WO2011028938A1 - Methods for lowering serum cholestrol in a subject using inhibition of pcsk9 - Google Patents

Abstract

The invention relates to a method of lowering serum cholesterol using a double- stranded ribonucleic acid (dsRNA) for inhibiting the expression of the PCSK9 gene (PCSK9 gene) and an antigen binding protein (ABP) that binds to and inhibits a PCSK9 protein. Also provided are methods of treating or preventing conditions associated with elevated serum cholesterol using the dsRNAs and ABPs of the invention.

Description

METHODS FOR LOWERING SERUM CHOLESTEROL IN A SUBJECT USING

INHIBITION OF PCSK9

Cross Reference to Related Application's)

[0001] This application claims the benefit of U.S. Provisional Application No. 61/239,378, filed September 2, 2009, the entire disclosure of which is hereby incorporated by reference in its entirety for all purposes.

Reference to a Sequence Listing

[0002] This application includes a Sequence Listing submitted electronically as a text file named 17028PCT_sequencelisting.txt, created on Month, XX, 2010, with a size of

ΧΧΧ,ΧΧΧ bytes. The sequence listing is incorporated by reference.

Field of the invention

[0003] The invention relates to methods and compositions for lowering serum cholesterol in a subject via administration of an antigen binding protein (ABP), e.g., an anti-PCSK9 antibody, and an RNA agent, e.g., an siRNA which inhibits the expression of a PCSK9 gene.

Description of the Related Art

[0004] 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 PCl/3, PC2, furin, PC4, PC5/6, PACE4, PC7, and SlP/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. Vase. 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).

[0005] 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. Ill, 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.

[0006] 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

[0007] Loss of function mutations in PCSK9 have been designed in mouse models (Rashid et.al, (2005) PNAS, 102, 5374-5379., and identified in human individuals Cohen et al,

(2005) , Nature Genetics., 37, 161-165. In both cases loss of PCSK9 function lead to lowering of total and LDLc cholesterol. In a retrospective outcome study over 15 years, loss of one copy of PCSK9 was shown to shift LDLc lower and to lead to an increased risk- benefit protection from developing cardiovascular heart disease (Cohen et.al., 2006 N. Engl. J. Med., 354., 1264-1272.). Clearly the evidence to date indicates that lowering of PCSK9 levels will lower LDLc.

[0008] 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.

[0009] 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 and methods that can inhibit PCSK9 gene expression and/or block PCSK9 protein and that can treat diseases and/or conditions associated with PCSK9 expression such as hyperlipidemia.

[0010] PCSK9 targeting siRNA are described in U.S. Ser. No. 12/554,231, filed on Sept. 4, 2009; U.S. Ser. No. 12/478,452, filed on June 4, 2009; U.S. Ser. No. 11/746,864, filed on May 10, 2007; and PCT/US 10/038679, filed on June 15, 2010; all of which are herein incorporated by reference.

[0011] Examples of antigen binding proteins that bind PCSK9 and methods of their use are included in U.S. Pat. App. Pub. 20090142352, herein incorporated by reference.

SUMMARY

[0012] The presently claimed invention is directed, at least in part, to methods, compositions and kits for treating or preventing conditions and/or diseases that can be modulated by down regulating and blocking the proprotein convertase subtilisin kexin 9 (PCSK9) by using an RNA effector agent, e.g., a double-stranded ribonucleic acid (dsRNA) to silence PCSK9 expression, and antigen binding proteins, e.g., antibodies, to inhibit PCSK9 full-length protein or fragments thereof.

[0013] In an embodiment, the invention includes a method of lowering serum cholesterol in a subject, the method including administering to said subject an effective amount of: an antigen binding protein that selectively binds to and inhibits a PCSK9 protein; and an RNA effector agent which inhibits the expression of a PCSK9 gene in a cell, wherein administration of the antigen binding protein and said R A effector agent lowers serum cholesterol levels in the subject. In an embodiment, the antigen binding protein is selected from the group consisting of 21B12, 31H4, and 3C4 and the RNA effector agent is a dsRNA including an antisense strand which consists of the nucleotide sequence of SEQ ID NO: 1228 and a substantially complementary sense strand thereof.

[0014] Also described herein is a method for treating or preventing a condition associated with an elevated serum cholesterol level in a subject, including administering to the subject in need thereof an effective amount of an antigen binding protein that selectively binds and inhibits a PCSK9 protein, and an RNA effector agent which inhibits the expression of a PCSK9 gene in a cell, wherein administration of the antigen binding protein and the RNA effector agent lowers serum cholesterol levels in the subject.

[0015] Also described herein is a method for treating or preventing a condition associated with an elevated serum cholesterol level in a subject, including administering to the subject in need thereof an effective amount of an antigen binding protein that selectively binds to and inhibits a PCSK9 protein, an RNA effector agent which inhibits the expression of a human PCSK9 gene in a cell and a chemical agent that elevates the availability of LDLR protein, thereby lowering serum cholesterol levels in the subject. In an embodiment, the chemical agent is a statin. In an embodiment, the statin is selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, and some combination thereof.

[0016] In an embodiment, the antigen binding protein binds to PCSK9 with a Kd that is less than ΙΟΟρΜ. In an embodiment, antigen binding protein binds to PCSK9 with a Kd that is less than lOpM. In an embodiment, antigen binding protein binds to PCSK9 with a Kd that is less than 5pM.

[0017] In an embodiment, the PCSK9 protein includes an amino acid sequence which is 90% or more identical to the amino acid sequence shown in Table 8. In an embodiment, the PCSK9 protein includes the amino acid sequence shown in Table 8.

[0018] In an embodiment, the antigen binding protein is an antibody. In an embodiment, the antibody is a humanized antibody. In an embodiment, the antibody is a human antibody. In an embodiment, the antibody binds to an epitope within residues 31-449 of the amino acid sequence shown in Table 8. In an embodiment, the antibody is selected from the group consisting of 30A4, 3C4, 23B5, 25G4, 31H4, 27B2, 25A7, 27H5, 26H5, 31D1.20D10, 27E7, 30B9, 19H9, 26E10, 21B12, 17C2, 23G1, 13H1, 9C9, 9H6, 31A4, 1A12, 16F12, 22E2, 27A6, 28B12, 28D6, 31G11, 13B5, 31B12 and 3B6. In an embodiment, the antibody is 2 IB 12. In an embodiment, the antibody is 31 H4. In an embodiment, the antibody is 3 C4.

[0019] In an embodiment, the RNA effector agent is an siRNA selected from the group consisting of the siRNAs of Tables 1 and 2. In an embodiment, the RNA effector agent binds to nucleotide residues 3530-3548 of the nucleotide sequence shown in Table 8. In an embodiment, the RNA effector agent binds to at least 15 contiguous nucleotides of nucleotide sequence in SEQ ID NO: 1523.

[0020] In an embodiment, RNA effector agent is a dsRNA comprising a first sequence and a second sequence that are complementary to each other. In an embodiment, the dsRNA includes a sense strand including a first sequence and an antisense strand including a second sequence having at least 15 contiguous nucleotides of SEQ ID NO: 1228. In an embodiment, the second sequence includes SEQ ID NO: 1228. In an embodiment, the antisense strand consists of SEQ ID NO: 1228. In an embodiment, the second sequence includes SEQ ID NO: 1228 and the first sequence includes SEQ ID NO: 1227. In an embodiment, the dsRNA includes a sense strand including a first sequence and an antisense strand including a second sequence, wherein the first sequence is selected from the group consisting of SEQ ID

NO: 1227, SEQ ID NO: 1229, SEQ ID NO: 1231, SEQ ID NO: 1233, SEQ ID NO: 1235, SEQ ID NO: 1237, SEQ ID NO: 1239, SEQ ID NO: 1241, SEQ ID NO: 1243, SEQ ID NO: 1245, SEQ ID NO: 1247, SEQ ID NO: 1249, SEQ ID NO: 1251, SEQ ID NO: 1253, SEQ ID

NO: 1255, and SEQ ID NO: 1257 and wherein the second sequence is selected from the group consisting of SEQ ID NO: 1228, SEQ ID NO: 1230, SEQ ID NO:1232, SEQ ID NO:1234, SEQ ID NO: 1236, SEQ ID NO: 1238, SEQ ID NO: 1240, SEQ ID NO: 1242, SEQ ID

NO: 1244, SEQ ID NO: 1246, SEQ ID NO: 1248, SEQ ID NO: 1250, SEQ ID NO: 1252, SEQ ID NO: 1254, SEQ ID NO: 1256, and SEQ ID NO: 1258.

[0021] In an embodiment, the RNA effector agent is administered in a delivery vehicle. In an embodiment, the delivery vehicle is a vector which expresses the RNA effector agent. In an embodiment, the delivery vehicle is a lipid formulation.

[0022] In an embodiment, the dsRNA comprises at least one modified nucleotide. In an embodiment, the modified nucleotide is chosen from the group of: a 2'-0-methyl modified nucleotide, a nucleotide comprising a S'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative of dodecanoic acid bisdecylamide group. In an

embodiment, the modified nucleotide is chosen from the group of: a 2'-0-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.

[0023] In an embodiment, the condition is hypercholesterolemia, atherosclerosis, or dyslipidemia.

[0024] In an embodiment, the antigen binding agent and the R A effector agent are administered concurrently. In an embodiment, the antigen binding agent and the RNA effector agent are administered separately.

[0025] In an embodiment, the RNA effector agent inhibits PCSK9 gene expression by at least 20% or by at least 80%.

[0026] In an embodiment, the RNA effector agent lowers serum LDL cholesterol in the subject by at least 20%>.

[0027] Also described herein is a method for lowering serum cholesterol levels in a subject in need thereof, including administering to the subject a therapeutically effective amount of an antibody selected from the group consisting of 21B12, 31H4 and 3C4 and a dsRNA including an antisense strand which consists of the nucleotide sequence of SEQ ID NO: 1230 and a substantially complementary sense strand thereof, wherein the administration of the antibody and the dsRNA lowers serum cholesterol levels in the subject.

[0028] Also described herein is a pharmaceutical composition for reducing serum cholesterol levels in a subject, the pharmaceutical composition including an effective amount of an antigen binding protein that selectively binds to and inhibits a PCSK9 protein, an RNA effector agent which inhibits the expression of a PCSK9 gene in a cell, and a

pharmaceutically acceptable carrier. In an embodiment, the antigen binding protein is an antibody selected from the group consisting of 21B12, 31H4 and 3C4, and the RNA effector agent is a dsRNA including a sense strand which consists of the nucleotide sequence of SEQ ID NO: 1229 and an antisense strand which consists of the nucleotide sequence of SEQ ID NO: 1230. In an embodiment, the pharmaceutically acceptable carrier includes a SNALP lipid formulation, a XTC lipid, a LNP01 lipid formulation, a MC3 lipid, a Lipid Formula A lipid, and/or a ALNY100 lipid.

[0029] Also described herein is a pharmaceutical kit for treating or preventing a condition associated with an elevated serum cholesterol level in a subject including, an antigen binding protein that binds to and inhibits a PCSK9 protein, an RNA effector agent which inhibits the expression of a human PCSK9 gene in a cell and a label or packaging insert containing instructions for use. In an embodiment, the antigen binding protein and the RNA effector agent are contained in separate intravenous pharmaceutical dosage forms. In an embodiment, the antigen binding protein is an antibody selected from the group consisting of 2 IB 12, 31H4 and 3C4, and the RNA effector agent is a dsRNA including a sense strand which consists of the nucleotide sequence of SEQ IDNO: 1229 and an antisense strand which consists of the nucleotide sequence of SEQ ID NO: 1230.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

[0031] FIG. 1 shows the structure of Formula 1.

[0032] 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).

[0033] FIG. 3 shows the results of the in vivo screen of 16 human/mouse/rat cross-reactive (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).

[0034] FIG. 4 shows the results of the in vivo screen of 16 mouse specific PCSK9 siRNAs (AL-DP-9327 through AL-DP-9342) 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).

[0035] FIG. 5 shows the results of the in vivo screen of 16 human/mouse/rat cross-reactive (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 siR A (blood coagulation factor VII).

[0036] FIGs. 6A and 6B compare in vitro and in vivo results, respectively, for silencing PCSK9.

[0037] FIG. 7A and FIG. 7B are an example of in vitro results for silencing PCSK9 using monkey primary hepatocytes.

[0038] FIG 7C show results for silencing of PCSK9 in monkey primary hepatocytes using

AL-DP-9680 and chemically modified version of AL-DP-9680.

[0039] FIG. 8 shows in vivo activity of LNP-01 formulated siRNAs to PCSK-9.

[0040] FIGs. 9A and 9B show in vivo activity of LNP-01 Formulated chemically modified

9314 and derivatives with chemical modifications such as AD-10792, AD-12382, AD-12384,

AD- 12341 at different times post a single dose in mice.

[0041] FIG. 10A shows the effect of PCSK9 siRNAs on PCSK9 transcript levels and total serum cholesterol levels in rats after a single dose of formulated AD-10792. FIG. 10B shows the effect of PCSK9 siRNAs on serum total cholesterol levels in the experiment as 10A. A single dose of formulated AD-10792 results in an -60% lowering of total cholesterol in the rats that returns to baseline by ~3-4 weeks. FIG. IOC shows the effect of PCSK9 siRNAs on hepatic cholesterol and triglyceride levels in the same experiment as 10A.

[0042] FIG. 11 is a Western blot showing that liver LDL receptor levels were upregulated following administration of PCSK9 siRNAs in rat.

[0043] FIGs. 12A-12D show the effects of PCSK9 siRNAs on LDLc and ApoB protein levels, total cholesterol/HDLc ratios, and PCSK9 protein levels, respectively, in nonhuman primates following a single dose of formulated AD-10792 or AD-9680.

[0044] FIG. 13A is a graph showing that unmodified siRNA-AD-AlA (AD-9314), but not 2'OMe modified siRNA-AD-lA2 (AD-10792), induced IFN-alpha in human primary blood monocytes. FIG. 13B is a graph showing that unmodified siRNA-AD-AlA (AD-9314), but not 2'OMe modified siRNA-AD-lA2 (AD-10792), also induced TNF-alpha in human primary blood monocytes.

[0045] FIG. 14A is a graph showing that the PCSK9 siRNA siRNA-AD-lA2 (a.k.a. LNP- PCS-A2 or a.k.a. "formulated AD-10792") decreased PCSK9 mRNA levels in mice liver in a dose-dependent manner. FIG. 14B is a graph showing that single administration of 5 mg/kg siRNA-AD-lA2 decreased serum total cholesterol levels in mice within 48 hours. [0046] FIG. 15A is a graph showing that PCSK9 siR As targeting human and monkey PCSK9 (LNP-PCS-C2) (a.k.a. "formulated AD-9736"), and PCSK9 siRNAs targeting mouse PCSK9 (LNP-PCS-A2) (a.k.a. "formulated AD- 10792"), reduced liver PCSK9 levels in transgenic mice expressing human PCSK9. FIG. 15B is a graph showing that LNP-PCS-C2 and LNP-PCS-A2 reduced plasma PCSK9 levels in the same transgenic mice.

[0047] FIG. 16 shows the structure of an siRNA conjugated to Chol-p-(GalNAc)3 via phosphate linkage at the 3 ' end.

[0048] FIG. 17 shows the structure of an siRNA conjugated to LCO(GalNAc)3 (a

(GalNAc)3 - 3'-Lithocholic-oleoyl siRNA Conjugate).

[0049] FIG. 18 is a graph showing the results of conjugated siRNAs on PCSK9 transcript levels and total serum cholesterol in mice.

[0050] FIG. 19 is a graph showing the results of lipid formulated siRNAs on PCSK9 transcript levels and total serum cholesterol in rats.

[0051] FIG. 20 is a graph showing the results of siRNA transfection on PCSK9 transcript levels in HeLa cells using AD-9680 and variations of AD-9680 as described in Table 6.

[0052] FIG. 21 is a graph showing the results of siRNA transfection on PCSK9 transcript levels in HeLa cells using AD- 14676 and variations of AD- 14676 as described in Table 6.

[0053] FIG. 22 shows the results of the SNALP and XTC2-PCSK9 siRNA dose response in rats.

[0054] FIG. 23 shows the results of treatment with a maintenance dose of PCSK9 targeted siRNA.

[0055] FIG. 24 the results of treatment with a maintenance dose of PCSK9 targeted siRNA.

[0056] FIG. 25 is the structure of C12-200.

DETAILED DESCRIPTION

[0057] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

Definitions

[0058] The prefixes "AD-" "DP-" "ALDP-" and "AL-DP-" and the like are used

interchangeably e.g., AL-DP-10792 and AD-10792 refer to the same siRNA.

[0059] As used herein, "PCSK9" refers to the proprotein convertase subtilisin kexin 9 gene or protein (also known as FH3, HCHOLA3, NARC-1, NARC1). Examples of mRNA sequences to PCSK9 include but are not limited to the following: human: NM l 74936; mouse: NM_153565, and rat: NM_199253. The term "PCSK9" can refer to a polypeptide as set forth in Tables 8 and/or 9 or fragments thereof, as well as related polypeptides, which include, but are not limited to, allelic variants, splice variants, derivative variants, substitution variants, deletion variants, and/or insertion variants including the addition of an N-terminal methionine, fusion polypeptides, and interspecies homologs. In certain embodiments, a PCSK9 polypeptide includes terminal residues, such as, but not limited to, leader sequence residues, targeting residues, amino terminal methionine residues, lysine residues, tag residues and/or fusion protein residues. "PCSK9" has also been referred to as proprotein convertase subtilisin/kexin type 9, and neural apoptosis regulated convertase 1. The PCSK9 gene encodes a proprotein convertase protein that belongs to the proteinase K subfamily of the secretory subtilase family. The term "PCSK9" denotes both the proprotein and the product generated following autocatalysis of the proprotein. When only the autocatalyzed product is being referred to (such as for an antigen binding protein that selectively binds to the cleaved PCSK9), the protein can be referred to as the "mature," "cleaved," "processed," or "active" PCSK9. When only the inactive form is being referred to, the protein can be referred to as the "inactive," "pro-form," or "unprocessed" form of PCSK9. The term PCSK9 as used herein also includes naturally occurring alleles, such as the mutations D374Y, S127R and F216L. The term PCSK9 also encompasses PCSK9 molecules incorporating post- translational modifications of the PCSK9 amino acid sequence, such as PCSK9 sequences that have been glycosylated, PEGylated, PCSK9 sequences from which its signal sequence has been cleaved, PCSK9 sequence from which its pro domain has been cleaved from the catalytic domain but not separated from the catalytic domain. Additional examples of PCSK9 genomic, mR A, and protein sequences are readily available using, e.g., GenBank.

[0060] Proprotein convertase subtilisin kexin type 9 (PCSK9) is a serine protease involved in regulating the levels of the low density lipoprotein receptor (LDLR) protein (Horton et al., 2007; Seidah and Prat, 2007). PCSK9 is a prohormone-proprotein convertase in the subtilisin (S8) family of serine proteases (Seidah et al., 2003). An exemplary, mature form human PCSK9 amino acid sequence is presented in Table 7 below. Table 8 shows amino acid and nucleic acid sequences of PCSK9, the start and stop codons in the nucleotide sequence are shown in bold. As described herein, PCSK9 proteins can also include fragments of the full length PCSK9 protein. The structure of the PCSK9 protein has recently been solved by two groups (Cunningham et al, Nature Structural & Molecular Biology, 2007, and Piper et al, Structure, 15: 1-8, 2007), the entireties of both of which are herein incorporated by reference. PCSK9 includes a signal sequence, a N-terminal prodomain, a subtilisin-like catalytic domain and a C-terminal domain.

[0061] The term "PCSK9 activity" includes any biological effect or expression of PCSK9. In certain embodiments, PCSK9 activity includes the ability of PCSK9 to interact or bind to a substrate or receptor. In some embodiments, PCSK9 activity is represented by the ability of PCSK9 to bind to a LDL receptor (LDLR). In some embodiments, PCSK9 binds to and catalyzes a reaction involving LDLR. In some embodiments, PCSK9 activity includes the ability of PCSK9 to alter (e.g., reduce) the availability of LDLR. In some embodiments, PCSK9 activity includes the ability of PCSK9 to increase the amount of LDL in a subject. In some embodiments, PCSK9 activity includes the ability of PCSK9 to decrease the amount of LDLR that is available to bind to LDL. In some embodiments, "PCSK9 activity" includes any biological activity resulting from PCSK9 signaling. Exemplary activities include, but are not limited to, PCSK9 binding to LDLR, PCSK9 enzyme activity that cleaves LDLR or other proteins, PCSK9 binding to proteins other than LDLR that facilitate PCSK9 action, PCSK9 altering APOB secretion (Sun X-M et al, "Evidence for effect of mutant PCSK9 on apoliprotein B secretion as the cause of unusually severe dominant hypercholesterolemia, Human Molecular Genetics 14: 1161-1169, 2005 and Ouguerram K et al, "Apolipoprotein B100 metabolism in autosomal-dominant hypercholesterolemia related to mutations in PCSK9, Arterioscler thromb Vase Biol. 24: 1448-1453, 2004), PCSK9^*s role in liver regeneration and neuronal cell differentiation (Seidah N G et al, "The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): Liver regeneration and neuronal differentiation" PNAS 100: 928-933, 2003), and PCSK9s role in hepatic glucose metabolism (Costet et al, "Hepatic PCSK9 expression is regulated by nutritional status via insulin and sterol regulatory element-binding protein lc" J. Biol. Chem. 281(10):6211-18, 2006).

[0062] The term "serum cholesterol" refers to cholesterol or total cholesterol that travels in the bloodstream of a subject in distinct particles including lipids and proteins. Three major classes of lipoproteins are generally found in the serum of a fasting subject: low-density lipoprotein (LDL), high-density lipoprotein (HDL), and very-low-density lipoprotein (VLDL). Another lipoprotein class, intermediate-density lipoprotein (IDL), resides between VLDL and LDL; in clinical practice, IDL is generally included in the LDL measurement.

[0063] The term "condition associated with an elevated serum cholesterol level" refers to conditions in a subject related to increased serum levels of cholesterol relative to a normal range of serum cholesterol levels in a control subject or subjects. One of skill in the art will readily understand what normal levels of serum cholesterol are and how to measure serum cholesterol in subjects, e.g., using the methods described in U.S. Pat. No. 4,366,244, herein incorporated by reference; and/or Pignone M, Phillips C, Atkins D, Teutsch S, Mulrow C, Lohr K (2001). "Screening and treating adults for lipid disorders". Am J Prev Med 20 (3 Suppl): 77-89, herein incorporated by reference. Generally, elevated serum cholesterol can refer to a level of serum cholesterol that is greater than that considered to be in the normal range for a given age in a population, e.g., about 5.25 mmoles/L or greater, i.e.,

approximately one standard deviation or more away from the age-adjusted mean. An example of such a condition associated with an elevated serum cholesterol level is hypercholesterolemia. The term "hypercholesterolemia," as used herein, refers to a condition in which cholesterol levels are elevated above a desired level. In some embodiments, this denotes that serum cholesterol levels are elevated. In some embodiments, the desired level takes into account various "risk factors" that are known to one of skill in the art (and are described or referenced herein).

