTsT 156 AD-9655 140 659-677 UGGUGAAGAUGAGUGGCGATsT 157 UCGCCACUCAUCUUCACCATsT 158 AD-9605 27 31 0,27 659-677 uGGuGAAGAuGAGuGGcATsT 159 UCGCcACUuAUCUUcACcATsT 160 AD-9731 31 31 0,32 663-681 GAAGAUGAGUGGCGACCUGTsT 161 CAGUUCGCCACUCAUCUUCTsT 162 AD-9596 37 663-681 GAAGAuGAGuGGcGAccuGTsT 163 cAGGUCGCcACUcAUCUUCTsT 164 AD-9722 76 704-722 CCCAUGUCGACUACAUCGATsT 165 UCGAUGUAGUCGACAUGGCTsT 166 AD-9583 42 704-722 cccAuGucGAcuAcAucGATsT 167 UCGAUGuAGUCGAcAUGGGTsT 168 AD-9709 104 718-736 AUCGAGGAGGACUCCUCUGTsT 169 CAGAGGAGUCCUCCUCGAUTsT 170 AD-9579 113 718-736 AucGAGGAGGAcuccucuGTsT 171 cAGAGGAGUCCUCCUCGAUTsT 172 AD-9705 81 758-776 GGAACCUGGAGCGGAUUACTT 173 CUAAUCCGCUCCACGUUCCTT 174 AD-15394 32 759-777 GAACCUGGAGCGGAUUACCTT 175 GGUAAUCCGCUCCAGGUUCTT 176 AD-15196 72 760-778 AACCUGGAGCGGAUUACCCTT 177 GGGUAAUCCGCUCCAGGUUTT 178 AD-15197 85 777-795 CCCUCCACGGUACCGGGCGTT 179 CGCCCGGUACCGUGGAGGGTT 180 AD-15198 71 782-800 CACGGUACCGGGCGGAUGATsT 181 UCAUCCGCCCGGUACCGUGTsT 182 AD-9609 66 71 782-800 cAcGGuAccGGGcGGAuCATsT 183 UcAUCCGCCCGGuACCGUGTsT 184 AD-9735 115 783-801 ACGGUACCGGGCGGAUGAATsT 185 UUCAUCCGCCCGGUACCGUTsT 186 AD-9537 145 793-801 AcGGuAccGGGcGGAuCAATsT 187 UUcAUCCGCCCGGuACCGUTsT 188 AD-9663 102 784-802 CGGUACCGGGCGGAUGAAUTsT 189 AUUCAUCCGCCCGGUACCGTsT 190 AD-9528 113 784 -802 cGGuAccGGGcGGAuGAAuTsT 191 AUUcAUCCGCCCGGuACCGTsT 192 AD-9654 107 785-803 GGUACCGGGCGGAUGAAUATsT 193 UAUUCAUCCGCCCGGUACCTsT 194 AD-9515 49 785-803 GGuAccGGGcGGAuGAAuATsT 195 uAUUcAUCCGCCCGGuACCTsT 196 AD-9641 92 786-804 GUACCGGGCGGAUCAAUACTsT 197 GUAUUCAUCCGCCCGGUACTsT 198 AD-9514 57 786-804 GuAccGGGcGGAuGAAuAcTsT 199 GuAUUcAUCCGCCCGGuACTsT 200 AD-9640 89 788-806 ACCGGGCGGAUGAAUACCATsT 201 UGGUAUUCAUCCGCCCGGUTsT 202 AD-9530 75 788-806 AccGGGcGGAuGAAuAccATsT 203 UGGuAUUcAUCCGCCCGGUTsT 204 AD-9656 77 789-807 CCGGGCGGAUCAAUACCAGTsT 205 CUGGUAUUCAUCCGCCCGGTsT 206 AD-9538 79 80 789-807 ccGGGcGGAuGAAuAccAGTsT 207 CUGGuAUUcAUCCGCCCGGTsT 208 AD-9664 53 825-843 CCUGGUGGAGGUGUAUCUCTsT 209 GAGAUACACCUCCACCAGGTsT 210 AD-9598 69 83 825-843 ccuGGuGGAGGuGuAucucTsT 211 GAGAuAcACCUCcACcAGGTsT 212 AD-9724 127 826-844 CUGGUGGAUGGUGUAUCUCCTsT 213 GGACAUACACCUCCACCAGTsT 214 AD-9625 58 88 826-844 cuGGuGGAGGuGAcuccTsT 215 GGAGAuAcACCUCcACcAGTsT 216 AD-9751 60 827-845 UGGUGGAGGUGUAUCUCCUTsT 217 AGGAGAUACACCUCCACCATsT 218 AD-9556 46 827-845 uGGuGGAGGuGuAucuccuTsT 219 AGGAGAuAcACCUCcACcATsT 220 AD-9682 38 828-846 GGUGGAGGUGUAUCUCCUATsT 221 UAGGAGAUACACCUCCACCTsT 222 AD-9539 56 63 828-846 GGuGGAGGuGuAucuccuATsT 223 uAGGAGAuAcACCUCcACCTsT 224 AD-9665 83 831-849 GGAGGUGUAUCUCCUAGACTsT 225 GUCUAGGAGAUACACCUCCTsT 226 AD-9517 36 831-849 GGAGGuGuAucuccuAGAcTsT 227 GUCuAGCAGAuAcACCUCCTsT 228 AD-9643 40 833-851 AGGUGUAUCUCCUAGACACTsT 229 GUGUCUAGGAGAUACACCUTsT 230 AD-9610 36 34 0,04 833-851 AGGuGuAucuccuAGAcAcTsT 231 GUGUCuAGGAGAuAcACCUTsT 232 AD-9736 22 29 0,04 833-851 AfgGfuGfuAfuCfuCfcUfaGfaCfTsT 233 p-gUfgUfcUfaGfgfgAfuAfcAfcCfuTsT 234 AD-14681 33 833-851 AGGUfGUfAUfCfUfCfCfUfAGACfACfTsT 235 GUfGUfCfUfAGGAGAUfACfACfCfUfTsT 236 AD-14691 27 833-851 AgGuGuAuCuCcUaGaCaCTsT 237 p-gUfgUfcUfaGfgAfgAfuAfcAfcCfuTsT 238 AD-14701 32 AgGuGuAuCuCcUaGaCaCTsT 239 GUfGUfCfUfAGGAGAUfACfACfCfUfTsT 240 AD-14711 33 833-851 AfgGfuGfuAfuCfuCfcUfaGfaCfaCfTsT 241 GUGUCuaGGagAUACAccuTsT 242 AD-14721 22 833-851 AGGUfGUfAUfCfUfCfCfUfAGACfACfTsT 243 GUGUCuaGGagAUACAccuTsT 244 AD-14731 21 833-851 AgGuGuAuCuCcUaGaCaCTsT 245 GUGUCuaGGagAUACAccuTsT 246 AD-14741 22 833-851 GfcAfcCfcUfcAfuAfgGfcCfuGfgAfTsT 247 p-uCfcAfgGfcCfuAfuGfaGfgGfuGfcTsT 248 AD-15087 37 833-851 GCfACfCfCfUfCfAUfAGGCfCfUfGGATsT 249 UfCfCfAGGCfCfUfAUfGAGGGUfGCfTsT 250 AD-15097 51 833-851 GcAcCcUcAuAgGcCuGgATsT 251 p-uCfcAfgGfcCfuAfuGfaGfgGfuGfcTsT 252 AD-15107 26 833-851 GcAcCcUcAuAgGcCuGgATsT 253 UfCfCfAGGCfCfUfAUfGAGGGUfGCfTsT 254 AD-15117 28 833-851 GfcAfcCfcUfcAfuAfgGfcCfuGfgAfTsT 255 UCCAGgcCUauGAGGGugcTsT 256 AD-15127 33 833-851 GCfACfCfCfUfCfAUfAGGCfCfUfGGATsT 257 UCCAGgcCUauGAGGGugcTsT 258 AD-15137 54 833-851 GcAcCcUcAuAgGcCuCgATsT 259 UCCAGgcCUauGAGGGugcTsT 260 AD-15147 52 836-854 UCUAUCUCCtJAUACACCAGTsT 261 CUUGUCUCUAGCAGAUACATsT 262 AD-9516 94 836-854 uGuAucuccuAGAcAccAGTsT 263 CUGGUGUCuAGGAGAuAcA TsT 264 AD-9642 105 840-858 UCUCCUAGACACCAGCAUATsT 265 UAUGCUGGUGUCUAGGAGATsT 266 AD-9562 46 51 840-858 ucuccuAGAcAccACcAuATsT 267 uAUGCUGGUGUCuAGGAGATsT 268 AD-9688 26 34 4,20 840-858 UfcUfcCfuAfgAfcAfcCfaGfcAfuAfTsT 269 p-uAfuGfcUfgGfuGfuCfuAfgGfaGfaTsT 270 AD-14677 38 840-858 UfCfUfCfCfUfAGACfACfCfAGCfAUfATsT 271 UfAUfGCfUfGGUfGUfCfUfAGGAGATsT 272 AD-14687 52 840-858 UcUcCuAsAeAcCaGcAuATsT 273 p-uAfuGfcUfgGfuGfuCfuAfgGfaGfaTsT 274 AD-14697 35 840-858 UcUcCuAgAcAcCaGcAuATsT 275 UfAUfGCfUGGUfGUfGUfUfUfAGGAGATsT 276 AD-14707 58 840-858 UfcUfcCfuAafAfcAfcCfaUfcAfuAfTsT 277 UAUGCugGUguCUAGGagaTsT 278 AD-14717 42 840-858 UfCfUfCfCfUfAGACfACfCfAGCfAUfATsT 279 UAUGCugGUguCUAGGagaTsT 280 AD-14727 50 840-858 UcUcCuAgAcAcCaGcAuATsT 281 UAUGCugCUguCUAGGagaTsT 282 AD-14737 32 840-858 AfgGfcCfuGfgAfgUfuUfaCfgGITsT 283 p-cCfgAfaUfaAfaCfuCfcAfgGfcCfuTsT 284 AD-15083 16 840-858 AGGCfCfUfGGAGUfUfUfAUfUfCfGGTsT 285 CfCfGAAUfAAACfUfCfCfAGGCfCfUfTsT 286 AD-15093 24 840-858 AgGcCuGgAgUuUaUuCgGTsT 287 p-cCfgAfaUfaAfaCfuCfcAfgGfcCfuTsT 288 AD-15103 11 840-858 AgGcCuGgAgUuUaUuCgGTsT 289 CfCfGAAUfAAACfUfCfTAGGCfCfUfTsT 290 AD-15113 34 840-858 AfgGfcCfuGfgAfgUfuUfaUfuCfgGfTsT 291 CCGAAuaAAcuCCAGGccuTsT 292 AD-15123 19 840-858 AGGCfCfUfGGAGUfUfUfAUfUfCfGGTsT 293 CCGAAuaAAcuCCAGGccuTsT 294 AD-15133 15 840-858 AgGcCuGgAgUuUaUuCgGTsT 295 CCCAAuaAAcuCCAGGccuTsT 296 AD-15143 16 841-859 CUCCUAGACACCAGCAUACTsT 297 GUAUGCUGGUGUCUAGGAGTsT 298 AD-9521 50 841-859 cuccuACAcAccAGcAuAcTsT 299 GuAUGCUGGUGUCuAGGAGTsT 300 AD-9647 62 842-860 UCCUACAGACCAGCAUACATsT 301 UGUAUGCUGGUGUCUAGGATsT 302 AD-9611 48 842-860 uccuAGAcAccAGcAuAcATsT 303 UGuAUGCUGGUGUCuAGGATsT 304 AD-9737 68 843-861 CCUAGACACCAGCAUACAUTsT 305 UGUAUGCUGGUGUCUAGGTsT 306 AD-9592 46 55 843-861 ccuAGAcAccAGcAuAcAGTsT 307 CUGuAUGCUGGUGUCuAGGTsT 308 AD-9718 78 847-865 GACACCAGCAUACAUAGUGTsT 309 CACUCUGUAUGCUGGUGUCTsT 