[0064] The term "LDLR protein" and "LDL receptor protein" are used interchangeably and refer to low-density lipoprotein particle receptor protein. LDLR protein is a protein that mediates the endocytosis of cholesterol-rich LDL. LDLR protein is a cell-surface receptor that recognizes the apoprotein B 100 which is embedded in the phospholipid outer layer of LDL particles. LDLR protein also recognizes the apoE protein found in chylomicron remnants and VLDL remnants (IDL).

[0065] The term "statin" refers to chemical agents that lower cholesterol levels in a subject with or at risk of a disease, e.g., cardiovascular disease. Statins generally lower cholesterol by inhibiting the enzyme HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase or HMGR), which is the rate-limiting enzyme of the mevalonate pathway of cholesterol synthesis. Inhibition of HMG-CoA reductase in the liver results in decreased cholesterol synthesis as well as increased synthesis of LDL receptors, resulting in an increased clearance of low-density lipoprotein (LDL) from the bloodstream. Examples of statins include atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.

[0066] The term "antigen binding protein" or "ABP" as used herein means any protein that binds a specified target antigen. In the instant application, the specified target antigen is the PCSK9 protein or fragment thereof. "Antigen binding protein" includes but is not limited to antibodies and binding parts thereof, such as immunologically functional fragments. ABPs include 30A4, 3C4, 23B5, 25G4, 31H4, 27B2, 25A7, 27H5, 26H5, 31D1.20D10, 27E7, 30B9, 19H9, 26E10, 21B12, 17C2, 23G1, 13H1, 9C9, 9H6, 31A4, 1A12, 16F12, 22E2, 27A6, 28B12, 28D6, 31G11, 13B5, 3 IB 12 and 3B6 which are described in more detail below.

[0067] The term "RNA effector agent" refers to an agent that modulates RNA. An RNA effector agent is capable of inhibiting or "silencing" the expression of a target gene with one or more target sequences. In certain embodiments, the RNA effector agent is capable of preventing complete processing (e.g, the full translation and/or expression) of a mRNA molecule through a post-transcriptional silencing mechanism. RNA effector agents include small (<50 b.p.), noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA effector agents include dsRNAs, siRNAs, miRNAs, siRNA-like duplexes, antisense oligonucleotides, and dual-function oligonucleotides as well as precursors thereof. In one embodiment, the RNA effector agent is capable of inducing RNA interference. In another embodiment, the RNA effector agent is capable of mediating translational repression. In one embodiment the RNA effector agent is an siRNA, e.g., AD- 9680.

[0068] "G," "C," "A" and "U" each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. "T" and "dT" are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the term "ribonucleotide" or

"nucleotide" or "deoxyribonucleotide" 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. [0069] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an m NA molecule formed during the transcription of the PCSK9 gene, including mRNA that is a product of RNA processing of a primary transcription product.

[0070] 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.

[0071] 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.

[0072] This includes base-pairing of the oligonucleotide or polynucleotide having the first nucleotide sequence to the oligonucleotide or polynucleotide having the second nucleotide sequence over the entire length of the first and second nucleotide sequences. Such sequences can be referred to as "fully complementary" with respect to each other. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA having one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide has a sequence of 21 nucleotides that is fully

complementary to the shorter oligonucleotide, may yet be referred to as "fully

complementary." [0073] "Complementary" sequences, as used herein, may also include, or be formed entirely from, non- Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non- Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogstein base pairing.

[0074] 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.

[0075] As used herein, a polynucleotide which is "substantially complementary to at least part of a messenger RNA (mRNA) refers to a polynucleotide that is substantially

complementary to a contiguous portion of the mRNA of interest (e.g., encoding PCSK9) including a 5' UTR, an open reading frame (ORF), or a 3' UTR. For example, a

polynucleotide is complementary to at least a part of a PCSK9 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding PCSK9 .

[0076] The term "double-stranded RNA" or "dsRNA", as used herein, refers a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. In general, the majority of nucleotides of each strand are

ribonucleotides, but as described in detail herein, each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide. 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.

[0077] 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 dsR A 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 general, the majority of nucleotides of each strand are

ribonucleotides, but as described in detail herein, each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide. 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 siR A type molecule, are encompassed by "dsRNA" for the purposes of this specification and claims.

[0078] 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.

[0079] 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 may be in the internal or terminal regions of the molecule. Generally the most tolerated mismatches are in the terminal regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5' and/or 3' terminus.

[0080] 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.

[0081] "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 dsR A 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.

[0082] The terms "silence," "inhibit the expression of," "down-regulate the expression of," "suppress the expression of," and the like, 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 PCSK9 mRNA which may be isolated or detected 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 cells)

— · 100%

(mRNA in control cells)

[0083] Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to PCSK9 gene expression, e.g. the amount of protein encoded by the PCSK9 gene which is produced by a cell, or the number of cells displaying a certain phenotype.. In principle, target gene silencing can 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 assays provided in the Examples below shall serve as such reference.

[0084] For example, in certain instances, expression of a PCSK9 gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the double-stranded oligonucleotide of the invention. In some embodiment, a 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, a PCSK9 gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention.

[0085] The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects as well as those with formally diagnosed disorders, those without formally recognized disorders, those receiving medical attention, those at risk of developing the disorders, etc.

[0086] The term "administering" refers to to the act of giving a composition to a subject or otherwise making such composition available to a subject or the subject taking a

composition.

[0087] The term "delivery vehicle" refers to to a composition that complexes with and facilitates the delivery of a R A effector agent through a cell membrane to a target site. Delivery vehicles in accordance with the present invention are "pharmaceutically

acceptable," which, as used herein, refers to the compatibility of the delivery vehicles with biological materials, for example, for use in pharmaceutical formulations and in other applications, either in vivo or in vitro, where they are in contact with biological materials, such as living cells or tissues.

[0088] The term "vector" means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus) used to transfer genetic information into a host cell.

[0089] The term "modified nucleotide" refers to a non-standard nucleotide, including non- naturally occurring ribonucleotides or deoxyribonucleotides. Nucleotides can be modified at any position so as to alter certain properties of the nucleotide yet can retain the ability of the nucleotide to perform its intended function. Examples of modified nucleotides include 2'-0- methyl modified nucleotide, a nucleotide including a S'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative of dodecanoic acid bisdecylamide group, a 2'-0- 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 including nucleotide.

[0090] 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 the 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.

[0091] 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 that can be mediated by down regulating the PCSK9 gene 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 an ordinary medical practitioner, and may vary depending on factors known in the art, such as, e.g., the type of pathological processes that can be mediated by down regulating the PCSK9 gene, the patient's history and age, the stage of pathological processes that can be mediated by down regulating PCSK9 gene expression, and the administration of other anti-pathological processes that can be mediated by down regulating PCSK9 gene expression.

[0092] As used herein, a "pharmaceutical composition" includes a pharmacologically effective amount of a dsRNA and/or a pharmacologically effective amount of an antigen binding protein 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 and/or antigen binding protein 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. Examples, of antigen binding proteins include 21B12, 31H4 and 3C4 (described in more detail below). An examples of an RNA effector agent is a dsRNA including a sense strand which includes the nucleotide sequence of SEQ ID NO: 1229 and an antisense strand which includes the nucleotide sequence of SEQ ID NO: 1230

[0093] 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.

[0094] As used herein, a "transformed cell" is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed. [0095] The term "in vivo" refers to processes that occur in a living organism.

[0096] The term "mammal" as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0097] The term "sufficient amount" means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.

[0098] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

DOUBLE-STRANDED RIBONUCLEIC ACID (DSRNA)

[0099] As described in more detail below, the invention provides methods and compositions having an RNA effector agent. In some embodiments, the R A effector agent is an siR A, e.g., a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the PCSK9 gene in a cell or mammal, wherein the dsRNA includes an antisense strand having a region of complementarity that 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. In some embodiments, the dsRNA, upon contact with a cell expressing the PCSK9 gene, inhibits the expression of said PCSK9 gene, e.g., as measured such as by an assay described herein. The dsRNA of the invention can further include one or more single-stranded nucleotide overhangs.

[00100] 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.

[00101] The dsRNA includes two nucleic acid strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) can have 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) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions.

[00102] Generally, the duplex structure is between 15 and 30, or between 25 and 30, or between 18 and 25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 base pairs in length. The duplex region can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 basepairs in length. In one embodiment the duplex is 19 base pairs in length. In another embodiment the duplex is 21 base pairs in length. When two different siRNAs are used in combination, the duplex lengths can be identical or can differ.

[00103] Each strand of the dsRNA of invention is generally between 15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. Each strand of the dsRNA can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 basepairs in length. In other embodiments, each is strand is 25-30 nucleotides in length. In one embodiment, each strand is 21 nucleotides in length. Each strand of the duplex can be the same length or of different lengths. When two different siRNAs are used in combination, the lengths of each strand of each siRNA can be identical or can differ.

[00104] The dsRNA of the invention can include one or more single-stranded overhang(s) of one or more nucleotides. In one embodiment, at least one end of the dsRNA has a single- stranded nucleotide overhang of 1 to 4, or 1, 2, 3 or 4 nucleotides. In another embodiment, the antisense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3' end and the 5' end over the sense strand. In further embodiments, the sense strand of the dsRNA has 1- 10 nucleotides overhangs each at the 3' end and the 5' end over the antisense strand. In one embodiment each strand has a 2 nucleotide overhang at the 3' end of both the sense and antisense strands. In one embodiment each strand has a TsT overhang at the 3' end of both the sense and antisense strands.

[00105] A dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties than the blunt-ended counterpart. In some embodiments the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. A 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 can also have a blunt end, generally located at the 5 '-end of the antisense strand. Such dsRNAs can 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. [00106] In one embodiment, the PCSK9 gene is a human PCSK9 gene. In other embodiments, the sense strand of the dsR A is one of the sense sequences of Table 1, Table 2, and Table 5a, and an antisense strand is one of the antisense sequences of Table 1, Table 2, and Table 5a. Alternative antisense agents that target elsewhere in the target sequence provided in Table 1, Table 2, and Table 5a, can readily be determined using the target sequence and the flanking PCSK9 sequence.

[00107] For example, the dsRNA AD-9680 (from Table 1) targets the PCSK9 gene at 3530-3548; therefore the target sequence is as follows: 5' UUCUAGACCUGUUUUGCUU 3' (SEQ ID NO: 1523).. The dsRNA AD-10792 (from Table 1) targets the PCSK9 gene at 1091-1109; therefore the target sequence is as follows: 5' GCCUGGAGUUUAUUCGGAA 3' (SEQ ID NO: 1524). Included in the invention are dsRNAs with antisense strands that have regions of complementarity to SEQ ID NO: 1523 or SEQ ID NO: 1524, or are

complementary to at least 15 contiguous nucleotides of SEQ ID NO: 1523 or SEQ ID

NO: 1524..

[00108] The skilled person is well aware that dsRNAs having 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 Table 1 , Table 2, and Table 5a the dsRNAs of the invention can include at least one strand of a length of a length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Table 1, Table 2, and Table 5a minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, 21, or 22 or more contiguous nucleotides from one of the sequences of Table 1, Table 2, and Table 5a and differing in their ability to inhibit the expression of the PCSK9 gene in an 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 Table 1, Table 2, and Table 5a can readily be made using the PCSK9 sequence and the target sequence provided.

[00109] In addition, the dsRNAs provided in Table 1, Table 2, and Table 5a identify a site in the PCSK9 mRNA that is susceptible to RNAi based cleavage. As such the present invention further includes dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Table 1, Table 2, and Table 5a 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 Table 1, Table 2, and Table 5a.

[00110] The dsRNA of the invention can contain one or more mismatches to the target sequence. In one embodiment, the dsRNA of the invention contains no more than 1, no more than 2, or no more than 3 mismatches. In one embodiment, the antisense strand of the dsRNA contains mismatches to the target sequence, and the area of mismatch is not located in the center of the region of complementarity. In another embodiment, the antisense strand of the dsRNA contains mismatches to the target sequence and the mismatch is 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 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.

Modifications

[00111] 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. Specific examples of dsRNA compounds useful in this invention include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsR As that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[00112] Preferred modified dsR A 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.

[00113] Representative U.S. patents that teach the preparation of the above phosphorus- containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference

[00114] Preferred modified dsRNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or ore or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

[00115] Representative U.S. patents that teach the preparation of the above

oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315;

5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.

[00116] In other certain dsRNA mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, a dsRNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of a dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.

[00117] Other embodiments of the invention are dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular— CH₂— NH— CH₂-, -CH₂-N(CH₃)-0--CH₂-[known as a methylene (methylimino) or MMI backbone], - -CH₂-0-N(CH₃)-CH₂-, -CH₂-N(CH₃)-N(CH₃)-CH₂-- and -N(CH₃)-CH₂-CH₂- [wherein the native phosphodiester backbone is represented as --0--P--0— CH₂— ] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAs having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[00118] Modified dsRNAs may also contain one or more substituted sugar moieties.

Preferred dsRNAs comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are 0[(CH₂)_nO]_mCH₃, 0(CH₂)_nOCH₃, 0(CH₂)_nNH₂, 0(CH₂)_nCH₃, 0(CH₂)_nONH₂, and 0(CH₂)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2' position: Ci to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH_3, OCN, CI, Br, CN, CF_3, OCF_3, SOCH_3, S0₂CH_3, ON0_2, N0_2, N_3, NH_2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy (2'-0— CH₂CH₂OCH₃, also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al, Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group. A further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a 0(CH₂)₂0N(CH₃)₂ group, also known as 2'- DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-0— CI¾— O— CH₂~N(CH₂)2, also described in examples herein below.

[00119] Other preferred modifications include 2'-methoxy (2'-OCH₃), 2'-aminopropoxy (2'-Ο ¾ ¾ ¾ΝΗ₂) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucleotide. DsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and

5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[00120] dsRNAs may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5- bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3- deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al, Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1.2. degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0- methoxyethyl sugar modifications.

[00121] Other nucleotide substitutions, such as "Universal" bases can be incorporated into siRNA duplexes to increase the number of target sequences (or in this case, number of different Ebola strains) any particular siRNA might have complementarity to and activity against. Universal bases are non-canonical synthetic molecules that mimic structures of traditional nucleotides (the genetic building blocks of DNA and RNA). However, instead of selectively pairing according to Watson/Crick rules (A with T or U, C with G), universal bases 'stack' equally well with all natural bases. Incorporating universal bases into siRNAs may enable the siRNA to tolerate a mutation at that specific site in its target mRNA. Thus, by decreasing the need for absolute complementarity between siRNA and its mRNA target, universal-base containing siRNAs may be an approach to (1) prevent drug resistance caused by site-specific viral mutations and (2) create siRNAs able to be broadly reactive across viral species with similar, but not absolutely conserved, targets. Among the modifications that can be used as universal basaes are: 3-Nitropyrrole, 5-Nitroindole, Imidazole-4-Carboxamide, 2,4-difluorotoluyl, and Inosine.

[00122] Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and

5,681,941, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference. Synthesis

[00123] 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'-0-alkyl guanosine and related compounds, including 2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No.

5,608,046, both drawn to conjugated 4'-desmethyl nucleoside analogs; U.S. Pat. Nos.

5,602,240, and 5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods of synthesizing 2'-fluoro- oligonucleotides.

[00124] 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.

[00125] 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 ah, PCT Application WO 93/07883). In one embodiment, the oligonucleotides or linked nucleosides featured in 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.

[00126] The incorporation of a 2^*-0-methyl, 2^*-0-ethyl, 2^*-0-propyl, 2^*-0-allyl, 2^*-0- 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'-0-methyl, 2'-0-ethyl, 2'-0-propyl, 2'-0-aminoalkyl, 2'-0-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.

[00127] 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.

[00128] 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.

[00129] 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.

[00130] 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.

[00131] 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.

[00132] 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 l,2-di-0-hexadecyl-rac-glycero-3-H- phosphonate (Manoharan et al, Tetrahedron Lett., 1995, 36:3651; Shea et al, Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al,

Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al, Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such oligonucleotide conjugates have been listed above. Typical conjugation protocols involve the synthesis of oligonucleotides bearing an

aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the oligonucleotide still bound to the solid support or following cleavage of the oligonucleotide in solution phase. Purification of the oligonucleotide conjugate by HPLC typically affords the pure conjugate. The use of a cholesterol conjugate is particularly preferred since such a moiety can increase targeting liver cells, a site of PCSK9 expression.

Conjugates

[00133] Another modification of the dsRNAs of the invention involves chemically linking to the dsRNA one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan et al, Biorg. Med. Chem. Let., 1994 4 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al, Ann. N.Y. Acad. Sci., 1992, 660, 306- 309; Manoharan et al, Biorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al, Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al, EMBO J, 1991, 10, 1111-1118; Kabanov et al, FEBS Lett., 1990, 259, 327-330; Svinarchuk et al, Biochimie, 1993, 75, 49-54), a

phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1 ,2-di-O-hexadecyl-rac- glycero-3-Hphosphonate (Manoharan et al, Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al, Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino- carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Prefered conjugates will assist in targeting cells infected by Ebola virus such as dendritic cells and macrophages which are involved in early stages of infection and epatocytes and other parenchymal cells which are involved in later phases of the infection. Such conjugates include, but are not limited to, mannose and folate conjugates.

[00134] Representative U.S. patents that teach the preparation of such dsRNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979: 4,948,882: 5,218,105: 5,525,465:

5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941 ; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.

[00135] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds. "Chimeric" dsRNA compounds or "chimeras," in the context of this invention, are dsRNA compounds, particularly dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an dsRNA compound. These dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[00136] In certain instances, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, 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 l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al, Tetrahedron Lett., 1995, 36:3651; Shea et al, Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs 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 dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase.

Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.

Vector encoded dsRNAs

[00137] In another aspect of the invention, PCSK9 specific dsRNA molecules that modulate PCSK9 gene expression activity are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al. , Proc. Natl. Acad. Sci. USA (1995) 92: 1292).

[00138] 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 one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

[00139] 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. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al, 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al, 1990, Proc. Natl. Acad. Sci. USA

87:61416145; Huber t al, 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al, 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al, 1991, Science 254: 1802- 1805; van Beusechem. et al, 1992, Proc. Nad. Acad. Sci. USA 89:7640-19 ; Kay et al, 1992, Human Gene Therapy 3:641-647; Dai et al, 1992, Proc. Natl.Acad. Sci. USA 89: 10892- 10895; Uwu 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. Natl. Acad. Sci. USA 81 :6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts {e.g., rat, hamster, dog, and chimpanzee) (Hsu et al, 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.

[00140] 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.

[00141] 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.

[00142] 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.

[00143] 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 having, for example, either the U6 or HI RNA promoters, or the cytomegalovirus (CMV) promoter.

[00144] 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.

[00145] 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.

[00146] 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 Ul 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)).

[00147] 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-Dl -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.

[00148] 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.

[00149] 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.

[00150] The PCSK9 specific dsR A 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 include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

ANTIGEN BINDING PROTEINS

[00151] The invention provides methods and compositions using an RNA effector agent, e.g., a PCSK9 dsRNA, and an antigen binding protein that binds PCSK9, e.g., a PCSK9 antibody. Examples of an antigen binding proteins that bind PCSK9 and methods of use are included in U.S. Pat. App. Pub. 20090142352, filed on Aug. 22, 2008, which is herein incorporated by reference in its entirety for all purposes.

[00152] An "antigen binding protein" ("ABP") as used herein means any protein that binds a specified target antigen. In the instant application, the specified target antigen is the PCSK9 protein or fragment thereof. "Antigen binding protein" includes but is not limited to antibodies and binding parts thereof, such as immunologically functional fragments.

Peptibodies are another example of antigen binding proteins. The term "immunologically functional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain) antigen binding protein, as used herein, is a species of antigen binding protein comprising a portion (regardless of how that portion is obtained or synthesized) of an antibody that lacks at least some of the amino acids present in a full-length chain but which is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to the target antigen and can compete with other antigen binding proteins, including intact antibodies, for binding to a given epitope. In some embodiments, the fragments are neutralizing fragments. In some embodiments, the fragments can block or reduce the likelihood of the interaction between LDLR and PCSK9. In one aspect, such a fragment will retain at least one CDR present in the full-length light or heavy chain, and in some embodiments will comprise a single heavy chain and/or light chain or portion thereof. These biologically active fragments can be produced by recombinant DNA techniques, or can be produced by enzymatic or chemical cleavage of antigen binding proteins, including intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, a diabody (heavy chain variable domain on the same polypeptide as a light chain variable domain, connected via a short peptide linker that is too short to permit pairing between the two domains on the same chain), Fab', F(ab')₂, Fv, domain antibodies and single-chain antibodies, and can be derived from any mammalian source, including but not limited to human, mouse, rat, camelid or rabbit. It is further contemplated that a functional portion of the antigen binding proteins disclosed herein, for example, one or more CDRs, could be covalently bound to a second protein or to a small molecule to create a therapeutic agent directed to a particular target in the body, possessing bifunctional therapeutic properties, or having a prolonged serum half-life. As will be appreciated by one of skill in the art, an antigen binding protein can include nonprotein components. In some sections of the present disclosure, examples of ABPs are described herein in terms of

"number/letter/number" (e.g., 25A7). In these cases, the exact name denotes a specific antibody. That is, an ABP named 25 A7 is not necessarily the same as an antibody named 25A7.1, (unless they are explicitly taught as the same in the specification, e.g., 25 A7 and 25A7.3). As will be appreciated by one of skill in the art, in some embodiments LDLR is not an antigen binding protein. In some embodiments, binding subsections of LDLR are not antigen binding proteins, e.g., EGFa. In some embodiments, other molecules through which PCSK9 signals in vivo are not antigen binding proteins. Such embodiments will be explicitly identified as such.

[00153] Certain antigen binding proteins described herein are antibodies or are derived from antibodies. In certain embodiments, the polypeptide structure of the antigen binding proteins is based on antibodies, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof, respectively. In some embodiments, the ABP comprises or consists of avimers (tightly binding peptide). These various antigen binding proteins are further described herein.

[00154] An "Fc" region comprises two heavy chain fragments comprising the C_Hi and C_H2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the C_H3 domains.

[00155] A "Fab fragment" comprises one light chain and the Cmand variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[00156] A "Fab' fragment" comprises one light chain and a portion of one heavy chain that contains the VH domain and the Cmdomain and also the region between the Cmand C_R2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')₂ molecule.

[00157] A "F(ab')₂ fragment" contains two light chains and two heavy chains containing a portion of the constant region between the C_HI and C_H2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab')₂ fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.

[00158] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[00159] "Single-chain antibodies" are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen binding region. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649 and U.S. Pat. No. 4,946,778 and No. 5,260,203, the disclosures of which are incorporated by reference.

[00160] A "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more V_H regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two V_H regions of a bivalent domain antibody can target the same or different antigens.

[00161] A "bivalent antigen binding protein" or "bivalent antibody" comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificities. Bivalent antigen binding proteins and bivalent antibodies can be bispecific, see, infra. A bivalent antibody other than a "multispecific" or "multifunctional" antibody, in certain embodiments, typically is understood to have each of its binding sites identical.