310 AD-9561 64 847-865 GAcAccAGcAuAcAGAGuGTsT 311 cACUCUGuAUCCUUGUCUCTsT 312 AD-9687 84 855-873 CAUACAGAGUGACCACCGGTsT 313 CCGGUGGUCACUCUGUAUGTsT 314 AD-9636 42 41 2,10 855-873 cAuAcACAGuUAccAccGGTsT 315 CCGGUGGUcACUCUCuAUCTsT 316 AD-9762 9 28 0,40 860-878 AGAGUGACCACCGGGAAAUTsT 317 AUUUCCCGGUGGUCACUCUTsT 318 AD-9540 45 860-878 AGAGuGAccAccGGGAAAuTsT 319 AUUUCCCUGUGGUcACUCUTsT 320 AD-9666 81 861-879 GAGUGACCACCGGGAAAUCTsT 321 GAUUUCCCGGUGGUCACUCTsT 322 AD-9535 48 73 861-879 GAGuGAccAccGGGAAAucTsT 323 GAUUUCCCGGUGGUcACUCTsT 324 AD-9661 83 863-881 GUGACCACCGGGAAA UCGATsT 325 UCUAUUUCCCGGUGGUCACTsT 326 AD-9559 35 863-881 GuGAccAccGGGAAAucGATsT 327 UCGAUUUCCCGGUGGUcACTsT 328 AD-9685 77 865-883 GACCACCGGGAAAUCGAGGTsT 329 CCUCGAUUUCCCOGUGGUCTsT 330 AD-9533 100 865-883 GAccAccGGGAAAucGAGGTsT 331 CCUCGAUUUCCCGGUGGUCTsT 332 AD-9659 88 866.884 ACCACCGGGAAAUCGAGGGTsT 333 CCCUCGAUUUCCCGGUGGUTsT 334 AD-9612 122 866-884 AccAccGGGAAAucGAGGGTsT 335 CCCUCGAUUUCCCGGUGGUTsT 336 AD-9738 83 867-885 CCACCGGGAAAUCGAGGGCTsT 337 GCCCUCGAUUUCCCGGUGGTsT 338 AD-9557 75 96 867-885 ccAccGGGAAAucGAGGGcTsT 339 GCCCUCGAUUUCCCGGUGGTsT 340 AD-9683 48 875-893 AAAUCGAGGGCAGGGAUTsT 341 AUGACCCUGCCCUCGAUUUTsT 342 AD-9531 31 32 0,53 875-893 AAAucGAGGGcAGGGucAuTsT 343 AUGACCCUGCCCUCGAUUUTsT 344 AD-9657 23 29 0,66 875-893 AfaAfuCfgAfgGfgCfaGfgGfuCfaUfTsT 345 p-aUfgAfcCfcUfgCfcCfuCfgAfuUfuTsT 346 AD-14673 81 875-893 AAAUfCfGAGGGCfAGGGUfCfAUfTsT 347
[0154] The invention furthermore comprises the following items: 1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human PCSK9 gene in a cell, wherein said dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding PCSK9, and wherein said region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said PCSK9, inhibits expression of said PCSK9 gene. 2. The dsRNA of item 1, wherein said first sequence is selected from the group consisting of Tables 1 and 2 and said second sequence is selected from the group consisting of Tables 1 and 2. 3. The dsRNA of item 1, wherein said dsRNA comprises at least one modified nucleotide. 4. The dsRNA of item 2, wherein said dsRNA comprises at least one modified nucleotide. 5. The dsRNA of item 3, wherein said modified nucleotide is chosen from the group of: a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. 6. The dsRNA of item 3, wherein said modified nucleotide is chosen from the group of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. 7. The dsRNA of item 3, wherein said first sequence is selected from the group consisting of Tables 1 and 2 and said second sequence is selected from the group consisting of Tables 1 and 2 8. The dsRNA of item 6, wherein said first sequence is selected from the group consisting of Tables 1 and 2, and said second sequence is selected from the group consisting of Tables 1 and 2. 9. A cell comprising the dsRNA of item 1. 