[00162] A "multispecific antigen binding protein" or "multispecific antibody" is one that targets more than one antigen or epitope.

[00163] A "bispecific," "dual-specific" or "bifunctional" antigen binding protein or antibody is a hybrid antigen binding protein or antibody, respectively, having two different antigen binding sites. Bispecific antigen binding proteins and antibodies are a species of multispecific antigen binding protein antibody and can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148: 1547-1553. The two binding sites of a bispecific antigen binding protein or antibody will bind to two different epitopes, which can reside on the same or different protein targets.

An antigen binding protein is said to "specifically bind" its target antigen when the dissociation constant (IQ) is <10^"7 M. The ABP specifically binds antigen with "high affinity" when the IQ is <5xl0^~9 M, and with "very high affinity" when the IQ is <5xl0 ^"10 M. In one embodiment, the ABP has a IQ of <10 ⁹ M. In one embodiment, the off-rate is <lxl0 ^{" 5}. In other embodiments, the ABPs will bind to human PCSK9 with a IQ of between about 10^~9 M and 10^"13 M, and in yet another embodiment the ABPs will bind with a IQ <5xl0^"10. As will be appreciated by one of skill in the art, in some embodiments, any or all of the antigen binding fragments can specifically bind to PCSK9. An antigen binding protein is "selective" when it binds to one target more tightly than it binds to a second target. "Antigen binding region" means a protein, or a portion of a protein, that specifically binds a specified antigen (e.g., a paratope). For example, that portion of an antigen binding protein that contains the amino acid residues that interact with an antigen and confer on the antigen binding protein its specificity and affinity for the antigen is referred to as "antigen binding region." An antigen binding region typically includes one or more "complementary binding regions" ("CDRs"). Certain antigen binding regions also include one or more "framework" regions. A "CDR" is an amino acid sequence that contributes to antigen binding specificity and affinity. "Framework" regions can aid in maintaining the proper conformation of the CDRs to promote binding between the antigen binding region and an antigen. Structurally, framework regions can be located in antibodies between CDRs. Examples of framework and CDR regions can be found in U.S. Pat. App. Pub. 20090142352. In some embodiments, the sequences for CDRs for the light chain of antibody 3B6 are as follows: CDR1

TLSSGYSSYEVD (SEQ ID NO: 1644); CDR2 VDTGGIVGSKGE (SEQ ID NO:1645); CDR3 GADHGSGTNFVVV (SEQ ID NO: 1646), and the FRs are as follows: FR1

QPVLTQPLFASASLGASVTLTC (SEQ ID NO: 1647); FR2 WYQQRPGKGPRFVMR (SEQ ID NO: 1648); FR3 GIPDRFSVLGSGLNRYLTIKNIQEEDESDYHC (SEQ ID NO: 1649); and FR4 FGGGTKLTVL (SEQ ID NO: 1650).

[00164] In certain aspects, recombinant antigen binding proteins that bind PCSK9, for example human PCSK9, are provided. In this context, a "recombinant antigen binding protein" is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as described herein. Methods and techniques for the production of recombinant proteins are well known in the art.

[00165] The term "antibody" refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes, for instance, chimeric, humanized, fully human, and bispecific antibodies. An "antibody" is a species of an antigen binding protein. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but in some instances can include fewer chains such as antibodies naturally occurring in camelids which can comprise only heavy chains. Antibodies can be derived solely from a single source, or can be "chimeric," that is, different portions of the antibody can be derived from two different antibodies as described further below. The antigen binding proteins, antibodies, or binding fragments can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof, respectively. In some embodiments, the term also encompasses peptibodies.

[00166] Naturally occurring antibody structural units typically comprise a tetramer. Each such tetramer typically is composed of two identical pairs of polypeptide chains, each pair having one full-length "light" (in certain embodiments, about 25 kDa) and one full-length "heavy" chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically includes a variable region of about 100 to 10 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that can be responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgMl and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2. Within full-length light and heavy chains, typically, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair typically form the antigen binding site.

[00167] The variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which can enable binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat

Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk, J. Mol. Biol, 196:901-917 (1987); Chothia et al, Nature, 342:878-883 (1989).

[00168] In certain embodiments, an antibody heavy chain binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody light chain binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an individual variable region specifically binds to an antigen in the absence of other variable regions. [00169] In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by solving the structure of the antibody and/or solving the structure of the antibody-ligand complex. In certain embodiments, that can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition and the contact definition.

[00170] The Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28: 214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account positions of certain structural loop regions. See, e.g., Chothia et al, J. Mol. Biol, 196: 901-17 (1986); Chothia et al, Nature, 342: 877-83 (1989). The AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g., Martin et al, Proc Natl Acad Sci (USA), 86:9268-9272 (1989); "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics SuppL, 3: 194-198 (1999). The contact definition is based on an analysis of the available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

[00171] By convention, the CDR regions in the heavy chain are typically referred to as HI, H2, and H3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus. The CDR regions in the light chain are typically referred to as LI, L2, and L3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus.

[00172] The term "light chain" includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, V_L, and a constant region domain, C_L. The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains. [00173] The term "heavy chain" includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes a variable region domain, V_H, and three constant region domains, C_Hi, C_H2, and C_H3- The V_H domain is at the amino-terminus of the polypeptide, and the C_H domains are at the carboxyl-terminus, with the C_R3 being closest to the carboxy -terminus of the polypeptide. Heavy chains can be of any isotype, including IgG (including IgGl, IgG2, IgG3 and IgG4 subtypes), IgA (including IgAl and IgA2 subtypes), IgM and IgE.

[00174] A bispecific or bifunctional antibody typically is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai et al, Clin. Exp. Immunol., 79: 315-321 (1990); Kostelny et al, J. Immunol, 148: 1547-1553 (1992).

[00175] Some species of mammals also produce antibodies having only a single heavy chain.

[00176] Each individual immunoglobulin chain is typically composed of several

"immunoglobulin domains," each consisting of roughly 90 to 110 amino acids and having a characteristic folding pattern. These domains are the basic units of which antibody polypeptides are composed. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains five heavy chains and five light chains. The heavy chain C region typically comprises one or more domains that can be responsible for effector function. The number of heavy chain constant region domains will depend on the isotype. IgG heavy chains, for example, contain three C region domains known as C_HI, C _H2 and C _H3. The antibodies that are provided can have any of these isotypes and subtypes. In certain embodiments of the present invention, an anti-PCSK9 antibody is of the IgG2 or IgG4 subtype.

[00177] The term "variable region" or "variable domain" refers to a portion of the light and/or heavy chains of an antibody, typically including approximately the amino-terminal 120 to 130 amino acids in the heavy chain and about 100 to 110 amino terminal amino acids in the light chain. In certain embodiments, variable regions of different antibodies differ extensively in amino acid sequence even among antibodies of the same species. The variable region of an antibody typically determines specificity of a particular antibody for its target [00178] The term "neutralizing antigen binding protein" or "neutralizing antibody" refers to an antigen binding protein or antibody, respectively, that binds to a ligand and prevents or reduces the biological effect of that ligand. This can be done, for example, by directly blocking a binding site on the ligand or by binding to the ligand and altering the ligand's ability to bind through indirect means (such as structural or energetic alterations in the ligand). In some embodiments, the term can also denote an antigen binding protein that prevents the protein to which it is bound from performing a biological function. In assessing the binding and/or specificity of an antigen binding protein, e.g., an antibody or

immunologically functional fragment thereof, an antibody or fragment can substantially inhibit binding of a ligand to its binding partner when an excess of antibody reduces the quantity of binding partner bound to the ligand by at least about 1-20, 20-30%, 30-40%, 40- 50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-98%, 98-99% or more (as measured in an in vitro competitive binding assay). In some embodiments, in the case of PCSK9 antigen binding proteins, such a neutralizing molecule can diminish the ability of PCSK9 to bind the LDLR. In some embodiments, the neutralizing ability is characterized and/or described via a competition assay. In some embodiments, the neutralizing ability is described in terms of an IC₅₀ or EC₅₀ value. In some embodiments, ABPs 27B2, 13H1, 13B5 and 3C4 are non-neutralizing ABPs, 3B6, 9C9 and 31A4 are weak neutralizers, and the remaining ABPs in Table 2 of U.S. Pat. App. Pub. 20090142352 (or Table 10, herein) are strong neutralizers. In some embodiments, the antibodies or antigen binding proteins neutralize by binding to PCSK9 and preventing PCSK9 from binding to LDLR (or reducing the ability of PCSK9 to bind to LDLR). In some embodiments, the antibodies or ABPs neutralize by binding to PCSK9, and while still allowing PCSK9 to bind to LDLR, preventing or reducing the PCSK9 mediated degradation of LDLR. Thus, in some embodiments, a neutralizing ABP or antibody can still permit PCSK9/LDLR binding, but will prevent (or reduce) subsequent PCSK9 involved degradation of LDLR.

[00179] The term "target" refers to a molecule or a portion of a molecule capable of being bound by an antigen binding protein. In certain embodiments, a target can have one or more epitopes. In certain embodiments, a target is an antigen. The use of "antigen" in the phrase "antigen binding protein" simply denotes that the protein sequence that comprises the antigen can be bound by an antibody. In this context, it does not require that the protein be foreign or that it be capable of inducing an immune response. [00180] The term "compete" when used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand, or a reference antibody) to a common antigen (e.g., PCSK9 or a fragment thereof). Numerous types of competitive binding assays can be used to determine if one antigen binding protein competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme

immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al, 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al, 1986, J. Immunol. 137:3614-3619) solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al, 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al, 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabelled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by

determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Usually the test antigen binding protein is present in excess. Antigen binding proteins identified by competition assay (competing antigen binding proteins) include antigen binding proteins binding to the same epitope as the reference antigen binding proteins and antigen binding proteins binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding protein for steric hindrance to occur. Additional details regarding methods for determining competitive binding are provided in the examples herein. Usually, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of a reference antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or more. In some instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[00181] The term "antigen" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antigen binding protein (including, e.g., an antibody or immunological functional fragment thereof). In some embodiments, the antigen is capable of being used in an animal to produce antibodies capable of binding to that antigen. An antigen can possess one or more epitopes that are capable of interacting with different antigen binding proteins, e.g., antibodies.

[00182] The term "epitope" includes any determinant capable being bound by an antigen binding protein, such as an antibody or to a T-cell receptor. An epitope is a region of an antigen that is bound by an antigen binding protein that targets that antigen, and when the antigen is a protein, includes specific amino acids that directly contact the antigen binding protein. Most often, epitopes reside on proteins, but in some instances can reside on other kinds of molecules, such as nucleic acids. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three dimensional structural characteristics, and/or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

Antigen Binding Proteins (ABPs) to PCSK9

[00183] Antigen binding proteins (ABPs) that bind PCSK9, including human PCSK9, are provided herein. In some embodiments, the antigen binding proteins provided are

polypeptides which comprise one or more complementary determining regions (CDRs), as described herein. In some antigen binding proteins, the CDRs are embedded into a

"framework" region, which orients the CDR(s) such that the proper antigen binding properties of the CDR(s) is achieved. In some embodiments, antigen binding proteins provided herein can interfere with, block, reduce or modulate the interaction between PCSK9 and LDLR. Such antigen binding proteins are denoted as "neutralizing." In some

embodiments, binding between PCSK9 and LDLR can still occur, even though the antigen binding protein is neutralizing and bound to PCSK9. For example, in some embodiments, the ABP prevents or reduces the adverse influence of PCSK9 on LDLR without blocking the LDLR binding site on PCSK9. Thus, in some embodiments, the ABP modulates or alters PCSK9's ability to result in the degradation of LDLR, without having to prevent the binding interaction between PCSK9 and LDLR. Such ABPs can be specifically described as "non- competitively neutralizing" ABPs. In some embodiments, the neutralizing ABP binds to PCSK9 in a location and/or manner that prevents PCSK9 from binding to LDLR. Such ABPs can be specifically described as "competitively neutralizing" ABPs. Both of the above neutralizers can result in a greater amount of free LDLR being present in a subject, which results in more LDLR binding to LDL (thereby reducing the amount of LDL in the subject). In turn, this results in a reduction in the amount of serum cholesterol present in a subject.

[00184] In some embodiments, the antigen binding proteins provided herein are capable of inhibiting PCSK9-mediated activity (including binding). In some embodiments, antigen binding proteins binding to these epitopes inhibit, inter alia, interactions between PCSK9 and LDLR and other physiological effects mediated by PCSK9. In some embodiments, the antigen binding proteins are human, such as fully human antibodies to PCSK9.

[00185] In some embodiments, the ABP binds to the catalytic domain of PCSK9. In some embodiments, the ABP binds to the mature form of PCSK9. In some embodiments the ABP binds in the prodomain of PCSK9. In some embodiments, the ABP selectively binds to the mature form of PCSK9. In some embodiments, the ABP binds to the catalytic domain in a manner such that PCSK9 cannot bind or bind as efficiently to LDLR. In some embodiments, the antigen binding protein does not bind to the c-terminus of the cataylytic domain. In some embodiments, the antigen binding protein does not bind to the n-terminus of the catalytic domain. In some embodiments, the ABP does not bind to the n- or c-terminus of the PCSK9 protein. In some embodiments, the ABP binds to any one of the epitopes bound by the antibodies discussed herein. In some embodiments, this can be determined by competition assays between the antibodies disclosed herein and other antibodies. In some embodiments, the ABP binds to an epitope bound by one of the antibodies described in Table 2 of U.S. Pat. App. Pub. 20090142352, which is herein incorporated by reference in its entirety for all purposes, or Table 10, herein. In some embodiments, the antigen binding proteins bind to a specific conformational state of PCSK9 so as to prevent PCSK9 from interacting with LDLR. In some embodiments, the ABP binds to the V domain of PCSK9. In some embodiments, the ABP binds to the V domain of PCSK9 and prevents (or reduces) PCSK9 from binding to LDLR. In some embodiments, the ABP binds to the V domain of PCSK9, and while it does not prevent (or reduce) the binding of PCSK9 to LDLR, the ABP prevents or reduces the adverse activities mediated through PCSK9 on LDLR.

[00186] The antigen binding proteins that are disclosed herein have a variety of utilities. Some of the antigen binding proteins, for instance, are useful in specific binding assays, affinity purification of PCSK9, in particular human PCSK9 or its ligands and in screening assays to identify other antagonists of PCSK9 activity. Some of the antigen binding proteins are useful for inhibiting binding of PCSK9 to LDLR, or inhibiting PCSK9-mediated activities.

[00187] The antigen binding proteins can be used in a variety of therapeutic applications, as explained herein. For example, in some embodiments the PCSK9 antigen binding proteins are useful for treating conditions associated with PCSK9, such as cholesterol related disorders (or "serum cholesterol related disorders") such as hypercholesterolemia, as further described herein. Other uses for the antigen binding proteins include, for example, diagnosis of PCSK9-associated diseases or conditions and screening assays to determine the presence or absence of PCSK9. Some of the antigen binding proteins described herein are useful in treating consequences, symptoms, and/or the pathology associated with PCSK9 activity.

[00188] In some embodiments, the antigen binding proteins that are provided comprise one or more CDRs (e.g., 1, 2, 3, 4, 5 or 6 CDRs). In some embodiments, the antigen binding protein comprises (a) a polypeptide structure and (b) one or more CDRs that are inserted into and/or joined to the polypeptide structure. The polypeptide structure can take a variety of different forms. For example, it can be, or comprise, the framework of a naturally occurring antibody, or fragment or variant thereof, or can be completely synthetic in nature. Examples of various polypeptide structures are further described below.

[00189] In certain embodiments, the polypeptide structure of the antigen binding proteins is an antibody or is derived from an antibody, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and portions or fragments of each, respectively. In some instances, the antigen binding protein is an immunological fragment of an antibody (e.g., a Fab, a Fab', a F(ab')₂, or a scFv). The various structures are further described and defined herein.

[00190] Certain of the antigen binding proteins as provided herein specifically and/or selectively bind to human PCSK9. In some embodiments, the antigen binding protein specifically and/or selectively binds to human PCSK9 protein having and/or consisting of residues 153-692 of human PCSK9. In some embodiments the ABP specifically and/or selectively binds to human PCSK9 having and/or consisting of residues 31-152 of human PCSK9. In some embodiments, the ABP selectively binds to a human PCSK9 protein. In some embodiments, the antigen binding protein specifically binds to at least a fragment of the PCSK9 protein and/or a full length PCSK9 protein, with or without a signal sequence. [00191] In embodiments where the antigen binding protein is used for therapeutic applications, an antigen binding protein can inhibit, interfere with or modulate one or more biological activities of PCSK9. In one embodiment, an antigen binding protein binds specifically to human PCSK9 and/or substantially inhibits binding of human PCSK9 to LDLR by at least about 20%-40%, 40-60%, 60-80%, 80-85%, or more (for example, by measuring binding in an in vitro competitive binding assay). Some of the antigen binding proteins that are provided herein are antibodies. In some embodiments, the ABP has a Kd of less (binding more tightly) than 10^"7, 10^"8, 10^"9, 10^"10, 10 ¹¹, 10^"12, 10^"13 M. In some embodiments, the ABP has an IC₅₀ for blocking the binding of LDLR to PCSK9 (D374Y, high affinity variant) of less than 1 microM, 1000 nM to 100 nM, lOOmM to 10 nM, 1 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM.

[00192] One example of an IgG2 heavy chain constant domain of an anti-PCSK9 antibody of the present invention has the amino acid sequence as shown in Table 9 below.

[00193] One example of an IgG4 heavy chain constant domain of an anti-PCSK9 antibody of the present invention has the amino acid sequence as shown in Table 9 below.

[00194] One example of a kappa light chain constant domain of an anti-PCSK9 antibody has the amino acid sequence as shown in Table 9 below.

[00195] One example of a lambda light chain constant domain of an anti-PC SK9 antibody has the amino acid sequence as shown in Table 9 below.

[00196] Variable regions of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FR) joined by three hypervariable regions, more often called "complementarity determining regions" or CDRs. The CDRs from the two chains of each heavy chain/light chain pair mentioned above typically are aligned by the framework regions to form a structure that binds specifically with a specific epitope on the target protein (e.g., PCSK9). From N-terminal to C-terminal, naturally-occurring light and heavy chain variable regions both typically conform with the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been devised for assigning numbers to amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.), or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al, 1989, Nature 342:878-883. [00197] Various heavy chain and light chain variable regions are provided in U.S. Pat. App. Pub. 20090142352 (see FIGS. 2A-3JJ and 3LL-3BBB of U.S. Pat. App. Pub.

20090142352). In some embodiments, each of these variable regions can be attached to the above heavy and light chain constant regions to form a complete antibody heavy and light chain, respectively. Further, each of the so generated heavy and light chain sequences can be combined to form a complete antibody structure.

[00198] Specific examples of some of the variable regions of the light and heavy chains of the antibodies that are provided and their corresponding amino acid sequences are

summarized in Table 2 of U.S. Pat. App. Pub. 20090142352, and Table 10, herein. These antibodies include: 30A4, 3C4, 23B5, 25G4, 31H4, 27B2, 25A7, 27H5, 26H5, 31D1.20D10, 27E7, 30B9, 19H9, 26E10, 21B12, 17C2, 23G1, 13H1, 9C9, 9H6, 31A4, 1A12, 16F12, 22E2, 27A6, 28B12, 28D6, 31G11, 13B5, 31B12 and 3B6.

[00199] Again, each of the exemplary variable heavy chains listed in Table 2 of U.S. Pat. App. Pub. 20090142352 (or Table 10, herein) can be combined with any of the exemplary variable light chains shown in Table 2 of U.S. Pat. App. Pub. 20090142352 (or Table 10, herein) to form an antibody. Table 2 of U.S. Pat. App. Pub. 20090142352 and Table 10, herein, shows exemplary light and heavy chain pairings found in several of the antibodies disclosed herein. In some instances, the antibodies include at least one variable heavy chain and one variable light chain from those listed in Table 2 of U.S. Pat. App. Pub. 20090142352 or Table 10, herein. In other instances, the antibodies contain two identical light chains and two identical heavy chains. As an example, an antibody or antigen binding protein can include a heavy chain and a light chain, two heavy chains, or two light chains. In some embodiments the antigen binding protein comprises (and/or consists) of 1, 2, and/or 3 heavy and/or light CDRs from at least one of the sequences listed in Table 2 of U.S. Pat. App. Pub. 20090142352 or Table 10, herein. In some embodiments, all 6 CDRs (CDRl-3 from the light (CDRLl, CDRL2, CDRL3) and CDRl-3 from the heavy (CDRH1, CDRH2, and CDRH3)) are part of the ABP. In some embodiments, 1, 2, 3, 4, 5, or more CDRs are included in the ABP. In some embodiments, one heavy and one light CDR from the CDRs in the sequences in Table 2 of U.S. Pat. App. Pub. 20090142352 (or Table 10, herein) is included in the ABP. In some embodiments, additional sections are also included in the ABP. Optional light chain variable sequences (including CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4) can be selected from the following of Table 2 of U.S. Pat. App. Pub. 20090142352 (or Table 10, herein): 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. Optional heavy chain variable sequences (including CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4) can be selected from the following of Table 2 of U.S. Pat. App. Pub. 20090142352 (or Table 10, herein): 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60. As will be appreciated by one of skill in the art, no more than one such sequence need actually be used in the creation of an antibody or ABP. Indeed, in some embodiments, only one or neither of the specific heavy or light chain nucleic acids need be present.

[00200] In some embodiments, the ABP is encoded by a nucleic acid sequence that can encode any of the protein sequences in Table 2 of U.S. Pat. App. Pub. 20090142352, or Table 10, herein.

[00201] In some embodiments, the ABP binds selectively to the form of PCSK9 that binds to LDLR (e.g., the autocatalyzed form of the molecule). In some embodiments, the antigen binding protein does not bind to the c-terminus of the cataylytic domain (e.g., the 5, 5-10, 10- 15, 15-20, 20-25, 25-30, 30-40 most amino acids in the c-terminus). In some embodiments, the antigen binding protein does not bind to the n-terminus of the catalytic domain (e.g., the 5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40 most amino acids in the n-terminus). In some embodiments, the ABP binds to amino acids within amino acids 1-100 of the mature form of PCSK9. In some embodiments, the ABP binds to amino acids within (and/or amino acid sequences consisting of) amino acids 31-100, 100-200, 31-152, 153-692, 200-300, 300-400, 452-683, 400-500, 500-600, 31-692, 31-449, and/or 600-692. In some embodiments, the ABP binds to the catalytic domain. In some embodiments, the neutralizing and/or non-neutralizing ABP binds to the prodomain. In some embodiments, the ABP binds to both the catalytic and pro domains. In some embodiments, the ABP binds to the catalytic domain so as to obstruct an area on the catalytic domain that interacts with the pro domain. In some embodiments, the ABP binds to the catalytic domain at a location or surface that the pro-domain interacts with as outlined in Piper et al. (Structure 15: 1-8 (2007), the entirety of which is hereby

incorporated by reference, including the structural representations therein). In some embodiments, the ABP binds to the catalytic domain and restricts the mobility of the prodomain. In some embodiments, the ABP binds to the catalytic domain without binding to the pro-domain. In some embodiments, the ABP binds to the catalytic domain, without binding to the pro-domain, while preventing the pro-domain from reorienting to allow PCSK9 to bind to LDLR. In some embodiments, the ABP binds in the same epitope as those surrounding residues 149-152 of the pro-domain in Piper et al. In some embodiments, the ABPs bind to the groove (as outlined in Piper et al.) on the V domain. In some embodiments, the ABPs bind to the histidine-rich patch proximal to the groove on the V domain. In some embodiments, such antibodies (that bind to the V domain) are not neutralizing. In some embodiments, antibodies that bind to the V domain are neutralizing. In some embodiments, the neutralizing ABPs prevent the binding of PCSK9 to LDLR. In some embodiments, the neturalizing ABPs, while preventing the PCSK9 degradation of LDLR, do not prevent the binding of PCSK9 to LDLR (for example ABP 31A4). In some embodiments, the ABP binds to or blocks at least one of the histidines depicted in FIG. 4 of the Piper et al. paper. In some embodiments, the ABP blocks the catalytic triad in PCSK9.

[00202] In some embodiments, the antibody binds selectively to variant PCSK9 proteins, e.g., D374Y over wild type PCSK9. In some embodiments, these antibodies bind to the variant at least twice as strongly as the wild type, and preferably 2-5, 5-10, 10-100, 100-1000, 1000-10,000 fold or more to the mutant than the wild type (as measured via a K.sub.d). In some embodiments, the antibody selectively inhibits variant D374Y PCSK9 from interacting with LDLR over wild type PCSK9's ability to interact with LDLR. In some embodiments, these antibodies block the variant's ability to bind to LDLR more strongly than the wild type's ability, e.g., at least twice as strongly as the wild type, and preferably 2-5, 5-10, 10-100, 100- 1000 fold or more to the mutant than the wild type (as measured via an IC₅₀). In some embodiments, the antibody binds to and neutralizes both wild type PCSK9 and variant forms of PCSK9, such as D374Y at similar levels. In some embodiments, the antibody binds to PCSK9 to prevent variants of LDLR from binding to PCSK9. In some embodiments, the variants of LDLR are at least 50% identical to human LDLR. It is noted that variants of LDLR are known to those of skill in the art (e.g., Brown M S et al, "Calcium cages, acid baths and recycling receptors" Nature 388: 629-630, 1997). In some embodiments, the ABP can raise the level of effective LDLR in heterozygote familial hypercholesterolemia (where a loss-of function variant of LDLR is present).

[00203] In some embodiments, the ABP binds to (but does not block) variants of PCSK9 that are at least 50%, 50-60, 60-70, 70-80, 80-90, 90-95, 95-99, or greater percent identity to a form of PCSK9. In some embodiments, the ABP binds to (but does not block) variants of PCSK9 that are at least 50%, 50-60, 60-70, 70-80, 80-90, 90-95, 95-99, or greater percent identity to a mature form of PCSK9. In some embodiments, the ABP binds to and prevents variants of PCSK9 that are at least 50%, 50-60, 60-70, 70-80, 80-90, 90-95, 95-99, or greater percent identity to a form of PCSK9 from interacting with LDLR. In some embodiments, the ABP binds to and prevents variants of PCSK9 that are at least 50, 50-60, 60-70, 70-80, 80-90, 90-95, 95-99, or greater percent identity to a mature form of PCSK9 from interacting with LDLR. In some embodiments, the variant of PCSK9 is a human variant, such as variants at position 474, E620G, and/or E670G. In some embodiments, the amino acid at position 474 is valine (as in other humans) or threonine (as in cyno and mouse). Given the cross-reactivity data presented herein, it is believed that the present antibodies will readily bind to the above variants.

[00204] In some embodiments, the ABP binds to an epitope bound by one of the antibodies described in Table 2 of U.S. Pat. App. Pub. 20090142352 or Table 10, herein. In some embodiments, the antigen binding proteins bind to a specific conformational state of PCSK9 so as to prevent PCSK9 from interacting with LDLR.

[00205] In certain embodiments, the effective amount of an antigen binding protein to PCSK9, with or without at least one additional therapeutic agent, to be employed will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, will thus vary depending, in part, upon the molecule delivered, the indication for which an antigen binding protein to PCSK9, with or without at least one additional therapeutic agent, is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. In certain embodiments, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. In certain embodiments, a typical dosage can range from about 0.1 μg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In certain embodiments, the dosage can range from 0.1 μg/kg up to about 100 mg/kg; or 1 μg/kg up to about 100 mg/kg; or 5 μg/kg up to about 100 mg/kg. For example, the ABP can be administered at 0.01 mg/kg, 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.

[00206] In another embodiment, the ABP dosage is between 0.01 and 0.2 mg/kg. For example, the ABP can be administered at a dose of 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg 0.08 mg/kg 0.09 mg/kg , 0.10 mg/kg, 0.11 mg/kg, 0.12 mg/kg, 0.13 mg/kg, 0.14 mg/kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg, 0.18 mg/kg, 0.19 mg/kg, or 0.20 mg/kg.

[00207] In one embodiment, the ABP dosage is between 0.2 mg/kg and 2.0 mg/kg. For example, the ABP can be administered at a dose of 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mg/kg.

[00208] The ABP can be administered at a dose of 0.03, 0.1, 0.3, or 1.3, or 3.0 mg/kg.

[00209] In certain embodiments, the frequency of dosing will take into account the pharmacokinetic parameters of an antigen binding protein to PCSK9 and/or any additional therapeutic agents in a formulation used. In certain embodiments, a clinician will administer the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data. In some embodiments, the amount and frequency of administration can take into account the desired cholesterol level (serum and/or total) to be obtained and the subject's present cholesterol level, LDL level, and/or LDLR levels, all of which can be obtained by methods that are well known to those of skill in the art.

Humanized Antigen Binding Proteins (e.g. Antibodies)

[00210] As described herein, an antigen binding protein to PCSK9 can comprise a humanized antibody and/or part thereof. An important practical application of such a strategy is the "humanization" of the mouse humoral immune system.

[00211] In certain embodiments, a humanized antibody is substantially non-immunogenic in humans. In certain embodiments, a humanized antibody has substantially the same affinity for a target as an antibody from another species from which the humanized antibody is derived. See, e.g., U.S. Pat. No. 5,530,101, U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No. 5,585,089.

[00212] In certain embodiments, amino acids of an antibody variable domain that can be modified without diminishing the native affinity of the antigen binding domain while reducing its immunogenicity are identified. See, e.g., U.S. Pat. Nos. 5,766,886 and 5,869,619.

[00213] In certain embodiments, modification of an antibody by methods known in the art is typically designed to achieve increased binding affinity for a target and/or to reduce immunogenicity of the antibody in the recipient. In certain embodiments, humanized antibodies are modified to eliminate glycosylation sites in order to increase affinity of the antibody for its cognate antigen. See, e.g., Co et al, Mol. Immunol, 30: 1361-1367 (1993). In certain embodiments, techniques such as "reshaping," "hyperchimerization," or

"veneering/resurfacing" are used to produce humanized antibodies. See, e.g., Vaswami et al, Annals of Allergy, Asthma, & Immunol. 81 : 105 (1998); Roguska et al, Prot. Engineer., 9:895-904 (1996); and U.S. Pat. No. 6,072,035. In certain such embodiments, such techniques typically reduce antibody immunogenicity by reducing the number of foreign residues, but do not prevent anti-idiotypic and anti-allotypic responses following repeated administration of the antibodies. Certain other methods for reducing immunogenicity are described, e.g., in Gilliland et al, J. Immunol, 62(6): 3663-71 (1999).

[00214] In certain instances, humanizing antibodies results in a loss of antigen binding capacity. In certain embodiments, humanized antibodies are "back mutated." In certain such embodiments, the humanized antibody is mutated to include one or more of the amino acid residues found in the donor antibody. See, e.g., Saldanha et al, Mol Immunol 36:709-19 (1999).

[00215] In certain embodiments the complementarity determining regions (CDRs) of the light and heavy chain variable regions of an antibody to PCSK9 can be grafted to framework regions (FRs) from the same, or another, species. In certain embodiments, the CDRs of the light and heavy chain variable regions of an antibody to PCSK9 can be grafted to consensus human FRs. To create consensus human FRs, in certain embodiments, FRs from several human heavy chain or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. In certain embodiments, the FRs of an antibody to PCSK9 heavy chain or light chain are replaced with the FRs from a different heavy chain or light chain. In certain embodiments, rare amino acids in the FRs of the heavy and light chains of an antibody to PCSK9 are not replaced, while the rest of the FR amino acids are replaced. Rare amino acids are specific amino acids that are in positions in which they are not usually found in FRs. In certain embodiments, the grafted variable regions from an antibody to PCSK9 can be used with a constant region that is different from the constant region of an antibody to PCSK9. In certain embodiments, the grafted variable regions are part of a single chain Fv antibody. CDR grafting is described, e.g., in U.S. Pat. Nos. 6,180,370, 6,054,297, 5,693,762, 5,859,205, 5,693,761, 5,565,332, 5,585,089, and 5,530,101, and in Jones et al, Nature, 321 : 522-525 (1986); Riechmann et al, Nature, 332: 323-327 (1988); Verhoeyen et al, Science, 239: 1534- 1536 (1988), Winter, FEBS Letts., 430:92-94 (1998), which are hereby incorporated by reference for any purpose. Human Antigen Binding Proteins (e.g., Antibodies)

[00216] As described herein, an antigen binding protein that binds to PCSK9 can comprise a human (i.e., fully human) antibody and/or part thereof. In certain embodiments, nucleotide sequences encoding, and amino acid sequences comprising, heavy and light chain

immunoglobulin molecules, particularly sequences corresponding to the variable regions are provided. In certain embodiments, sequences corresponding to complementarity determining regions (CDR's), specifically from CDRl through CDR3, are provided. According to certain embodiments, a hybridoma cell line expressing such an immunoglobulin molecule is provided. According to certain embodiments, a hybridoma cell line expressing such a monoclonal antibody is provided. In certain embodiments a hybridoma cell line is selected from at least one of the cell lines described in Table 2 of U.S. Pat. App. Pub. 20090142352 (or Table 10, herein), e.g., 21B12, 16F12 and 31H4. In certain embodiments, a purified human monoclonal antibody to human PCSK9 is provided.

[00217] One can engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci in anticipation that such mice would produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can preserve the large variable gene diversity as well as the proper regulation of antibody production and expression. By exploiting the mouse machinery for antibody diversification and selection and the lack of immunological tolerance to human proteins, the reproduced human antibody repertoire in these mouse strains can yield high affinity fully human antibodies against any antigen of interest, including human antigens. Using the hybridoma technology, antigen- specific human MAbs with the desired specificity can be produced and selected. Certain exemplary methods are described in WO 98/24893, U.S. Pat. No. 5,545,807, EP 546073, and EP 546073.

[00218] In certain embodiments, one can use constant regions from species other than human along with the human variable region(s).

[00219] The ability to clone and reconstruct megabase sized human loci in yeast artificial chromosomes (YACs) and to introduce them into the mouse germline provides an approach to elucidating the functional components of very large or crudely mapped loci as well as generating useful models of human disease. Furthermore, the utilization of such technology for substitution of mouse loci with their human equivalents could provide insights into the expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression. [00220] Human antibodies avoid some of the problems associated with antibodies that possess murine or rat variable and/or constant regions. The presence of such murine or rat derived proteins can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by a patient. In order to avoid the utilization of murine or rat derived antibodies, fully human antibodies can be generated through the introduction of functional human antibody loci into a rodent, other mammal or animal so that the rodent, other mammal or animal produces fully human antibodies.

[00221] Humanized antibodies are those antibodies that, while initially starting off containing antibody amino acid sequences that are not human, have had at least some of these nonhuman antibody amino acid sequences replaced with human antibody sequences. This is in contrast with human antibodies, in which the antibody is encoded (or capable of being encoded) by genes possessed a human.

Antigen Binding Protein Variants

[00222] Other antibodies that are provided are variants of the ABPs listed above formed by combination or subparts of the variable heavy and variable light chains shown in Table 2 of U.S. Pat. App. Pub. 20090142352 (or Table 10, herein) and comprise variable light and/or variable heavy chains that each have at least 50%, 50-60, 60-70, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or above 99% identity to the amino acid sequences of the sequences in Table 2 of U.S. Pat. App. Pub. 20090142352 or Table 10, herein (either the entire sequence or a subpart of the sequence, e.g., one or more CDR). In some instances, such antibodies include at least one heavy chain and one light chain, whereas in other instances the variant forms contain two identical light chains and two identical heavy chains (or subparts thereof). In some embodiments, a sequence comparison can be used in order to identify sections of the antibodies that can be modified by observing those variations that impact binding and those variations that do not appear to impact binding. For example, by comparing similar sequences, one can identify those sections (e.g., particular amino acids) that can be modified and how they can be modified while still retaining (or improving) the functionality of the ABP. CDRs can be defined based upon a hybrid combination of the Chothia method (based on the location of the structural loop regions, see, e.g., "Standard conformations for the canonical structures of immunoglobulins," Bissan Al-Lazikani, Arthur M. Lesk and Cyrus Chothia, Journal of Molecular Biology, 273(4): 927-948, 7 November (1997)) and the Kabat method (based on sequence variability, see, e.g., Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242, Kabat et al, (1991)). Each residue determined by either method, can be included in the final list of CDR residues.

[00223] In light of the present disclosure, a skilled artisan will be able to determine suitable variants of the ABPs as set forth herein using well-known techniques. In certain embodiments, one skilled in the art can identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In certain embodiments, one can identify residues and portions of the molecules that are conserved among similar polypeptides. In certain embodiments, even areas that can be important for biological activity or for structure can be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

[00224] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues which are important for activity or structure in similar proteins. One skilled in the art can opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[00225] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar ABPs. In view of such information, one skilled in the art can predict the alignment of amino acid residues of an antibody with respect to its three dimensional structure. In certain embodiments, one skilled in the art can choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues can be involved in important interactions with other molecules. Moreover, one skilled in the art can generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change can be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations. [00226] A number of scientific publications have been devoted to the prediction of secondary structure. See Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996), Chou et al, Biochemistry, 13(2):222-245 (1974); Chou et al, Biochemistry, 113(2):211-222 (1974); Chou et al, Adv. Enzymol. Relat. Areas Mol. Biol, 47:45-148 (1978); Chou et al, Ann. Rev. Biochem., 47:251-276 and Chou et al, Biophys. J., 26:367-384 (1979). Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural database (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure. See Holm et al, Nucl. Acid. Res., 27(l):244-247 (1999). It has been suggested (Brenner et al, Curr. Op. Struct. Biol, 7(3):369-376 (1997)) that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved, structural prediction will become dramatically more accurate.

[00227] Additional methods of predicting secondary structure include "threading" (Jones, D., Curr. Opin. Struct. Biol, 7(3):377-87 (1997); Sippl et al, Structure, 4(1): 15-19 (1996)), "profile analysis" (Bowie et al, Science, 253:164-170 (1991); Gribskov et al, Meth. Enzym., 183: 146-159 (1990); Gribskov et al, Proc. Nat. Acad. Sci. USA, 84(13):4355-4358 (1987)), and "evolutionary linkage" (See Holm, supra (1999), and Brenner, supra (1997)).

[00228] In certain embodiments, antigen binding protein variants include glycosylation variants wherein the number and/or type of glycosylation site has been altered compared to the amino acid sequences of a parent polypeptide. In certain embodiments, protein variants comprise a greater or a lesser number of N-linked glycosylation sites than the native protein. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created. Additional preferred antibody variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the parent amino acid sequence. Cysteine variants can be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.

[00229] According to certain embodiments, amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and/or (4) confer or modify other physiocochemical or functional properties on such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be made in the naturally-occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). In certain embodiments, a conservative amino acid substitution typically may not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden & J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al, Nature, 354: 105 (1991), which are each incorporated herein by reference.

[00230] In some embodiments, the variants are variants of the nucleic acid sequences of the ABPs disclosed herein. One of skill in the art will appreciate that the above discussion can be used for identifying, evaluating, and/creating ABP protein variants and also for nucleic acid sequences that can encode for those protein variants. Thus, nucleic acid sequences encoding for those protein variants (as well as nucleic acid sequences that encode for the ABPs in Table 2 of U.S. Pat. App. Pub. 20090142352, or Table 10, herein, but are different from those explicitly disclosed herein) are contemplated.

[00231] In some embodiments, the antibody (or nucleic acid sequence encoding it) is a variant if the nucleic acid sequence that encodes the particular ABP (or the nucleic acid sequence itself) can selectively hybridize to any of the nucleic acid sequences that encode the proteins in Table 2 of U.S. Pat. App. Pub. 20090142352 (or Table 10, herein) under stringent conditions. In one embodiment, suitable moderately stringent conditions include prewashing in a solution of 5xSSC; 0.5% SDS, 1.0 mM EDTA (pH 8:0); hybridizing at 50°C, -65 °C, 5xSSC, overnight or, in the event of cross-species homology, at 45 °C with 0.5xSSC;

followed by washing twice at 65 °C for 20 minutes with each of 2x, 0.5x and 0.2xSSC containing 0.1% SDS. Such hybridizing DNA sequences are also within the scope of this invention, as are nucleotide sequences that, due to code degeneracy, encode an antibody polypeptide that is encoded by a hybridizing DNA sequence and the amino acid sequences that are encoded by these nucleic acid sequences. In some embodiments, variants of CDRs include nucleic acid sequences and the amino acid sequences encoded by those sequences, that hybridize to one or more of the CDRs within the sequences noted in U.S. Pat. App. Pub. 20090142352. The phrase "selectively hybridize" referred to in this context means to detectably and selectively bind. Polynucleotides, oligonucleotides and fragments thereof in accordance with the invention selectively hybridize to nucleic acid strands under

hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions as known in the art and discussed herein. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments of the invention and a nucleic acid sequence of interest will be at least 80%, and more typically with preferably increasing homologies of at least 85%, 90%, 95%, 99%, and 100%. Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O., in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, pp. 1-10. The two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program. The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TAT AC" corresponds to a reference sequence "TAT AC" and is complementary to a reference sequence "GTATA."

Preparation of Antigen Binding Proteins (e.g., Antibodies)

[00232] In certain embodiments, antigen binding proteins (such as antibodies) are produced by immunization with an antigen (e.g., PCSK9). In certain embodiments, antibodies can be produced by immunization with full-length PCSK9, a soluble form of PCSK9, the catalytic domain alone, a mature form of PCSK9, a splice variant form of PCSK9, or a fragment thereof. In certain embodiments, the antibodies of the invention can be polyclonal or monoclonal, and/or can be recombinant antibodies. In certain embodiments, antibodies of the invention are human antibodies prepared, for example, by immunization of transgenic animals capable of producing human antibodies (see, for example, PCT Published Application No. WO 93/12227).

[00233] In certain embodiments, certain strategies can be employed to manipulate inherent properties of an antibody, such as the affinity of an antibody for its target. Such strategies include, but are not limited to, the use of site-specific or random mutagenesis of the polynucleotide molecule encoding an antibody to generate an antibody variant. In certain embodiments, such generation is followed by screening for antibody variants that exhibit the desired change, e.g. increased or decreased affinity.

[00234] In certain embodiments, the amino acid residues targeted in mutagenic strategies are those in the CDRs. In certain embodiments, amino acids in the framework regions of the variable domains are targeted. In certain embodiments, such framework regions have been shown to contribute to the target binding properties of certain antibodies. See, e.g., Hudson, Curr. Opin. Biotech., 9:395-402 (1999) and references therein.

[00235] In certain embodiments, smaller and more effectively screened libraries of antibody variants are produced by restricting random or site-directed mutagenesis to hyper- mutation sites in the CDRs, which are sites that correspond to areas prone to mutation during the somatic affinity maturation process. See, e.g., Chowdhury & Pastan, Nature Biotech., 17: 568-572 (1999) and references therein. In certain embodiments, certain types of DNA elements can be used to identify hyper-mutation sites including, but not limited to, certain direct and inverted repeats, certain consensus sequences, certain secondary structures, and certain palindromes. For example, such DNA elements that can be used to identify hyper- mutation sites include, but are not limited to, a tetrabase sequence comprising a purine (A or G), followed by guainine (G), followed by a pyrimidine (C or T), followed by either adenosine or thymidine (A or T) (i.e., A/G-G-C/T-A/T). Another example of a DNA element that can be used to identify hyper-mutation sites is the serine codon, A-G-C/T.

Preparation of Fully Human ABPs (e.g., Antibodies)

[00236] In certain embodiments, a phage display technique is used to generate monoclonal antibodies. In certain embodiments, such techniques produce fully human monoclonal antibodies. In certain embodiments, a polynucleotide encoding a single Fab or Fv antibody fragment is expressed on the surface of a phage particle. See, e.g., Hoogenboom et al, J. Mol. Biol, 227: 381 (1991); Marks et al, J Mol Biol 222: 581 (1991); U.S. Pat. No. 5,885,793. In certain embodiments, phage are "screened" to identify those antibody fragments having affinity for target. Thus, certain such processes mimic immune selection through the display of antibody fragment repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to target. In certain such procedures, high affinity functional neutralizing antibody fragments are isolated. In certain such embodiments

(discussed in more detail below), a complete repertoire of human antibody genes is created by cloning naturally rearranged human V genes from peripheral blood lymphocytes. See, e.g., Mullinax et al, Proc Natl Acad Sci (USA), 87: 8095-8099 (1990).

[00237] According to certain embodiments, antibodies of the invention are prepared through the utilization of a transgenic mouse that has a substantial portion of the human antibody producing genome inserted but that is rendered deficient in the production of endogenous, murine antibodies. Such mice, then, are capable of producing human

immunoglobulin molecules and antibodies and are deficient in the production of murine immunoglobulin molecules and antibodies. Technologies utilized for achieving this result are disclosed in the patents, applications and references disclosed in the specification, herein. In certain embodiments, one can employ methods such as those disclosed in PCT Published Application No. WO 98/24893 or in Mendez et al, Nature Genetics, 15:146-156 (1997), which are hereby incorporated by reference for any purpose.

[00238] Generally, fully human monoclonal ABPs (e.g., antibodies) specific for PCSK9 can be produced as follows. Transgenic mice containing human immunoglobulin genes are immunized with the antigen of interest, e.g. PCSK9, lymphatic cells (such as B-cells) from the mice that express antibodies are obtained. Such recovered cells are fused with a myeloid- type cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. In certain embodiments, the production of a hybridoma cell line that produces antibodies specific to PCSK9 is provided.