10. A pharmaceutical composition for inhibiting the expression of the PCSK9 gene in an organism, comprising a dsRNA and a pharmaceutically acceptable carrier, wherein the dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding PCSK9, and wherein said region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said PCSK9, inhibits expression of said PCSK9 gene. 11. The pharmaceutical composition of item 10, wherein said first sequence of said dsRNA is selected from the group consisting of Tables 1 and 2, and said second sequence of said dsRNA is selected from the group consisting of Tables 1 and 2. 12. The pharmaceutical composition of item 10, wherein said first sequence of said dsRNA is selected from the group consisting of Tables 1 and 2 and said second sequence of said dsRNA is selected from the group consisting of Tables 1 and 2. 13. A method for inhibiting the expression of the PCSK9 gene in a cell, the method comprising: (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 and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding PCSK9, and wherein said region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said PCSK9, inhibits expression of said PCSK9 gene; 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. 14. A method of treating, preventing or managing pathological processes which can be mediated by down regulating PCSK9 gene expression comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of a dsRNA, wherein the dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding PCSK9, and wherein said region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said PCSK9, inhibits expression of said PCSK9 gene. 15. A vector for inhibiting the expression of the PCSK9 gene in a cell, said vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of a dsRNA, wherein one of the strands of said dsRNA is substantially complementary to at least a part of a mRNA encoding PCSK9 and wherein said dsRNA is less than 30 base pairs in length and wherein said dsRNA, upon contact with a cell expressing said PCSK9, inhibits the expression of said PCSK9 gene. 16. A cell comprising the vector of item 15. 17. A double-stranded ribonucleic acid (dsRNA) for reducing the expression level of a human PCSK9 gene in a cell, wherein said dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding PCSK9, and wherein said dsRNA, upon contact with a cell expressing said PCSK9, reduces the expression level of said PCSK9 gene. 18. The dsRNA of item 17, wherein said contact reduces the expression level of said PCSK9 gene. 19. The dsRNA of item 17, wherein said contact is perfonned in vitro at 30 nM or less. 20. A pharmaceutical composition for reducing the expression level of the PCSK9 gene in an organism, comprising the dsRNA of item 17 and a pharmaceutically acceptable carrier, 21. A method of treating a PCSK9 associated disorder comprising administering to a patient in need of such treatment, a therapeutically effective amount of a dsRNA of item 17. 22. A method of treating a PCSK9-associated disorder comprising administering to a patient in need of such treatment, a therapeutically effective amount of a dsRNA of item 17.