[00239] In certain embodiments, fully human antibodies are produced by exposing human splenocytes (B or T cells) to an antigen in vitro, and then reconstituting the exposed cells in an immunocompromised mouse, e.g. SCID or nod/SCID. See, e.g., Brams et al, J. Immunol. 160: 2051-2058 (1998); Carballido et al, Nat. Med., 6: 103-106 (2000). In certain such approaches, engraftment of human fetal tissue into SCID mice (SCID-hu) results in long-term hematopoiesis and human T-cell development. See, e.g., McCune et al, Science, 241 : 1532- 1639 (1988); lfversen et al., Sem. Immunol., 8:243-248 (1996). In certain instances, humoral immune response in such chimeric mice is dependent on co-development of human T-cells in the animals. See, e.g., Martensson et al., Immunol., 83: 1271-179 (1994). In certain approaches, human peripheral blood lymphocytes are transplanted into SCID mice. See, e.g., Mosier et al, Nature, 335:256-259 (1988). In certain such embodiments, when such transplanted cells are treated either with a priming agent, such as Staphylococcal Enterotoxin A (SEA), or with anti-human CD40 monoclonal antibodies, higher levels of B cell production is detected. See, e.g., Martensson et al, Immunol, 84: 224-230 (1995); Murphy et al, Blood, 86: 1946-1953 (1995).

[00240] Thus, in certain embodiments, fully human antibodies can be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells. In other embodiments, antibodies can be produced using the phage display techniques described herein.

[00241] The antibodies described herein were prepared through the utilization of the XenoMouse™ technology, as described herein. Such mice, then, are capable of producing human immunoglobulin molecules and antibodies and are deficient in the production of murine immunoglobulin molecules and antibodies. Technologies utilized for achieving the same are disclosed in the patents, applications, and references disclosed in the background section herein. In particular, however, a preferred embodiment of transgenic production of mice and antibodies therefrom is disclosed in U.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996 and International Patent Application Nos. WO 98/24893, published Jun. 11, 1998 and WO 00/76310, published Dec. 21, 2000, the disclosures of which are hereby incorporated by reference. See also Mendez et al, Nature Genetics, 15 :146-156 (1997), the disclosure of which is hereby incorporated by reference.

[00242] Through the use of such technology, fully human monoclonal antibodies to a variety of antigens have been produced. Essentially, XenoMouse™ lines of mice are immunized with an antigen of interest (e.g. PCSK9), lymphatic cells (such as B-cells) are recovered from the hyper-immunized mice, and the recovered lymphocytes are fused with a myeloid-type cell line to prepare immortal hybridoma cell lines. These hybridoma cell lines are screened and selected to identify hybridoma cell lines that produced antibodies specific to the antigen of interest. Provided herein are methods for the production of multiple hybridoma cell lines that produce antibodies specific to PCSK9 Further, provided herein are

characterization of the antibodies produced by such cell lines, including nucleotide and amino acid sequence analyses of the heavy and light chains of such antibodies.

[00243] The production of the XenoMouse™ strains of mice is further discussed and delineated in U.S. patent application Ser. Nos. 07/466,008, filed Jan. 12, 1990, 07/610,515, filed Nov. 8, 1990, 07/919,297, filed Jul. 24, 1992, 07/922,649, filed Jul. 30, 1992,

08/031,801, filed Mar. 15, 1993, 08/112,848, filed Aug. 27, 1993, 08/234,145, filed Apr. 28, 1994, 08/376,279, filed Jan. 20, 1995, 08/430, 938, filed Apr. 27, 1995, 08/464,584, filed Jun. 5, 1995, 08/464,582, filed Jun. 5, 1995, 08/463,191, filed Jun. 5, 1995, 08/462,837, filed Jun. 5, 1995, 08/486,853, filed Jun. 5, 1995, 08/486,857, filed Jun. 5, 1995, 08/486,859, filed Jun. 5, 1995, 08/462,513, filed Jun. 5, 1995, 08/724,752, filed Oct. 2, 1996, 08/759,620, filed Dec. 3, 1996, U.S. Publication 2003/0093820, filed Nov. 30, 2001 and U.S. Pat. Nos.

6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See also European Patent No., EP 0 463 151 Bl, grant published Jun. 12, 1996, International Patent Application No., WO 94/02602, published Feb. 3, 1994, International Patent Application No., WO 96/34096, published Oct. 31, 1996, WO 98/24893, published Jun. 11, 1998, WO 00/76310, published Dec. 21, 2000. The disclosures of each of the above-cited patents, applications, and references are hereby incorporated by reference in their entirety.

[00244] In an alternative approach, others, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more V_H genes, one or more DH genes, one or more JH genes, a mu constant region, and usually a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. This approach is described in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each to Lonberg & Kay, U.S. Pat. Nos. 5,591,669 and 6,023.010 to Krimpenfort & Berns, U.S. Pat. Nos.

5,612,205, 5,721,367, and 5,789,215 to Bems et al, and U.S. Pat. No. 5,643,763 to Choi & Dunn, and GenPharm International U.S. patent application Ser. No. 07/574,748, filed Aug. 29, 1990, 07/575,962, filed Aug. 31, 1990, 07/810,279, filed Dec. 17, 1991, 07/853,408, filed Mar. 18, 1992, 07/904,068, filed Jun. 23, 1992, 07/990,860, filed Dec. 16, 1992, 08/053,131, filed Apr. 26, 1993, 08/096,762, filed Jul. 22, 1993, 08/155,301, filed Nov. 18, 1993, 08/161,739, filed Dec. 3, 1993, 08/165,699, filed Dec. 10, 1993, 08/209,741, filed Mar. 9, 1994, the disclosures of which are hereby incorporated by reference. See also European Patent No. 0 546 073 Bl, International Patent Application Nos. WO 92/03918, WO

92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No. 5,981,175, the disclosures of which are hereby incorporated by reference in their entirety. See further Taylor et al., 1992, Chen et al, 1993, TuaiUon et al, 1993, Choi et al, 1993, Lonberg et al, (1994), Taylor et al, (1994), and TuaiUon et al, (1995), Fishwild et al, (1996), the disclosures of which are hereby incorporated by reference in their entirety.

[00245] Kirin has also demonstrated the generation of human antibodies from mice in which, through microcell fusion, large pieces of chromosomes, or entire chromosomes, have been introduced. See European Patent Application Nos. 773 288 and 843 961, the disclosures of which are hereby incorporated by reference. Additionally, KM™ mice, which are the result of cross-breeding of Kirin' s Tc mice with Medarex's minilocus (Humab) mice have been generated. These mice possess the human IgH transchromosome of the Kirin mice and the kappa chain transgene of the Genpharm mice (Ishida et al., Cloning Stem Cells, (2002) 4:91-102).

[00246] Human antibodies can also be derived by in vitro methods. Suitable examples include but are not limited to phage display (CAT, Morphosys, Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed) ribosome display (CAT), yeast display, and the like.

[00247] In some embodiments, the antibodies described herein possess human IgG4 heavy chains as well as IgG2 heavy chains. Antibodies can also be of other human isotypes, including IgGl . The antibodies possessed high affinities, typically possessing a Kj of from about 10^~6 through about 10^~13 M or below, when measured by various techniques.

[00248] As will be appreciated, antibodies can be expressed in cell lines other than hybridoma cell lines. Sequences encoding particular antibodies can be used to transform a suitable mammalian host cell. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by reference). The transformation procedure used depends upon the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

[00249] Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture

Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and a number of other cell lines. Cell lines of particular preference are selected through determining which cell lines have high expression levels and produce antibodies with constitutive PCSK9 binding properties.

[00250] In certain embodiments, antibodies and/or ABP are produced by at least one of the following hybridomas: 21B12, 31H4, 16F12, any the other hybridomas listed in Table 2 of U.S. Pat. App. Pub. 20090142352 (or Table 10, herein) or disclosed in the examples. In certain embodiments, antigen binding proteins bind to PCSK9 with a dissociation constant (K_D) of less than approximately 1 nM, e.g., 1000 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM, and/or 1 pM to 0.1 pM or less.

[00251] In certain embodiments, antigen binding proteins comprise an immunoglobulin molecule of at least one of the IgGl, IgG2, IgG3, IgG4, Ig E, IgA, IgD, and IgM isotype. In certain embodiments, antigen binding proteins comprise a human kappa light chain and/or a human heavy chain. In certain embodiments, the heavy chain is of the IgGl, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM isotype. In certain embodiments, antigen binding proteins have been cloned for expression in mammalian cells. In certain embodiments, antigen binding proteins comprise a constant region other than any of the constant regions of the IgGl, IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM isotype.

[00252] In certain embodiments, antigen binding proteins comprise a human lambda light chain and a human IgG2 heavy chain. In certain embodiments, antigen binding proteins comprise a human lambda light chain and a human IgG4 heavy chain. In certain

embodiments, antigen binding proteins comprise a human lambda light chain and a human IgGl, IgG3, IgE, IgA, IgD or IgM heavy chain. In other embodiments, antigen binding proteins comprise a human kappa light chain and a human IgG2 heavy chain. In certain embodiments, antigen binding proteins comprise a human kappa light chain and a human IgG4 heavy chain. In certain embodiments, antigen binding proteins comprise a human kappa light chain and a human IgGl, IgG3, IgE, IgA, IgD or IgM heavy chain. In certain embodiments, antigen binding proteins comprise variable regions of antibodies ligated to a constant region that is neither the constant region for the IgG2 isotype, nor the constant region for the IgG4 isotype. In certain embodiments, antigen binding proteins have been cloned for expression in mammalian cells.

[00253] In certain embodiments, conservative modifications to the heavy and light chains of antibodies from at least one of the hybridoma lines: 2 IB 12, 31H4 and 16F12 (and corresponding modifications to the encoding nucleotides) will produce antibodies to PCSK9 having functional and chemical characteristics similar to those of the antibodies from the hybridoma lines: 21B12, 31H4 and 16F12. In contrast, in certain embodiments, substantial modifications in the functional and/or chemical characteristics of antibodies to PCSK9 can be accomplished by selecting substitutions in the amino acid sequence of the heavy and light chains that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[00254] For example, a "conservative amino acid substitution" can involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide can also be substituted with alanine, as has been previously described for "alanine scanning mutagenesis." [00255] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. In certain embodiments, amino acid substitutions can be used to identify important residues of antibodies to PCSK9, or to increase or decrease the affinity of the antibodies to PCSK9 as described herein.

[00256] In certain embodiments, antibodies of the present invention can be expressed in cell lines other than hybridoma cell lines. In certain embodiments, sequences encoding particular antibodies can be used for transformation of a suitable mammalian host cell.

According to certain embodiments, transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by reference for any purpose). In certain embodiments, the transformation procedure used can depend upon the host to be transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the

polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

[00257] Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines. In certain embodiments, cell lines can be selected through determining which cell lines have high expression levels and produce antibodies with constitutive HGF binding properties.

Appropriate expression vectors for mammalian host cells are well known.

[00258] In certain embodiments, antigen binding proteins comprise one or more polypeptides. In certain embodiments, any of a variety of expression vector/host systems can be utilized to express polynucleotide molecules encoding polypeptides comprising one or more ABP components or the ABP itself. Such systems include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.

[00259] In certain embodiments, a polypeptide comprising one or more ABP components or the ABP itself is recombinantly expressed in yeast. Certain such embodiments use commercially available expression systems, e.g., the Pichia Expression System (Invitrogen, San Diego, Calif), following the manufacturer's instructions. In certain embodiments, such a system relies on the pre -pro-alpha sequence to direct secretion. In certain embodiments, transcription of the insert is driven by the alcohol oxidase (AOX1) promoter upon induction by methanol.

[00260] In certain embodiments, a secreted polypeptide comprising one or more ABP components or the ABP itself is purified from yeast growth medium. In certain embodiments, the methods used to purify a polypeptide from yeast growth medium is the same as those used to purify the polypeptide from bacterial and mammalian cell supernatants.

[00261] In certain embodiments, a nucleic acid encoding a polypeptide comprising one or more ABP components or the ABP itself is cloned into a baculovirus expression vector, such as pVL1393 (PharMingen, San Diego, Calif). In certain embodiments, such a vector can be used according to the manufacturer's directions (PharMingen) to infect Spodoptera frugiperda cells in sF9 protein-free media and to produce recombinant polypeptide. In certain

embodiments, a polypeptide is purified and concentrated from such media using a heparin- Sepharose column (Pharmacia).

[00262] In certain embodiments, a polypeptide comprising one or more ABP components or the ABP itself is expressed in an insect system. Certain insect systems for polypeptide expression are well known to those of skill in the art. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. In certain embodiments, a nucleic acid molecule encoding a polypeptide can be inserted into a nonessential gene of the virus, for example, within the polyhedrin gene, and placed under control of the promoter for that gene. In certain embodiments, successful insertion of a nucleic acid molecule will render the nonessential gene inactive. In certain embodiments, that inactivation results in a detectable characteristic. For example, inactivation of the polyhedrin gene results in the production of virus lacking coat protein. [00263] In certain embodiments, recombinant viruses can be used to infect S. frugiperda cells or Trichoplusia larvae. See, e.g., Smith et al, J. Virol, 46: 584 (1983); Engelhard et al, Proc. Nat. Acad. Sci. (USA), 91 : 3224-7 (1994).

[00264] In certain embodiments, polypeptides comprising one or more ABP components or the ABP itself made in bacterial cells are produced as insoluble inclusion bodies in the bacteria. In certain embodiments, host cells comprising such inclusion bodies are collected by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg/ml lysozyme (Sigma, St. Louis, Mo.) for 15 minutes at room temperature. In certain embodiments, the lysate is cleared by sonication, and cell debris is pelleted by centrifugation for 10 minutes at 12,000xg. In certain embodiments, the polypeptide-containing pellet is resuspended in 50 mM Tris, pH 8, and 10 mM EDTA; layered over 50% glycerol; and centrifuged for 30 minutes at 6000xg. In certain embodiments, that pellet can be resuspended in standard phosphate buffered saline solution (PBS) free of Mg⁺⁺ and Ca⁺⁺. In certain embodiments, the polypeptide is further purified by fractionating the resuspended pellet in a denaturing SDS polyacrylamide gel {See, e.g., Sambrook et al, supra). In certain

embodiments, such a gel can be soaked in 0.4 M KC1 to visualize the protein, which can be excised and electroeluted in gel-running buffer lacking SDS. According to certain

embodiments, a Glutathione-S-Transferase (GST) fusion protein is produced in bacteria as a soluble protein. In certain embodiments, such GST fusion protein is purified using a GST Purification Module (Pharmacia).

[00265] In certain embodiments, it is desirable to "refold" certain polypeptides, e.g., polypeptides comprising one or more ABP components or the ABP itself. In certain embodiments, such polypeptides are produced using certain recombinant systems discussed herein. In certain embodiments, polypeptides are "refolded" and/or oxidized to form desired tertiary structure and/or to generate disulfide linkages. In certain embodiments, such structure and/or linkages are related to certain biological activity of a polypeptide. In certain embodiments, refolding is accomplished using any of a number of procedures known in the art. Exemplary methods include, but are not limited to, exposing the solubilized polypeptide agent to a pH typically above 7 in the presence of a chaotropic agent. An exemplary chaotropic agent is guanidine. In certain embodiments, the refolding/oxidation solution also contains a reducing agent and the oxidized form of that reducing agent. In certain

embodiments, the reducing agent and its oxidized form are present in a ratio that will generate a particular redox potential that allows disulfide shuffling to occur. In certain embodiments, such shuffling allows the formation of cysteine bridges. Exemplary redox couples include, but are not limited to, cysteine/cystamine, glutathione/dithiobisGSH, cupric chloride, dithiothreitol DTT/dithiane DTT, and 2-mercaptoethanol (bME)/dithio-bME. In certain embodiments, a co-solvent is used to increase the efficiency of refolding. Exemplary cosolvents include, but are not limited to, glycerol, polyethylene glycol of various molecular weights, and arginine.

[00266] In certain embodiments, one substantially purifies a polypeptide comprising one or more ABP components or the ABP itself. Certain protein purification techniques are known to those of skill in the art. In certain embodiments, protein purification involves crude fractionation of polypeptide fractionations from non-polypeptide fractions. In certain embodiments, polypeptides are purified using chromatographic and/or electrophoretic techniques. Exemplary purification methods include, but are not limited to, precipitation with ammonium sulphate; precipitation with PEG; immunoprecipitation; heat denaturation followed by centrifugation; chromatography, including, but not limited to, affinity chromatography (e.g., Protein-A-Sepharose), ion exchange chromatography, exclusion chromatography, and reverse phase chromatography; gel filtration; hydroxyapatite chromatography; isoelectric focusing; polyacrylamide gel electrophoresis; and combinations of such and other techniques. In certain embodiments, a polypeptide is purified by fast protein liquid chromatography or by high pressure liquid chromotography (HPLC). In certain embodiments, purification steps can be changed or certain steps can be omitted, and still result in a suitable method for the preparation of a substantially purified polypeptide.

[00267] In certain embodiments, one quantitates the degree of purification of a polypeptide preparation. Certain methods for quantifying the degree of purification are known to those of skill in the art. Certain exemplary methods include, but are not limited to, determining the specific binding activity of the preparation and assessing the amount of a polypeptide within a preparation by SDS/PAGE analysis. Certain exemplary methods for assessing the amount of purification of a polypeptide preparation comprise calculating the binding activity of a preparation and comparing it to the binding activity of an initial extract. In certain embodiments, the results of such a calculation are expressed as "fold purification." The units used to represent the amount of binding activity depend upon the particular assay performed.

[00268] In certain embodiments, a polypeptide comprising one or more ABP components or the ABP itself is partially purified. In certain embodiments, partial purification can be accomplished by using fewer purification steps or by utilizing different forms of the same general purification scheme. For example, in certain embodiments, cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "fold purification" than the same technique utilizing a low-pressure chromatography system. In certain embodiments, methods resulting in a lower degree of purification can have advantages in total recovery of polypeptide, or in maintaining binding activity of a polypeptide.

[00269] In certain instances, the electrophoretic migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE. See, e.g., Capaldi et al, Biochem. Biophys. Res. Comm., 76: 425 (1977). It will be appreciated that under different electrophoresis conditions, the apparent molecular weights of purified or partially purified polypeptide can be different.

PHARMACEUTICAL COMPOSITIONS

[00270] The methods of the invention include administering an RNA effector agent, e.g., a PCSK9 siRNA, and an antigen binding protein (ABP), e.g., a PCS9 antibody. The RNA effector agent and ABP can be administered concurrently or sequentially. In some embodiments, the RNA effector agent and/or ABP are administered as pharmaceutical compositions, e.g., in a pharmaceutically acceptable carrier. Examples include lipid formulations and the like. The pharmaceutical composition is useful for treating a disease or disorder associated with the expression or activity of a PCSK9 gene, such as pathological processes mediated by PCSK9 expression, e.g., hyperlipidemia. Such pharmaceutical compositions are formulated based on the mode of delivery.

Dosage

[00271] The pharmaceutical compositions featured herein are administered in dosages such that the RNA effector agent, e.g., the siRNA, is sufficient to inhibit expression of target genes. In general, a suitable dose of siRNA will be in the range of 0.01 to 200.0 milligrams siRNA per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.01 mg/kg, 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.

[00272] In another embodiment, the dosage is between 0.01 and 0.2 mg/kg. For example, the dsRNA can be administered at a dose of 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg 0.08 mg/kg 0.09 mg/kg , 0.10 mg/kg, 0.11 mg/kg, 0.12 mg/kg, 0.13 mg/kg, 0.14 mg/kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg, 0.18 mg/kg, 0.19 mg/kg, or 0.20 mg/kg.

[00273] In one embodiment, the dosage is between 0.2 mg/kg and 2.0 mg/kg. For example, the pharmaceutical composition can be administered at a dose of 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mg/kg.

[00274] The pharmaceutical composition can be administered at a dose of 0.03, 0.1, 0.3, or 1.3, or 3.0 mg/kg.

[00275] The pharmaceutical compositions featured herein are administered in dosages such that an ABP is sufficient to interfere with PCSK9. In certain embodiments, the effective amount of a pharmaceutical composition comprising an antigen binding protein to PCSK9, with or without at least one additional therapeutic agent, to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, will thus vary depending, in part, upon the molecule delivered, the indication for which an antigen binding protein to PCSK9, with or without at least one additional therapeutic agent, is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. In certain embodiments, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. In certain embodiments, a typical dosage can range from about 0.1 μg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In certain embodiments, the dosage can range from 0.1 μg/kg up to about 100 mg/kg; or 1 μg/kg up to about 100 mg/kg; or 5 μg/kg up to about 100 mg/kg. In certain embodiments, the frequency of dosing will take into account the pharmacokinetic parameters of an antigen binding protein to PCSK9 and/or any additional therapeutic agents in the formulation used. In certain embodiments, a clinician will administer the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data. In some embodiments, the amount and frequency of administration can take into account the desired cholesterol level (serum and/or total) to be obtained and the subject's present cholesterol level, LDL level, and/or LDLR levels, all of which can be obtained by methods that are well known to those of skill in the art. Other dosage information for ABPs can be found in U.S. Pat. App No. 20090142352, herein incorporated by reference.

[00276] In one embodiment the lipid formulated pharmaceutical composition is administered at a first dose of about 3 mg/kg followed by administering at least one subsequent dose once a week, wherein the subsequent dose is lower than the first dose, e.g., the subsequent dose is about 1.0 mg/kg or about 0.3 mg/kg.

[00277] The pharmaceutical composition comprising an RNA effector agent, e.g., siRNA, and/or an ABP, can be administered once daily, or the pharmaceutical composition may be administered as two, three, or more sub-doses at appropriate intervals throughout the day. The effect of a single dose on target mRNA levels is long lasting, such that subsequent doses are administered at not more than 7 day intervals, or at not more than 1, 2, 3, or 4 week intervals.

[00278] The subsequent dose can be administered, e.g., once a week for four weeks. In some embodiments the pharmaceutical composition is administered using continuous infusion or delivery through a controlled release formulation. In that case, the

pharmaceutical composition 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 RNA effector agent, e.g., siRNA and/or ABP over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[00279] 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 RNA effector agent, e.g., siRNA and/or ABP 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.

[00280] Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by target gene expression. Such models are used for in vivo testing of pharmaceutical compositions, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a plasmid expressing a human target gene. Another suitable mouse model is a transgenic mouse carrying a transgene that expresses a human target gene.

[00281] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

[00282] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence {e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately to determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[00283] In addition to their administration, as discussed above, the dsR As featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression. In any event, the

administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein. Administration

[00284] The pharmaceutical compositions comprising an RNA effector agent, e.g., siRNA, and/or an ABP of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.

Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, and subdermal, oral or parenteral, e.g., subcutaneous.

[00285] For example, when treating a mammal with hyperlipidemia, the RNA effector agent, e.g., siRNA and/or ABP are administered systemically via parental means. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration. For example, a RNA effector agent, e.g., siRNA and/or ABP, conjugated or unconjugated or formulated with or without liposomes, can be administered intravenously to a patient. For such, a RNA effector agent, e.g., siRNA and/or ABP 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 RNA effector agent, e.g., siRNA and/or ABP 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). Formulations are described in more detail herein.

[00286] The pharmaceutical composition can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).

Formulations

[00287] The pharmaceutical formulations comprising an RNA effector agent, e.g., siRNA, and/or an ABP of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the

pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. [00288] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[00289] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. In one aspect are formulations that target the liver when treating hepatic disorders such as hyperlipidemia.