权利要求:
Claims (16)
[0001] A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human PCSK9 gene in a cell, wherein said dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is fully complementary to at least a part of an mRNA encoding PCSK9, and wherein said region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said PCSK9, inhibits expression of said PCSK9 gene.
[0002] The dsRNA of claim 1, wherein said part of an mRNA encoding PCSK9 consists of the sequence from positions 1085 to 1103 of Accession No. NM_174936 which sequence is UCAUAGGCCUGGAGUUUAU.
[0003] The dsRNA of claim 1 or 2, wherein said dsRNA comprises at least one modified nucleotide.
[0004] The dsRNA of any one of claims 1 or 3, wherein said modified nucleotide is chosen from the group of a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
[0005] The dsRNA of claim 3 or 4, wherein said first sequence is the sequence of SEQ ID NO: 453 and said second sequence is the sequence of SEQ ID NO: 454.
[0006] A pharmaceutical composition for inhibiting the expression of the PCSK9 gene in an organism, comprising a dsRNA and a pharmaceutically acceptable carrier, wherein the dsRNA is as defined in any one of claims 1 to 5.
[0007] An in vitro method for inhibiting the expression of the PCSK9 gene in a cell, the method comprising:
(a) introducing into the cell a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA is as defined in any one of claims 1 to 5; 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.
[0008] A dsRNA for treating, preventing or managing pathological processes which can be mediated by down regulating PCSK9 gene expression, wherein the dsRNA is as defined in any one of claims 1 to 5.
[0009] A vector for inhibiting the expression of the PCSK9 gene in a cell, said vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of a dsRNA, wherein said dsRNA is as defined in any one of claims 1 to 5.
[0010] A cell comprising the dsRNA of any one of claims 1 to 5 or the vector of claim 9.
[0011] A double-stranded ribonucleic acid (dsRNA) for reducing the expression level of a human PCSK9 gene in a cell, wherein said dsRNA is as defined in any one of claims 1 to 5.
[0012] The dsRNA of claim 11, wherein said contact reduces the expression level of said PCSK9 gene.
[0013] The dsRNA of claim 11, wherein said contact is performed in vitro at 30nM or less.
[0014] A pharmaceutical composition for reducing the expression level of the PCSK9 gene in an organism, comprising the dsRNA of claim 11 and a pharmaceutically acceptable carrier.
[0015] The dsRNA of claim 11 for treating a PCSK9 associated disorder.
[0016] The dsRNA of claim 15, wherein said PCSK9 associated disorder is hyperlipidemia.

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优先权:

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申请号 | 申请日 | 专利标题

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US79945806P| true| 2006-05-11|2006-05-11||

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US81720306P| true| 2006-06-27|2006-06-27||

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US84008906P| true| 2006-08-25|2006-08-25||

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US82991406P| true| 2006-10-18|2006-10-18||

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US90113407P| true| 2007-02-13|2007-02-13||

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EP07762085A|EP2021507A4|2006-05-11|2007-05-10|Compositions and methods for inhibiting expression of the pcsk9 gene|

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EP09015323A|EP2194128B1|2006-05-11|2007-05-10|Compositions and methods for inhibiting expression of the PCSK9 gene|EP17162101.4A| EP3249052B1|2006-05-11|2007-05-10|Compositions and methods for inhibiting expression of the pcsk9 gene|

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EP14173646.2A| EP2835429B1|2006-05-11|2007-05-10|Compositions and methods for inhibiting expression of the PCSK9 gene|

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EP19168142.8A| EP3578656B1|2006-05-11|2007-05-10|Compositions and methods for inhibiting expression of the pcsk9 gene|