[00290] In addition, R A effector agent, e.g., siRNA and/or ABP that target the target gene can be formulated into compositions containing the RNA effector agent, e.g., siRNA and/or ABP admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of nucleic acids. For example, a composition containing one or more RNA effector agent, e.g., siRNA and/or ABP that target the target gene can contain other therapeutic agents, such as other cancer therapeutics or one or more RNA effector agent, e.g., siRNA and/or ABP that target other target genes.

Oral, parenteral, topical, and biologic formulations

[00291] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or nonaqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which RNA effector agent, e.g., siRNA and/or ABP featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include

chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1- dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some

embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9- lauryl ether, polyoxyethylene-20-cetyl ether. RNA effector agent, e.g., siRNA and/or ABP featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. RNA effector agent, e.g., siRNA and/or ABP complexing agents include poly-amino acids; polyimines; polyacrylates;

polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates;

DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene

P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate),

poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Patent

6,887,906, U.S. Patent Publication. No. 20030027780, and U.S. Patent No. 6,747,014, each of which is incorporated herein by reference.

[00292] Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[00293] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Suitable topical formulations include those in which the RNA effector agent, e.g., siRNA and/or ABP featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and

dioleoylphosphatidyl ethanolamine DOTMA). RNA effector agent, e.g., siRNA and/or ABP featured in the invention may be encapsulated within liposomes or may form complexes thereto. Alternatively, RNA effector agent, e.g., siRNA and/or ABP may be complexed to lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, l-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C_1-10 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Patent No.

6,747,014, which is incorporated herein by reference. In addition, pharmaceutical compositions 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 RNA effector agent, e.g., siRNA and/or ABP provided herein and (2) complexing a RNA effector agent, e.g., siRNA and/or ABP with lipids or liposomes to form nucleic acid-lipid or nucleic acid- liposome complexes. The liposome can be composed of cationic and neutral lipids commonly used to transfect cells in vitro. Cationic lipids can complex (e.g., charge- associate) with negatively charged nucleic acids to form liposomes. Examples of cationic liposomes include, without limitation, lipofectin, lipofectamine, lipofectace, and DOTAP. Procedures for forming liposomes are well known in the art. Liposome compositions can be formed, for example, from phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, or dioleoyl

phosphatidylethanolamine. Numerous lipophilic agents are commercially available, including Lipofectin™ (Invitrogen/Life Technologies, Carlsbad, Calif.) and Effectene™ (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 ah, 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.

[00294] 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 herpes virus 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.

Liposomal formulations

[00295] The pharmaceutical comprising an RNA effector agent, e.g., siRNA, and/or an ABP can be in a liposomal formulation.

[00296] Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

[00297] In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

[00298] Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; and liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes. [00299] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

[00300] Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

[00301] Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis

[00302] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[00303] Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269- 274).

[00304] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl

phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[00305] Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405- 410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[00306] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/po- lyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin- A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al., S.T.P. Pharma. Sci., 1994, 4, 6, 466).

[00307] Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_MI, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[00308] Various liposomes comprising one or more glycolipids are known in the art.

Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_MI, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_MI or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1 ,2-sn-dimyristoylphosphat- idylcholine are disclosed in WO 97/13499 (Lim et al.).

[00309] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2Ci2i5G, that contains a PEG moiety. Ilium et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG- derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of

distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 Bl and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 Bl). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

[00310] A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating

oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.

[00311] Transfersomes are yet another type of liposomes and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes, it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[00312] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[00313] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxy ethylene surfactants are the most popular members of the nonionic surfactant class.

[00314] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[00315] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

[00316] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[00317] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic acid lipid particles

[00318] In one embodiment, a RNA effector agent, e.g., a dsRNA featured in the invention (e.g., a dsRNA targeting targeting PCSK9) is fully encapsulated in the lipid formulation, e.g., to form a nucleic acid-lipid particle. Nucleic acid-lipid particles typically contain a cationic lipid, a non-cationic lipid, a sterol, and a lipid that prevents aggregation of the particle {e.g., a PEG-lipid conjugate). Nucleic acid-lipid particles are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites {e.g., sites physically separated from the

administration site). In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT

Publication No. WO 96/40964.

[00319] Nucleic acid-lipid particles can further include one or more additional lipids and/or other components such as cholesterol. Other lipids may be included in the liposome compositions for a variety of purposes, such as to prevent lipid oxidation or to attach ligands onto the liposome surface. Any of a number of lipids may be present, including amphipathic, neutral, cationic, and anionic lipids. Such lipids can be used alone or in combination.

Specific examples of additional lipid components that may be present are described herein.

[00320] Additional components that may be present in a nucleic acid-lipid particle include bilayer stabilizing components such as polyamide oligomers (see, e.g., U.S. Patent

No. 6,320,017), peptides, proteins, detergents, lipid-derivatives, such as PEG coupled to phosphatidylethanolamine and PEG conjugated to ceramides (see, U.S. Patent

No. 5,885,613).

[00321] A nucleic acid-lipid particle can include one or more of a second amino lipid or cationic lipid, a neutral lipid, a sterol, and a lipid selected to reduce aggregation of lipid particles during formation, which may result from steric stabilization of particles which prevents charge-induced aggregation during formation.

[00322] Nucleic acid-lipid particles include, e.g., a SPLP, pSPLP, and SNALP. The term"SNALP" refers to a stable nucleic acid-lipid particle, including SPLP. The term "SPLP" refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SPLPs include "pSPLP," which include an encapsulated condensing agent- nucleic acid complex as set forth in PCT Publication No. WO 00/03683.

[00323] The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. The particles of the present invention can have a mean diameter of about less than 50, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1 10, 111, 1 12, 113, 1 14, 115, 1 16, 117, 118, 1 19, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 13 1 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, or more than 150 nm.

[00324] In one embodiment, the lipid to drug ratio (mass/mass ratio) {e.g., lipid to dsRNA ratio) will be in the range of from about 1 : 1 to about 50: 1, from about 1 : 1 to about 25 : 1 , from about 3 : 1 to about 15: 1, from about 4 : 1 to about 10: 1, from about 5 : 1 to about 9 : 1 , or about 6: 1 to about 9: 1, or about 6:1 , 7:1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 21 : 1, 22: 1, 23: 1, 24: 1, 25: 1, 26: 1, 27: 1, 28: 1, 29: 1, 30: 1, 31 : 1, 32: 1, or 33: 1. Cationic lipids

[00325] Cationic lipids can include ionizable cationic lipids and non-ionizable cationic lipids. A cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I -(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I -(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3- dioleyloxy propylamine (DODMA), 1 ,2-DiLinoleyloxy-N,N-dimethylaminopropane

(DLinDMA), l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2- Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1 ,2-Dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), l ,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-Dilinoleylthio-3- dimethylaminopropane (DLin-S-DMA), 1 -Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), l,2-Dilinoleyloxy-3- (N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-l,2-propanedio (DOAP), l,2-Dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), l,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[ 1 ,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca- 9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine (ALNY-100),

(6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), or a mixture thereof.

[00326] Other cationic lipids, which carry a net positive charge at about physiological pH, in addition to those specifically described above, may also be included in lipid particles of the invention. Such cationic lipids include, but are not limited to, N,N-dioleyl-N,N- dimethylammonium chloride ("DODAC"); N-(2,3-dioleyloxy)propyl-N,N-N- triethylammonium chloride ("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTAP"); l,2-Dioleyloxy-3-trimethylaminopropane chloride salt ("DOTAP.C1"); 3β-(Ν-(Ν',Ν'- dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol"), N-(l-(2,3-dioleyloxy)propyl)-N- 2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate ("DOSPA"), dioctadecylamidoglycyl carboxyspermine ("DOGS"), l,2-dileoyl-sn-3-phosphoethanolamine ("DOPE"), l,2-dioleoyl-3-dimethylammonium propane ("DODAP"), N, N-dimethyl-2,3- dioleyloxy propylamine ("DODMA"), and N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethyl ammonium bromide ("DMRIE"). Additionally, a number of commercial preparations of cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECT AMINE (comprising DOSPA and DOPE, available from GIBCO/BRL). In particular embodiments, a cationic lipid is an amino lipid.

[00327] As used herein, the term "amino lipid" is meant to include those lipids having one or two fatty acid or fatty alkyl chains and an amino head group (including an alkylamino or dialkylamino group) that may be protonated to form a cationic lipid at physiological pH.

[00328] Other amino lipids would include those having alternative fatty acid groups and other dialkylamino groups, including those in which the alkyl substituents are different {e.g., N-ethyl-N-methylamino-, N-propyl-N-ethylamino- and the like). For those embodiments in which R¹¹ and R¹² are both long chain alkyl or acyl groups, they can be the same or different. In general, amino lipids having less saturated acyl chains are more easily sized, particularly when the complexes must be sized below about 0.3 microns, for purposes of filter sterilization. Amino lipids containing unsaturated fatty acids with carbon chain lengths in the range of C₁₄ to C22 are preferred. Other scaffolds can also be used to separate the amino group and the fatty acid or fatty alkyl portion of the amino lipid. Suitable scaffolds are known to those of skill in the art.

[00329] In certain embodiments, amino or cationic lipids of the invention have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Lipids that have more than one protonatable or deprotonatable group, or which are zwiterrionic, are not excluded from use in the invention.

[00330] In certain embodiments, protonatable lipids according to the invention have a pKa of the protonatable group in the range of about 4 to about 11. Most preferred is pKa of about 4 to about 7, because these lipids will be cationic at a lower pH formulation stage, while particles will be largely (though not completely) surface neutralized at physiological pH around pH 7.4. One of the benefits of this pKa is that at least some nucleic acid associated with the outside surface of the particle will lose its electrostatic interaction at physiological H and be removed by simple dialysis; thus greatly reducing the particle's susceptibility to clearance.

[00331] One example of a cationic lipid is l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA). Synthesis and preparation of nucleic acid-lipid particles including DlinDMA is described in International application number PCT/CA2009/00496, filed April 15, 2009.

[00332] In one embodiment, the cationic lipid XTC (2,2-Dilinoleyl-4-dimethylaminoethyl- [l,3]-dioxolane) is used to prepare nucleic acid-lipid particles . Synthesis of XTC is described, e.g., in PCT/US 10/22614 filed on Jan. 29, 2010, which is hereby incorporated by reference.

[00333] In another embodiment, the cationic lipid MC3 ((6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,3 l-tetraen-19-yl 4-(dimethylamino)butanoate), (e.g., DLin-M-C3-DMA) is used to prepare nucleic acid-lipid particles . Synthesis of MC3 and MC3 comprising formulations are described, e.g., in U.S. Serial No. 12/813,448, filed June 10, 2010, which is hereby incorporated by reference.

[00334] In another embodiment, the cationic lipid ALNY- 100 ((3aR,5s,6aS)-N,N- dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5- amine) is used to prepare nucleic acid-lipid particles . Synthesis of ALNY- 100 is described in International patent application number PCT/US09/63933 filed on November 10, 2009, which is herein incorporated by reference.

[00335] In another embodiment, the cationic lipid Ι,Γ- (2-(4-(2-((2-(bis (2- hydroxydodecyl) amino) ethyl) (2-hydroxydodecyl) amino) ethyl) piperazin-l-yl)

ethylazanediyl) didodecan-2-ol (CI 2-200) is used to prepare nuceic acid lipid particles. CI 2- 200 is also known as Tech Gl . Synthesis of CI 2-200 and formulations using CI 2-200 are described in International patent application no. PCT/US 10/33777 filed May 5, 2010 and in Love et al (Love et al. (2010) PNAS 107(5); 1864-69). FIG. 25 illustrates the structure of C12-200.

[00336] The cationic lipid may comprise from about 20 mol % to about 70 mol % or about 45-65 mol % or about 40 mol % of the total lipid present in the particle. The cationic lipid may comprise the mol% of the total lipid present in the particle as indicated in Tables A-C. The cationic lipid may comprise about less than 20, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or more than 70 mol % of the total lipid present in the particle. Non-cationic lipids

[00337] The nucleic acid-lipid particles of the invention can include a non-cationic lipid. The non-cationic lipid may be an anionic lipid or a neutral lipid. Examples include but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG),

dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE),

dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1 -stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof.

[00338] Anionic lipids suitable for use in lipid particles of the invention include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine,

diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl

phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids.

[00339] Neutral lipids, when present in the lipid particle, can be any of a number of lipid species which exist either in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, for example diacylphosphatidylcholme, diacylphosphatidylethanolamme, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids for use in the particles described herein is generally guided by consideration of, e.g. , liposome size and stability of the liposomes in the bloodstream. Preferably, the neutral lipid component is a lipid having two acyl groups, {i.e., diacylphosphatidylcholme and diacylphosphatidylethanolamme). Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. In one group of embodiments, lipids containing saturated fatty acids with carbon chain lengths in the range of C₁₄ to C₂₂ are preferred. In another group of embodiments, lipids with mono- or di-unsaturated fatty acids with carbon chain lengths in the range of C₁₄ to C₂₂ are used. Additionally, lipids having mixtures of saturated and

unsaturated fatty acid chains can be used. Preferably, the neutral lipids used in the invention are DOPE, DSPC, POPC, or any related phosphatidylcholine. The neutral lipids useful in the invention may also be composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head groups, such as serine and inositol.

[00340] In one embodiment the non-cationic lipid is distearoylphosphatidylcholine (DSPC). In another embodiment the non-cationic lipid is dipalmitoylphosphatidylcholine (DPPC).

[00341] The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 5 mol % to about 10 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle. The non-cationic lipid may comprise the mol% of the total lipid present in the particle as indicated in Tables A-C. The non-cationic lipid may comprise about less than 5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, or more than 90 mol % of the total lipid present in the particle.

Conjugated lipids

[00342] Conjugated lipids can be used in nucleic acid-lipid particle to prevent

aggregation, including polyethylene glycol (PEG)-modified lipids, monosialoganglioside Gml, and polyamide oligomers ("PAO") such as (described in US Pat. No. 6,320,017).

Other compounds with uncharged, hydrophilic, steric-barrier moieties, which prevent aggregation during formulation, like PEG, Gml or ATT A, can also be coupled to lipids for use as in the methods and compositions of the invention. ATTA- lipids are described, e.g. , in U.S. Patent No. 6,320,017, and PEG-lipid conjugates are described, e.g., in U.S. Patent Nos. 5,820,873, 5,534,499 and 5,885,613. Typically, the concentration of the lipid component selected to reduce aggregation is about 1 to 15% (by mole percent of lipids).

[00343] Specific examples of PEG-modified lipids (or lipid-polyoxyethylene conjugates) that are useful in the invention can have a variety of "anchoring" lipid portions to secure the PEG portion to the surface of the lipid vesicle. Examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates {e.g., PEG-CerC14 or PEG-CerC20) which are described in co-pending USSN 08/486,214, incorporated herein by reference, PEG-modified dialkylamines and PEG- modified l,2-diacyloxypropan-3-amines. Particularly preferred are PEG-modified diacylglycerols and dialkylglycerols. [00344] In embodiments where a sterically-large moiety such as PEG or ATTA are conjugated to a lipid anchor, the selection of the lipid anchor depends on what type of association the conjugate is to have with the lipid particle. It is well known that mePEG (mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE) will remain associated with a liposome until the particle is cleared from the circulation, possibly a matter of days. Other conjugates, such as PEG-CerC20 have similar staying capacity. PEG-CerC14, however, rapidly exchanges out of the formulation upon exposure to serum, with a T ₂ less than 60 mins. in some assays. As illustrated in US Pat. Application SN 08/486,214, at least three characteristics influence the rate of exchange: length of acyl chain, saturation of acyl chain, and size of the steric-barrier head group. Compounds having suitable variations of these features may be useful for the invention. For some therapeutic applications, it may be preferable for the PEG-modified lipid to be rapidly lost from the nucleic acid-lipid particle in vivo and hence the PEG-modified lipid will possess relatively short lipid anchors. In other therapeutic applications, it may be preferable for the nucleic acid-lipid particle to exhibit a longer plasma circulation lifetime and hence the PEG-modified lipid will possess relatively longer lipid anchors. Exemplary lipid anchors include those having lengths of from about C₁₄ to about C₂2, preferably from about C₁₄ to about C₁₆. In some embodiments, a PEG moiety, for example an mPEG-NH₂, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.

[00345] It should be noted that aggregation preventing compounds do not necessarily require lipid conjugation to function properly. Free PEG or free ATTA in solution may be sufficient to prevent aggregation. If the particles are stable after formulation, the PEG or ATTA can be dialyzed away before administration to a subject.

[00346] The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG- distearyloxypropyl (C]g). Additional conjugated lipids include polyethylene glycol - didimyristoyl glycerol (C14-PEG or PEG-C14, where PEG has an average molecular weight of 2000 Da) (PEG-DMG); (R)-2,3-bis(octadecyloxy)propyll-(methoxy polyethylene glycol)2000)propylcarbamate) (PEG-DSG); PEG-carbamoyl- 1 ,2-dimyristyloxypropylamine, in which PEG has an average molecular weight of 2000 Da (PEG-cDMA); N- Acetylgalactosamine-((R)-2,3 -bis(octadecyloxy)propyl 1 -(methoxy poly(ethylene glycol)2000)propylcarbamate)) (GalNAc-PEG-DSG); and polyethylene glycol - dipalmitoylglycerol (PEG-DPG).

[00347] In one embodiment the conjugated lipid is PEG-DMG. In another embodiment the conjugated lipid is PEG-cDMA. In still another embodiment the conjugated lipid is PEG- DPG. Alternatively the conjugated lipid is GalNAc-PEG-DSG.

[00348] The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 0.5 to about 5.0 mol % or about 2 mol % of the total lipid present in the particle. The conjugated lipid may comprise the mol% of the total lipid present in the particle as indicated in Tables A-C. The conjugated lipid may comprise about 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 mol % of the total lipid present in the particle.

[00349] The sterol component of the lipid mixture, when present, can be any of those sterols conventionally used in the field of liposome, lipid vesicle or lipid particle preparation. A preferred sterol is cholesterol.

[00350] In some embodiments, the nucleic acid-lipid particle further includes a sterol, e.g., a cholesterol at, e.g., about 10 mol % to about 60 mol % or about 25 to about 40 mol % or about 48 mol % of the total lipid present in the particle. The sterol may comprise the mol% of the total lipid present in the particle as indicated in Tables A-C. The sterol may comprise about less than 10, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, or more than 60 mol % of the total lipid present in the particle.

Lipoproteins

[00351] In one embodiment, the formulations of the invention further comprise an apolipoprotein. As used herein, the term "apolipoprotein" or "lipoprotein" refers to apolipoproteins known to those of skill in the art and variants and fragments thereof and to apolipoprotein agonists, analogues or fragments thereof described below.

[00352] Suitable apolipoproteins include, but are not limited to, ApoA-I, ApoA-II, ApoA- IV, ApoA-V and ApoE, and active polymorphic forms, isoforms, variants and mutants as well as fragments or truncated forms thereof. In certain embodiments, the apolipoprotein is a thiol containing apolipoprotein. "Thiol containing apolipoprotein" refers to an

apolipoprotein, variant, fragment or isoform that contains at least one cysteine residue. The most common thiol containing apolipoproteins are ApoA-I Milano (APOA-I_M) and ApoA-I Paris (ApoA-I_P) which contain one cysteine residue (Jia et al, 2002, Biochem. Biophys. Res. Comm. 297: 206-13; Bielicki and Oda, 2002, Biochemistry 41 : 2089-96). ApoA-II, ApoE2 and ApoE3 are also thiol containing apolipoproteins. Isolated ApoE and/or active fragments and polypeptide analogues thereof, including recombinantly produced forms thereof, are described in U.S. Pat. Nos. 5,672,685; 5,525,472; 5,473,039; 5,182,364; 5,177,189;

5,168,045; 5,116,739; the disclosures of which are herein incorporated by reference. ApoE3 is disclosed in Weisgraber, et al, "Human E apoprotein heterogeneity: cysteine-arginine interchanges in the amino acid sequence of the apo-E isoforms," J. Biol. Chem. (1981) 256: 9077-9083; and Rail, et al., "Structural basis for receptor binding heterogeneity of apolipoprotein E from type III hyperlipoproteinemic subjects," Proc. Nat. Acad. Sci. (1982) 79: 4696-4700. (See also GenBank accession number K00396.)

[00353] In certain embodiments, the apolipoprotein can be in its mature form, in its preproapolipoprotein form or in its proapolipoprotein form. Homo- and heterodimers (where feasible) of pro- and mature ApoA-I (Duverger et al., 1996, Arterioscler. Thromb. Vase. Biol. 16(12): 1424-29), ApoA-I Milano (Klon et al, 2000, Biophys. J. 79:(3)1679-87;

Franceschini et al, 1985, J. Biol. Chem. 260: 1632-35), ApoA-I Paris (Daum et al, 1999, J. Mol. Med. 77:614-22), ApoA-II (Shelness et al, 1985, J. Biol. Chem. 260(14):8637-46; Shelness et al, 1984, J. Biol. Chem. 259(15):9929-35), ApoA-IV (Duverger et al, 1991, Euro. J. Biochem. 201(2):373-83), and ApoE (McLean et al, 1983, J. Biol. Chem.

258(14):8993-9000) can also be utilized within the scope of the invention.

[00354] In certain embodiments, the apolipoprotein can be a fragment, variant or isoform of the apolipoprotein. The term "fragment" refers to any apolipoprotein having an amino acid sequence shorter than that of a native apolipoprotein and which fragment retains the activity of native apolipoprotein, including lipid binding properties. By "variant" is meant substitutions or alterations in the amino acid sequences of the apolipoprotein, which substitutions or alterations, e.g., additions and deletions of amino acid residues, do not abolish the activity of native apolipoprotein, including lipid binding properties. Thus, a variant can comprise a protein or peptide having a substantially identical amino acid sequence to a native apolipoprotein provided herein in which one or more amino acid residues have been conservatively substituted with chemically similar amino acids.

Examples of conservative substitutions include the substitution of at least one hydrophobic residue such as isoleucine, valine, leucine or methionine for another. Likewise, the present invention contemplates, for example, the substitution of at least one hydrophilic residue such as, for example, between arginine and lysine, between glutamine and asparagine, and between glycine and serine (see U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166). The term "isoform" refers to a protein having the same, greater or partial function and similar, identical or partial sequence, and may or may not be the product of the same gene and usually tissue specific (see Weisgraber 1990, J. Lipid Res. 31(8): 1503-11 ; Hixson and Powers 1991, J. Lipid Res. 32(9): 1529-35; Lackner et al, 1985, J. Biol. Chem. 260(2):703-6; Hoeg et al, 1986, J. Biol. Chem. 261(9):3911-4; Gordon et al, 1984, J. Biol. Chem. 259(l):468-74; Powell et al, 1987, Cell 50(6):831-40; Aviram et al, 1998, Arterioscler. Thromb. Vase. Biol. 18(10): 1617-24; Aviram et al, 1998, J. Clin. Invest. 101(8): 1581-90; Billecke et al, 2000, Drug Metab. Dispos. 28(11): 1335-42; Draganov et al, 2000, J. Biol. Chem. 275(43):33435- 42; Steinmetz and Utermann 1985, J. Biol. Chem. 260(4):2258-64; Widler et al, 1980, J. Biol. Chem. 255(21): 10464-71; Dyer et al, 1995, J. Lipid Res. 36(l):80-8; Sacre et al, 2003, FEBS Lett. 540(1-3): 181-7; Weers, et al, 2003, Biophys. Chem. 100(l-3):481-92; Gong et al, 2002, J. Biol. Chem. 277(33):29919-26; Ohta et al, 1984, J. Biol. Chem. 259(23): 14888- 93 and U.S. Pat. No. 6,372,886).

[00355] In certain embodiments, the methods and compositions of the present invention include the use of a chimeric construction of an apolipoprotein. For example, a chimeric construction of an apolipoprotein can be comprised of an apolipoprotein domain with high lipid binding capacity associated with an apolipoprotein domain containing ischemia reperfusion protective properties. A chimeric construction of an apolipoprotein can be a construction that includes separate regions within an apolipoprotein {i.e., homologous construction) or a chimeric construction can be a construction that includes separate regions between different apolipoproteins {i.e., heterologous constructions). Compositions comprising a chimeric construction can also include segments that are apolipoprotein variants or segments designed to have a specific character {e.g., lipid binding, receptor binding, enzymatic, enzyme activating, antioxidant or reduction-oxidation property) (see Weisgraber 1990, J. Lipid Res. 31(8): 1503-11 ; Hixson and Powers 1991, J. Lipid Res. 32(9): 1529-35; Lackner et al, 1985, J. Biol. Chem. 260(2):703-6; Hoeg et al, 1986, J. Biol. Chem.

261(9):3911-4; Gordon et al, 1984, J. Biol. Chem. 259(l):468-74; Powell et al, 1987, Cell 50(6):831-40; Aviram et al, 1998, Arterioscler. Thromb. Vase. Biol. 18(10): 1617-24;

Aviram et al, 1998, J. Clin. Invest. 101(8): 1581-90; Billecke et al, 2000, Drug Metab.

Dispos. 28(l l): 1335-42; Draganov et al, 2000, J. Biol. Chem. 275(43):33435-42; Steinmetz and Utermann 1985, J. Biol. Chem. 260(4):2258-64; Widler et al, 1980, J. Biol. Chem. 255(21): 10464-71; Dyer et al, 1995, J. Lipid Res. 36(l):80-8; Sorenson et al, 1999, Arterioscler. Thromb. Vase. Biol. 19(9) :2214-25; Palgunachari 1996, Arterioscler. Throb. Vase. Biol. 16(2):328-38: Thurberg et al, J. Biol. Chem. 271(1 1):6062-70; Dyer 1991, J. Biol. Chem. 266(23): 150009-15; Hill 1998, J. Biol. Chem. 273(47):30979-84).

[00356] Apolipoproteins utilized in the invention also include recombinant, synthetic, semi-synthetic or purified apolipoproteins. Methods for obtaining apolipoproteins or equivalents thereof, utilized by the invention are well-known in the art. For example, apolipoproteins can be separated from plasma or natural products by, for example, density gradient centrifugation or immunoaffinity chromatography, or produced synthetically, semi- synthetically or using recombinant DNA techniques known to those of the art (see, e.g., Mulugeta et al, 1998, J. Chromatogr. 798(1-2): 83-90; Chung et al, 1980, J. Lipid Res. 21(3):284-91; Cheung et al, 1987, J. Lipid Res. 28(8):913-29; Persson, et al, 1998, J.

Chromatogr. 711 :97-109; U.S. Pat. Nos. 5,059,528, 5,834,596, 5,876,968 and 5,721,114; and PCT Publications WO 86/04920 and WO 87/02062).

[00357] Apolipoproteins utilized in the invention further include apolipoprotein agonists such as peptides and peptide analogues that mimic the activity of ApoA-I, ApoA-I Milano (ApoA-I_M), ApoA-I Paris (ApoA-I_P), ApoA-II, ApoA-IV, and ApoE. For example, the apolipoprotein can be any of those described in U.S. Pat. Nos. 6,004,925, 6,037,323, 6,046,166, and 5,840,688, the contents of which are incorporated herein by reference in their entireties.

[00358] Apolipoprotein agonist peptides or peptide analogues can be synthesized or manufactured using any technique for peptide synthesis known in the art including, e.g., the techniques described in U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166. For example, the peptides may be prepared using the solid-phase synthetic technique initially described by Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154). Other peptide synthesis techniques may be found in Bodanszky et al, Peptide Synthesis, John Wiley & Sons, 2d Ed., (1976) and other references readily available to those skilled in the art. A summary of polypeptide synthesis techniques can be found in Stuart and Young, Solid Phase Peptide. Synthesis, Pierce Chemical Company, Rockford, III, (1984). Peptides may also be synthesized by solution methods as described in The Proteins, Vol. II, 3d Ed., Neurath et al, Eds., p. 105- 237, Academic Press, New York, N.Y. (1976). Appropriate protective groups for use in different peptide syntheses are described in the above-mentioned texts as well as in McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, N.Y. (1973). The peptides of the present invention might also be prepared by chemical or enzymatic cleavage from larger portions of, for example, apolipoprotein A-I.

[00359] In certain embodiments, the apolipoprotein can be a mixture of apolipoproteins. In one embodiment, the apolipoprotein can be a homogeneous mixture, that is, a single type of apolipoprotein. In another embodiment, the apolipoprotein can be a heterogeneous mixture of apolipoproteins, that is, a mixture of two or more different apolipoproteins.

Embodiments of heterogenous mixtures of apolipoproteins can comprise, for example, a mixture of an apolipoprotein from an animal source and an apolipoprotein from a semisynthetic source. In certain embodiments, a heterogenous mixture can comprise, for example, a mixture of ApoA-I and ApoA-I Milano. In certain embodiments, a heterogeneous mixture can comprise, for example, a mixture of ApoA-I Milano and ApoA-I Paris. Suitable mixtures for use in the methods and compositions of the invention will be apparent to one of skill in the art.

[00360] If the apolipoprotein is obtained from natural sources, it can be obtained from a plant or animal source. If the apolipoprotein is obtained from an animal source, the apolipoprotein can be from any species. In certain embodiments, the apolipoprotien can be obtained from an animal source. In certain embodiments, the apolipoprotein can be obtained from a human source. In preferred embodiments of the invention, the apolipoprotein is derived from the same species as the individual to which the apolipoprotein is administered.

Other components

[00361] In numerous embodiments, amphipathic lipids are included in lipid particles of the invention. "Amphipathic lipids" refer to any suitable material, wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Such compounds include, but are not limited to, phospholipids, aminolipids, and sphingo lipids. Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatdylcholine,

lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or

dilinoleylphosphatidylcholine. Other phosphorus-lacking compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and β-acyloxyacids, can also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols.

[00362] Also suitable for inclusion in the lipid particles of the invention are programmable fusion lipids. Such lipid particles have little tendency to fuse with cell membranes and deliver their payload until a given signal event occurs. This allows the lipid particle to distribute more evenly after injection into an organism or disease site before it starts fusing with cells. The signal event can be, for example, a change in pH, temperature, ionic environment, or time. In the latter case, a fusion delaying or "cloaking" component, such as an ATTA-lipid conjugate or a PEG-lipid conjugate, can simply exchange out of the lipid particle membrane over time. Exemplary lipid anchors include those having lengths of from about C_M to about C₂₂, preferably from about C₁₄ to about C₁₆. In some embodiments, a PEG moiety, for example an mPEG-NH₂, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.

[00363] A lipid particle conjugated to a nucleic acid agent can also include a targeting moiety, e.g., a targeting moiety that is specific to a cell type or tissue. Targeting of lipid particles using a variety of targeting moieties, such as ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and monoclonal antibodies, has been previously described (see, e.g., U.S. Patent Nos. 4,957,773 and 4,603,044). The targeting moieties can include the entire protein or fragments thereof. Targeting mechanisms generally require that the targeting agents be positioned on the surface of the lipid particle in such a manner that the targeting moiety is available for interaction with the target, for example, a cell surface receptor. A variety of different targeting agents and methods are known and available in the art, including those described, e.g., in Sapra, P. and Allen, TM, Prog. Lipid Res. 42(5):439- 62 (2003); and Abra, RM et al., J. Liposome Res. 12: 1-3, (2002).

[00364] The use of lipid particles, i.e., liposomes, with a surface coating of hydrophilic polymer chains, such as polyethylene glycol (PEG) chains, for targeting has been proposed (Allen, et al., Biochimica et Biophysica Acta 1237: 99-108 (1995); DeFrees, et al., Journal of the American Chemistry Society 118: 6101-6104 (1996); Blume, et al., Biochimica et Biophysica Acta 1149: 180-184 (1993); Klibanov, et al., Journal of Liposome Research 2: 321-334 (1992); U.S. Patent No. 5,013556; Zalipsky, Bioconjugate Chemistry 4: 296-299 (1993); Zalipsky, FEBS Letters 353: 71-74 (1994); Zalipsky, in Stealth Liposomes Chapter 9 (Lasic and Martin, Eds) CRC Press, Boca Raton Fl (1995). In one approach, a ligand, such as an antibody, for targeting the lipid particle is linked to the polar head group of lipids forming the lipid particle. In another approach, the targeting ligand is attached to the distal ends of the PEG chains forming the hydrophilic polymer coating (Klibanov, et al., Journal of Liposome Research 2: 321-334 (1992); Kirpotin et al, FEBS Letters 388: 115-118 (1996)).

[00365] Standard methods for coupling the target agents can be used. For example, phosphatidylethanolamine, which can be activated for attachment of target agents, or derivatized lipophilic compounds, such as lipid-derivatized bleomycin, can be used.

Antibody-targeted liposomes can be constructed using, for instance, liposomes that incorporate protein A {see, Renneisen, et al, J. Bio. Chem., 265: 16337-16342 (1990) and Leonetti, et al, Proc. Natl. Acad. Sci. (USA), 87:2448-2451 (1990). Other examples of antibody conjugation are disclosed in U.S. Patent No. 6,027,726, the teachings of which are incorporated herein by reference. Examples of targeting moieties can also include other proteins, specific to cellular components, including antigens associated with neoplasms or tumors. Proteins used as targeting moieties can be attached to the liposomes via covalent bonds (see, Heath, Covalent Attachment of Proteins to Liposomes, 149 Methods in

Enzymology 111-119 (Academic Press, Inc. 1987)). Other targeting methods include the biotin-avidin system.

Production of nucleic acid-lipid particles

[00366] In one embodiment, the nucleic acid-lipid particle formulations of the invention are produced via an extrusion method or an in-line mixing method.

[00367] The extrusion method (also referred to as preformed method or batch process) is a method where the empty liposomes (i.e. no nucleic acid) are prepared first, followed by the addition of nucleic acid to the empty liposome. Extrusion of liposome compositions through a small-pore polycarbonate membrane or an asymmetric ceramic membrane results in a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome complex size distribution is achieved. The liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size. In some instances, the lipid-nucleic acid compositions which are formed can be used without any sizing. These methods are disclosed in the US 5,008,050; US 4,927,637; US 4,737,323; Biochim Biophys Acta. 1979 Oct 19;557(l):9-23; Biochim Biophys Acta. 1980 Oct 2;601(3):559-7; Biochim Biophys Acta. 1986 Jun 13;858(l): 161-8; and Biochim. Biophys. Acta 1985 812, 55-65, which are hereby incorporated by reference in their entirety. [00368] The in-line mixing method is a method wherein both the lipids and the nucleic acid are added in parallel into a mixing chamber. The mixing chamber can be a simple T- connector or any other mixing chamber that is known to one skill in the art. These methods are disclosed in US patent nos. 6,534,018 and US 6,855,277; US publication 2007/0042031 and Pharmaceuticals Research, Vol. 22, No. 3, Mar. 2005, p. 362-372, which are hereby incorporated by reference in their entirety.

[00369] It is further understood that the formulations of the invention can be prepared by any methods known to one of ordinary skill in the art.

Characterization of nucleic acid-lipid particles

[00370] Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA).

Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton- XI 00. The total siRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the "free" siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%. In one embodiment, the formulations of the invention are entrapped by at least 75%, at least 80% or at least 90%.

[00371] For nucleic acid-lipid particle formulations, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

Formulations of nucleic acid-lipid particles

LNP01 [00372] One example of synthesis of a nucleic acid-lipid particle is as follows. Nucleic acid-lipid particles are synthesized using the lipidoid ND98-4HC1 (MW 1487) (Formula 1; FIG. 1), Cholesterol (Sigma- Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids). This nucleic acid-lipid particle is sometimes referred to as a LNPOl particles. Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG- Ceramide CI 6, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48: 10 molar ratio. The combined lipid solution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-siRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

[00373] LNPOl formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

[00374] Additional exemplary nucleic acid-lipid particle formulations are described in the following table (Table A). It is to be understood that the name of the nucleic acid-lipid particle in the table is not meant to be limiting. For example, as used herein, the term

SNALP refers to formulations that include the cationic lipid DLinDMA.

Table A

XTC/DSPC/Cholesterol/PEG-DMG

57.5/7.5/31.5/3.5

LNP06

lipid: siRNA - 11:1

Process: Extrusion

XTC/DSPC/Cholesterol/PEG-DMG

60/7.5/31/1.5,

LNP07

lipid:siRNA ~ 6:l

Process: In-line mixing

XTC/DSPC/Cholesterol/PEG-DMG

60/7.5/31/1.5,

LNP08

lipid: siRNA - 11:1

Process: In-line mixing

XTC/DSPC/Cholesterol/PEG-DMG

50/10/38.5/1.5

LNP09

lipid:siRNA ~ 10:1

Process: In-line mixing

ALNY- 100/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5

LNP10

lipid:siRNA ~ 10:1

Process: In-line mixing

MC3/DSPC/Cholesterol/PEG-DMG

50/10/38.5/1.5

LNP11

lipid:siRNA ~ 10:1

Process: In-line mixing

C12-200/DSPC/Cholesterol/PEG-DMG

LNP12 50/10/38.5/1.5

lipid:siRNA ~ 10:1

TechGl/DSPC/Cholesterol/PEG-DMG

50/10/38.5/1.5

LNP12

Lipid:siRNA 10:1

Process: In-line mixing

XTC/DSPC/Cholesterol/PEG-DMG

LNP13 50/10/38.5/1.5

lipid:siRNA ~ 33:l

MC3/DSPC/Cholesterol/PEG-DMG

LNP14 40/15/40/5

lipid:siRNA ~l l: l

MC3/DSPC/Cholesterol/PEG-DSG/GalNAc-PEG-DSG

LNP15 50/10/35/4.5/0.5

lipid:siRNA ~l l: l

MC3/DSPC/Cholesterol/PEG-DMG

LNP16 50/10/38.5/1.5

lipid: siRNA -7: 1

MC3/DSPC/Cholesterol/PEG-DSG

LNP17 50/10/38.5/1.5

lipid:siRNA ~10: l

MC3/DSPC/Cholesterol/PEG-DMG

LNP18 50/10/38.5/1.5

lipid:siRNA ~12: l

MC3/DSPC/Cholesterol/PEG-DMG

LNP19 50/10/35/5

lipid:siRNA ~8: l

MC3/DSPC/Cholesterol/PEG-DPG

LNP20 50/10/38.5/1.5

lipid:siRNA ~10: l

C12-200/DSPC/Cholesterol/PEG-DSG

LNP21 50/10/38.5/1.5

lipid: siRNA -7: 1 XTC/DSPC/Cholesterol/PEG-DSG

LNP22 50/10/38.5/1.5

lipid:siRNA ~10: l

[00375] XTC comprising formulations are described, e.g., in PCT/US 10/22614 filed on Jan. 29, 2010, which is hereby incorporated by reference.

[00376] MC3 comprising formulations are described, e.g., in U.S. Serial No. 12/813,448, filed June 10, 2010, which is hereby incorporated by reference.

[00377] ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on November 10, 2009, which is hereby incorporated by reference.

[00378] CI 2-200 comprising formulations are described in International patent application number PCT/US 10/33777 filed May 5, 2010 and in Love et al (Love et al. (2010) PNAS 107(5); 1864-69) which are hereby incorporated by reference.

[00379] Additional representative formulations delineated in Tables B and C. Lipid refers to a cationic lipid.

Table B: Composition of exemplary nucleic acid-lipid particle (mole %) prepared via extrusion methods.

Lipid (mol %) DSPC (mol Choi (mol PEG (mol Lipid/ siRNA

20 35 40 5 3.00

20 35 40 5 3.32

20 35 40 5 3.05

20 35 40 5 3.67

20 35 40 5 4.71

30 25 40 5 2.47

30 25 40 5 2.98

30 25 40 5 3.29

30 25 40 5 4.99

30 25 40 5 7.15

40 15 40 5 2.79

40 15 40 5 3.29

40 15 40 5 4.33

40 15 40 5 7.05

40 15 40 5 9.63

45 10 40 5 2.44

45 10 40 5 3.21

45 10 40 5 4.29

45 10 40 5 6.50

45 10 40 5 8.67

20 35 40 5 4.10

20 35 40 5 4.83

30 25 40 5 3.86

30 25 40 5 5.38

30 25 40 5 7.07

40 15 40 5 3.85

40 15 40 5 4.88

40 15 40 5 7.22

40 15 40 5 9.75

45 10 40 5 2.83

45 10 40 5 3.85

45 10 40 5 4.88

45 10 40 5 7.05

45 10 40 5 9.29

45 20 30 5 4.01

45 20 30 5 3.70

50 15 30 5 4.75

50 15 30 5 3.80

55 10 30 5 3.85

55 10 30 5 4.13

60 5 30 5 5.09

60 5 30 5 4.67

65 0 30 5 4.75 Lipid (mol %) DSPC (mol Choi (mol PEG (mol Lipid/ siRNA

65 0 30 5 6.06

56.5 10 30 3.5 3.70

56.5 10 30 3.5 3.56

57.5 10 30 2.5 3.48

57.5 10 30 2.5 3.20

58.5 10 30 1.5 3.24

58.5 10 30 1.5 3.13

59.5 10 30 0.5 3.24

59.5 10 30 0.5 3.03

45 10 40 5 7.57

45 10 40 5 7.24

45 10 40 5 7.48

45 10 40 5 7.84

65 0 30 5 4.01

60 5 30 5 3.70

55 10 30 5 3.65

50 10 35 5 3.43

50 15 30 5 3.80

45 15 35 5 3.70

45 20 30 5 3.75

45 25 25 5 3.85

55 10 32.5 2.5 3.61

60 10 27.5 2.5 3.65

60 10 25 5 4.07

55 5 38.5 1.5 3.75

60 10 28.5 1.5 3.43

55 10 33.5 1.5 3.48

60 5 33.5 1.5 3.43

55 5 37.5 2.5 3.75

60 5 32.5 2.5 4.52

60 5 32.5 2.5 3.52

45 15 (DMPC) 35 5 3.20

45 15 (DPPC) 35 5 3.43

45 15 (D0PC) 35 5 4.52

45 15 (P0PC) 35 5 3.85

55 5 37.5 2.5 3.96

55 10 32.5 2.5 3.56

60 5 32.5 2.5 3.80

60 10 27.5 2.5 3.75

60 5 30 5 4.19

60 5 33.5 1.5 3.48

60 5 33.5 1.5 6.64

60 5 30 5 3.90 Lipid (mol %) DSPC (mol Choi (mol PEG (mol Lipid/ siRNA

60 5 30 5 4.65

60 5 30 5 5.88

60 5 30 5 7.51

60 5 30 5 9.51

60 5 30 5 11.06

62.5 2.5 50 5 6.63

45 15 35 5 3.31

45 15 35 5 6.80

60 5 25 10 6.48

60 5 32.5 2.5 3.43

60 5 30 5 3.90

60 5 30 5 7.61

45 15 35 5 3.13

45 15 35 5 6.42

60 5 25 10 6.48

60 5 32.5 2.5 3.03

60 5 30 5 3.43

60 5 30 5 6.72

60 5 30 5 4.13

70 5 20 5 5.48

80 5 10 5 5.94

90 5 0 5 9.50

60 5 30 5 C12PEG 3.85

60 5 30 5 3.70

60 5 30 5 C16PEG 3.80

60 5 30 5 4.19

60 5 29 5 4.07

60 5 30 5 3.56

60 5 30 5 3.39

60 5 30 5 3.96

60 5 30 5 4.01

60 5 30 5 4.07

60 5 30 5 4.25

60 5 30 5 3.80

60 5 30 5 3.31

60 5 30 5 4.83

60 5 30 5 4.67

60 5 30 5 3.96

57.5 7.5 33.5 1.5 3.39

57.5 7.5 32.5 2.5 3.39

57.5 7.5 31.5 3.5 3.52

57.5 7.5 30 5 4.19

60 5 30 5 3.96 Lipid (mol %) DSPC (mol Choi (mol PEG (mol Lipid/ siRNA

60 5 30 5 3.96

60 5 30 5 3.56

60 5 33.5 1.5 3.52

60 5 25 10 5.18

60 5 (DPPC) 30 5 4.25

60 5 32.5 2.5 3.70

57.5 7.5 31.5 3.5 3.06

57.5 7.5 31.5 3.5 3.65

57.5 7.5 31.5 3.5 4.70

57.5 7.5 31.5 3.5 6.56

Table C: Composition of exemplary nucleic acid-lwid particles prepared via in-line mixing

Lipid (mol %) DSPC (mol %) Choi (mol %) PEG (mol %) Lipid A/ siRNA

60 7.5 31.5 1 3.56

60 10 29 1 3.80

70 5 24 1 3.70

70 7.5 21.5 1 4.13

70 10 19 1 3.85

60 5 34 1 3.52

60 5 34 1 3.70

60 5 34 1 3.52

60 7.5 27.5 5 5.18

60 7.5 29 3.5 4.45

60 5 31.5 3.5 4.83

60 7.5 31 1.5 3.48

57.5 7.5 30 5 4.75

57.5 7.5 31.5 3.5 4.83

57.5 5 34 3.5 4.67

57.5 7.5 33.5 1.5 3.43

55 7.5 32.5 5 4.38

55 7.5 34 3.5 4.13

55 5 36.5 3.5 4.38

55 7.5 36 1.5 3.35

Therapeutic Agent-Lipid Particle Compositions and Formulations

[00380] The invention includes compositions comprising a lipid particle of the invention and an active agent, wherein the active agent is associated with the lipid particle. In particular embodiments, the active agent is a therapeutic agent. In particular embodiments, the active agent is encapsulated within an aqueous interior of the lipid particle. In other embodiments, the active agent is present within one or more lipid layers of the lipid particle. In other embodiments, the active agent is bound to the exterior or interior lipid surface of a lipid particle.

[00381] "Fully encapsulated" as used herein indicates that the nucleic acid in the particles is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free DNA. In a fully encapsulated system, preferably less than 25% of particle nucleic acid is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than 10% and most preferably less than 5% of the particle nucleic acid is degraded. Alternatively, full encapsulation may be determined by an

Oligreen^® assay. Oligreen^® is an ultra-sensitive fluorescent nucleic acid stain for quantitating oligonucleotides and single-stranded DNA in solution (available from Invitrogen Corporation, Carlsbad, CA). Fully encapsulated also suggests that the particles are serum stable, that is, that they do not rapidly decompose into their component parts upon in vivo administration.

[00382] Active agents, as used herein, include any molecule or compound capable of exerting a desired effect on a cell, tissue, organ, or subject. Such effects may be biological, physiological, or cosmetic, for example. Active agents may be any type of molecule or compound, including e.g., nucleic acids, peptides and polypeptides, including, e.g., antibodies, such as, e.g., polyclonal antibodies, monoclonal antibodies, antibody fragments; humanized antibodies, recombinant antibodies, recombinant human antibodies, and

Primatized™ antibodies, cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell surface receptors and their ligands; hormones; and small molecules, including small organic molecules or compounds.

[00383] In one embodiment, the active agent is a therapeutic agent, or a salt or derivative thereof. Therapeutic agent derivatives may be therapeutically active themselves or they may be prodrugs, which become active upon further modification. Thus, in one embodiment, a therapeutic agent derivative retains some or all of the therapeutic activity as compared to the unmodified agent, while in another embodiment, a therapeutic agent derivative lacks therapeutic activity.

[00384] In various embodiments, therapeutic agents include any therapeutically effective agent or drug, such as anti-inflammatory compounds, anti-depressants, stimulants, analgesics, antibiotics, birth control medication, antipyretics, vasodilators, anti-angiogenics, cytovascular agents, signal transduction inhibitors, cardiovascular drugs, e.g., anti-arrhythmic agents, vasoconstrictors, hormones, and steroids.

[00385] In certain embodiments, the therapeutic agent is an oncology drug, which may also be referred to as an anti-tumor drug, an anti-cancer drug, a tumor drug, an antineoplastic agent, or the like. Examples of oncology drugs that may be used according to the invention include, but are not limited to, adriamycin, alkeran, allopurinol, altretamine, amifostine, anastrozole, araC, arsenic trioxide, azathioprine, bexarotene, biCNU, bleomycin, busulfan intravenous, busulfan oral, capecitabine (Xeloda), carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin, cladribine, cyclosporin A, cytarabine, cytosine arabinoside, daunorubicin, Cytoxan, daunorubicin, dexamethasone, dexrazoxane, dodetaxel, doxorubicin, doxorubicin, DTIC, epirubicin, estramustine, etoposide phosphate, etoposide and VP- 16, exemestane, FK506, fludarabine, fluorouracil, 5-FU, gemcitabine (Gemzar), gemtuzumab- ozogamicin, goserelin acetate, hydrea, hydroxyurea, idarubicin, ifosfamide, imatinib mesylate, interferon, irinotecan (Camptostar, CPT-111), letrozole, leucovorin, leustatin, leuprolide, levamisole, litretinoin, megastrol, melphalan, L-PAM, mesna, methotrexate, methoxsalen, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel, pamidronate, Pegademase, pentostatin, porfimer sodium, prednisone, rituxan, streptozocin, STI-571, tamoxifen, taxotere, temozolamide, teniposide, VM-26, topotecan (Hycamtin), toremifene, tretinoin, ATRA, valrubicin, velban, vinblastine, vincristine, VP 16, and vinorelbine. Other examples of oncology drugs that may be used according to the invention are ellipticin and ellipticin analogs or derivatives, epothilones, intracellular kinase inhibitors and camptothecins.

Additional formulations

Emulsions

[00386] The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μιη in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion.

Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

[00387] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[00388] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in

categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

[00389] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid

consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

[00390] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume l, p. 199).

[00391] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

[00392] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[00393] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been

administered orally as o/w emulsions.

[00394] In one embodiment of the present invention, the compositions of dsRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and

thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).

Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[00395] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions,

microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

[00396] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),

decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

[00397] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol, 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or dsRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of dsRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of dsRNAs and nucleic acids. [00398] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the dsRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories- surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

Penetration Enhancers

[00399] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly RNA effector agent, e.g., siRNA and/or ABP to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non- lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

[00400] Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non- surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

[00401] Surfactants: In connection with the present invention, surfactants (or "surface- active agents") are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of dsRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252). [00402] Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1- monocaprate, l-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C_1-10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al, J. Pharm. Pharmacol, 1992, 44, 651- 654).

[00403] Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term "bile salts" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1- 33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[00404] Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of dsRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones

(enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control ReL, 1990, 14, 43-51).

[00405] Non-chelating non- surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate

insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[00406] Agents that enhance uptake of RNA effector agent, e.g., siRNA and/or ABP at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No.

5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.

[00407] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

[00408] The RNA effector agent, e.g., siRNA and/or ABP 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).

[00409] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The co-administration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extra- circulatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially

phosphorothioate dsR A in hepatic tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'isothiocyano-stilbene- 2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

[00410] In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars,

micro crystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc); disintegrants {e.g., starch, sodium starch glycolate, etc.); and wetting agents {e.g., sodium lauryl sulphate, etc).

[00411] Pharmaceutically acceptable organic or inorganic excipients suitable for non- parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin,

hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[00412] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

[00413] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Other Components

[00414] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation. [00415] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Methods of preparing lipid particles

[00416] The methods and compositions of the invention make use of certain cationic lipids, the synthesis, preparation and characterization of which is described below and in the accompanying Examples. In addition, the present invention provides methods of preparing lipid particles, including those associated with a therapeutic agent, e.g., a nucleic acid. In the methods described herein, a mixture of lipids is combined with a buffered aqueous solution of nucleic acid and/or protein to produce an intermediate mixture containing nucleic acid and/or protein encapsulated in lipid particles wherein the encapsulated nucleic acids and/or protein are present in a ratio of about 3 wt% to about 25 wt%, preferably 5 to 15 wt%. The intermediate mixture may optionally be sized to obtain lipid-encapsulated nucleic acid particles wherein the lipid portions are unilamellar vesicles, preferably having a diameter of 30 to 150 nm, more preferably about 40 to 90 nm. The pH is then raised to neutralize at least a portion of the surface charges on the lipid particles, thus providing an at least partially surface-neutralized lipid-encapsulated composition.

[00417] As described above, several of these cationic lipids are amino lipids that are charged at a pH below the pK_a of the amino group and substantially neutral at a pH above the pK_a. These cationic lipids are termed titratable cationic lipids and can be used in the formulations of the invention using a two-step process. First, lipid vesicles can be formed at the lower pH with titratable cationic lipids and other vesicle components in the presence of nucleic acids and/or proteins. In this manner, the vesicles will encapsulate and entrap the nucleic acids and/or proteins. Second, the surface charge of the newly formed vesicles can be neutralized by increasing the pH of the medium to a level above the pK_a of the titratable cationic lipids present, i.e., to physiological pH or higher. Particularly advantageous aspects of this process include both the facile removal of any surface adsorbed nucleic acid and/or protein and a resultant nucleic acid and/or protein delivery vehicle which has a neutral surface. Liposomes or lipid particles having a neutral surface are expected to avoid rapid clearance from circulation and to avoid certain toxicities which are associated with cationic liposome preparations. Additional details concerning these uses of such titratable cationic lipids in the formulation of lipid particles are provided in US Patent 6,287,591 and US Patent 6,858,225, incorporated herein by reference. [00418] It is further noted that the vesicles formed in this manner provide formulations of uniform vesicle size with high content of nucleic acids and/or proteins. Additionally, the vesicles have a size range of from about 30 to about 150 nm, more preferably about 30 to about 90 nm.

[00419] Without intending to be bound by any particular theory, it is believed that the very high efficiency of nucleic acid encapsulation is a result of electrostatic interaction at low pH. At acidic pH (e.g. pH 4.0) the vesicle surface is charged and binds a portion of the nucleic acids through electrostatic interactions. When the external acidic buffer is exchanged for a more neutral buffer (e.g.. pH 7.5) the surface of the lipid particle or liposome is neutralized, allowing any external nucleic acid to be removed. More detailed information on the formulation process is provided in various publications (e.g., US Patent 6,287,591 and US Patent 6,858,225).

[00420] In view of the above, the present invention provides methods of preparing lipid/nucleic acid and/or protein formulations. In the methods described herein, a mixture of lipids is combined with a buffered aqueous solution of nucleic acid and/or protein to produce an intermediate mixture containing nucleic acid and/or protein encapsulated in lipid particles, e.g., wherein the encapsulated nucleic acids and/or proteins are present in a ratio of about 10 wt% to about 20 wt%. The intermediate mixture may optionally be sized to obtain lipid- encapsulated nucleic acid and/or protein particles wherein the lipid portions are unilamellar vesicles, preferably having a diameter of 30 to 150 nm, more preferably about 40 to 90 nm. The pH is then raised to neutralize at least a portion of the surface charges on the lipid particles, thus providing an at least partially surface-neutralized lipid-encapsulated nucleic acid and/or protein composition.

[00421] In certain embodiments, the mixture of lipids includes at least two lipid components: a first amino lipid component of the present invention that is selected from among lipids which have a pKa such that the lipid is cationic at pH below the pKa and neutral at pH above the pKa, and a second lipid component that is selected from among lipids that prevent particle aggregation during lipid particle formation. In particular embodiments, the amino lipid is a novel cationic lipid of the present invention.

[00422] In preparing the lipid particles of the invention, the mixture of lipids is typically a solution of lipids in an organic solvent. This mixture of lipids can then be dried to form a thin film or lyophilized to form a powder before being hydrated with an aqueous buffer to form liposomes. Alternatively, in a preferred method, the lipid mixture can be solubilized in a water miscible alcohol, such as ethanol, and this ethanolic solution added to an aqueous buffer resulting in spontaneous liposome formation. In most embodiments, the alcohol is used in the form in which it is commercially available. For example, ethanol can be used as absolute ethanol (100%), or as 95% ethanol, the remainder being water. This method is described in more detail in US Patent 5,976,567).

[00423] In accordance with the invention, the lipid mixture is combined with a buffered aqueous solution that may contain the nucleic acids and/or proteins. The buffered aqueous solution of is typically a solution in which the buffer has a pH of less than the pK_a of the protonatable lipid in the lipid mixture. Examples of suitable buffers include citrate, phosphate, acetate, and MES. A particularly preferred buffer is citrate buffer. Preferred buffers will be in the range of 1-1000 mM of the anion, depending on the chemistry of the nucleic acid being encapsulated, and optimization of buffer concentration may be significant to achieving high loading levels (see, e.g., US Patent 6,287,591 and US Patent 6,858,225). Alternatively, pure water acidified to pH 5-6 with chloride, sulfate or the like may be useful. In this case, it may be suitable to add 5% glucose, or another non-ionic solute which will balance the osmotic potential across the particle membrane when the particles are dialyzed to remove ethanol, increase the pH, or mixed with a pharmaceutically acceptable carrier such as normal saline. The amount of nucleic acid and/or protein in buffer can vary, but will typically be from about 0.01 mg/mL to about 200 mg/mL, more preferably from about 0.5 mg/mL to about 50 mg/mL.

[00424] The mixture of lipids and the buffered aqueous solution of therapeutic nucleic acids and/or proteins are combined to provide an intermediate mixture. The intermediate mixture is typically a mixture of lipid particles having encapsulated nucleic acids and/or proteins. Additionally, the intermediate mixture may also contain some portion of nucleic acids and/or proteins which are attached to the surface of the lipid particles (liposomes or lipid vesicles) due to the ionic attraction of the negatively-charged nucleic acids and positively-charged lipids on the lipid particle surface (the amino lipids or other lipid making up the protonatable first lipid component are positively charged in a buffer having a pH of less than the pKa of the protonatable group on the lipid). In one group of preferred embodiments, the mixture of lipids is an alcohol solution of lipids and the volumes of each of the solutions is adjusted so that upon combination, the resulting alcohol content is from about 20% by volume to about 45% by volume. The method of combining the mixtures can include any of a variety of processes, often depending upon the scale of formulation produced. For example, when the total volume is about 10-20 mL or less, the solutions can be combined in a test tube and stirred together using a vortex mixer. Large-scale processes can be carried out in suitable production scale glassware.

[00425] Optionally, the lipid-encapsulated therapeutic agent (e.g., nucleic acid) complexes which are produced by combining the lipid mixture and the buffered aqueous solution of therapeutic agents (nucleic acids and/or proteins) can be sized to achieve a desired size range and relatively narrow distribution of lipid particle sizes. Preferably, the compositions provided herein will be sized to a mean diameter of from about 70 to about 200 nm, more preferably about 90 to about 130 nm. Several techniques are available for sizing liposomes to a desired size. One sizing method is described in U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles (SUVs) less than about 0.05 microns in size. Homogenization is another method which relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, multilamellar vesicles are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size determination. For certain methods herein, extrusion is used to obtain a uniform vesicle size.

[00426] Extrusion of liposome compositions through a small-pore polycarbonate membrane or an asymmetric ceramic membrane results in a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome complex size distribution is achieved. The liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size. In some instances, the lipid compositions which are formed can be used without any sizing.

[00427] In particular embodiments, methods of the present invention further comprise a step of neutralizing at least some of the surface charges on the lipid portions of the lipid compositions. By at least partially neutralizing the surface charges, unencapsulated nucleic acid and/or protein is freed from the lipid particle surface and can be removed from the composition using conventional techniques. Preferably, unencapsulated and surface adsorbed nucleic acids and/or proteins are removed from the resulting compositions through exchange of buffer solutions. For example, replacement of a citrate buffer (pH about 4.0, used for forming the compositions) with a HEPES -buffered saline (HBS pH about 7.5) solution, results in the neutralization of liposome surface and nucleic acid and/or protein release from the surface. The released nucleic acid and/or protein can then be removed via

chromatography using standard methods, and then switched into a buffer with a pH above the pKa of the lipid used.

[00428] Optionally the lipid vesicles (i.e., lipid particles) can be formed by hydration in an aqueous buffer and sized using any of the methods described above prior to addition of the nucleic acid and/or protein. As described above, the aqueous buffer should be of a pH below the pKa of the amino lipid. A solution of the nucleic acids and/or proteins can then be added to these sized, preformed vesicles. To allow encapsulation of nucleic acids and/or proteins into such "pre-formed" vesicles the mixture should contain an alcohol, such as ethanol. In the case of ethanol, it should be present at a concentration of about 20% (w/w) to about 45% (w/w). In addition, it may be necessary to warm the mixture of pre-formed vesicles and nucleic acid in the aqueous buffer-ethanol mixture to a temperature of about 25° C to about 50° C depending on the composition of the lipid vesicles and the nature of the nucleic acid and/or protein. It will be apparent to one of ordinary skill in the art that optimization of the encapsulation process to achieve a desired level of nucleic acid and/or protein in the lipid vesicles will require manipulation of variable such as ethanol concentration and temperature. Examples of suitable conditions for nucleic acid and/or protein encapsulation are provided in the Examples. Once the nucleic acids and/or proteins are encapsulated within the preformed vesicles, the external pH can be increased to at least partially neutralize the surface charge. Unencapsulated and surface adsorbed nucleic acids and/or proteins can then be removed as described above.

Method of Use

[00429] The lipid particles of the invention may be used to deliver a therapeutic agent to a cell, in vitro or in vivo. In particular embodiments, the therapeutic agent is a nucleic acid, which is delivered to a cell using a nucleic acid-lipid particle and/or ABP of the invention. While the following description of various methods of using the lipid particles and related pharmaceutical compositions of the invention are exemplified by description related to nucleic acid-lipid particles, it is understood that these methods and compositions may be readily adapted for the delivery of any therapeutic agent for the treatment of any disease or disorder that would benefit from such treatment, e.g., an ABP. [00430] In certain embodiments, the invention provides methods for introducing a nucleic acid into a cell. Preferred nucleic acids for introduction into cells are siR A, immune- stimulating oligonucleotides, plasmids, antisense and ribozymes. These methods may be carried out by contacting the particles or compositions of the invention with the cells for a period of time sufficient for intracellular delivery to occur.

[00431] The compositions of the invention can be adsorbed to almost any cell type. Once adsorbed, the nucleic acid-lipid particles can either be endocytosed by a portion of the cells, exchange lipids with cell membranes, or fuse with the cells. Transfer or incorporation of the nucleic acid portion of the complex can take place via any one of these pathways. Without intending to be limited with respect to the scope of the invention, it is believed that in the case of particles taken up into the cell by endocytosis the particles then interact with the endosomal membrane, resulting in destabilization of the endosomal membrane, possibly by the formation of non-bilayer phases, resulting in introduction of the encapsulated nucleic acid into the cell cytoplasm. Similarly in the case of direct fusion of the particles with the cell plasma membrane, when fusion takes place, the liposome membrane is integrated into the cell membrane and the contents of the liposome combine with the intracellular fluid. Contact between the cells and the lipid-nucleic acid compositions, when carried out in vitro, will take place in a biologically compatible medium. The concentration of compositions can vary widely depending on the particular application, but is generally between about 1 μιηοΐ and about 10 mmol. In certain embodiments, treatment of the cells with the lipid-nucleic acid compositions will generally be carried out at physiological temperatures (about 37°C) for periods of time from about 1 to 24 hours, preferably from about 2 to 8 hours. For in vitro applications, the delivery of nucleic acids can be to any cell grown in culture, whether of plant or animal origin, vertebrate or invertebrate, and of any tissue or type. In preferred embodiments, the cells will be animal cells, more preferably mammalian cells, and most preferably human cells.

[00432] In one group of embodiments, a lipid-nucleic acid particle suspension is added to 60-80% confluent plated cells having a cell density of from about 10³ to about 10⁵ cells/mL, more preferably about 2 x 10⁴ cells/mL. The concentration of the suspension added to the cells is preferably of from about 0.01 to 20 μg/mL, more preferably about 1 μg/mL.

[00433] Typical applications include using well known procedures to provide intracellular delivery of siRNA to knock down or silence specific cellular targets. Alternatively applications include delivery of DNA or mRNA sequences that code for therapeutically useful polypeptides. In this manner, therapy is provided for genetic diseases by supplying deficient or absent gene products (i.e., for Duchenne's dystrophy, see Kunkel, et al., Brit. Med. Bull. 45(3):630-643 (1989), and for cystic fibrosis, see Goodfellow, Nature 341 : 102- 103 (1989)). Other uses for the compositions of the invention include introduction of antisense oligonucleotides in cells (see, Bennett, et al, Mol. Pharm. 41 :1023-1033 (1992)).

[00434] Alternatively, the compositions of the invention can also be used for deliver of nucleic acids to cells in vivo, using methods which are known to those of skill in the art. With respect to application of the invention for delivery of DNA or mRNA sequences, Zhu, et al, Science 261 :209-211 (1993), incorporated herein by reference, describes the intravenous delivery of cytomegalovirus (CMV)-chloramphenicol acetyltransferase (CAT) expression plasmid using DOTMA-DOPE complexes. Hyde, et al, Nature 362:250-256 (1993), incorporated herein by reference, describes the delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to epithelia of the airway and to alveoli in the lung of mice, using liposomes. Brigham, et al, Am. J. Med. Sci. 298:278-281 (1989), incorporated herein by reference, describes the in vivo transfection of lungs of mice with a functioning prokaryotic gene encoding the intracellular enzyme, chloramphenicol acetyltransferase (CAT). Thus, the compositions of the invention can be used in the treatment of infectious diseases.

[00435] For in vivo administration, the pharmaceutical compositions are preferably administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally,

subcutaneously, or intramuscularly. In particular embodiments, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection. For one example, see Stadler, et al, U.S. Patent No. 5,286,634, which is incorporated herein by reference. Intracellular nucleic acid delivery has also been discussed in Straubringer, et al, METHODS IN ENZYMOLOGY, Academic Press, New York. 101 :512-527 (1983); Mannino, et al, Biotechniques 6:682-690 (1988); Nicolau, et al, Crit. Rev. Ther. Drug Carrier Syst. 6:239-271 (1989), and Behr, Acc. Chem. Res. 26:274-278 (1993). Still other methods of administering lipid-based therapeutics are described in, for example, Rahman et al, U.S. Patent No. 3,993,754; Sears, U.S. Patent No. 4,145,410; Papahadjopoulos et al, U.S. Patent No. 4,235,871; Schneider, U.S. Patent No. 4,224,179; Lenk et al, U.S. Patent No. 4,522,803; and Fountain et al, U.S. Patent No. 4,588,578.

[00436] In other methods, the pharmaceutical preparations may be contacted with the target tissue by direct application of the preparation to the tissue. The application may be made by topical, "open" or "closed" procedures. By "topical," it is meant the direct application of the pharmaceutical preparation to a tissue exposed to the environment, such as the skin, oropharynx, external auditory canal, and the like. "Open" procedures are those procedures which include incising the skin of a patient and directly visualizing the underlying tissue to which the pharmaceutical preparations are applied. This is generally accomplished by a surgical procedure, such as a thoracotomy to access the lungs, abdominal laparotomy to access abdominal viscera, or other direct surgical approach to the target tissue. "Closed" procedures are invasive procedures in which the internal target tissues are not directly visualized, but accessed via inserting instruments through small wounds in the skin. For example, the preparations may be administered to the peritoneum by needle lavage.

Likewise, the pharmaceutical preparations may be administered to the meninges or spinal cord by infusion during a lumbar puncture followed by appropriate positioning of the patient as commonly practiced for spinal anesthesia or metrazamide imaging of the spinal cord. Alternatively, the preparations may be administered through endoscopic devices.

[00437] The lipid-nucleic acid compositions can also be administered in an aerosol inhaled into the lungs (see, Brigham, et ah, Am. J. Sci. 298(4):278-281 (1989)) or by direct injection at the site of disease (Culver, Human Gene Therapy, Mary Ann Liebert, Inc., Publishers, New York, pp.70-71 (1994)).

[00438] The methods of the invention may be practiced in a variety of hosts. Preferred hosts include mammalian species, such as humans, non-human primates, dogs, cats, cattle, horses, sheep, and the like.

[00439] Dosages for the lipid-therapeutic agent particles of the invention will depend on the ratio of therapeutic agent to lipid and the administrating physician's opinion based on age, weight, and condition of the patient.

[00440] In one embodiment, the invention provides a method of modulating the expression of a target polynucleotide or polypeptide. These methods generally comprise contacting a cell with a lipid particle of the invention that is associated with a nucleic acid capable of modulating the expression of a target polynucleotide or polypeptide. As used herein, the term "modulating" refers to altering the expression of a target polynucleotide or polypeptide. In different embodiments, modulating can mean increasing or enhancing, or it can mean decreasing or reducing. Methods of measuring the level of expression of a target

polynucleotide or polypeptide are known and available in the arts and include, e.g., methods employing reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical techniques. In particular embodiments, the level of expression of a target polynucleotide or polypeptide is increased or reduced by at least 10%, 20%>, 30%>, 40%, 50%, or greater than 50% as compared to an appropriate control value. For example, if increased expression of a polypeptide desired, the nucleic acid may be an expression vector that includes a polynucleotide that encodes the desired polypeptide. On the other hand, if reduced expression of a polynucleotide or polypeptide is desired, then the nucleic acid may be, e.g., an antisense oligonucleotide, siRNA, or microRNA that comprises a polynucleotide sequence that specifically hybridizes to a polynucleotide that encodes the target polypeptide, thereby disrupting expression of the target polynucleotide or polypeptide. Alternatively, the nucleic acid may be a plasmid that expresses such an antisense oligonucleotide, siRNA, or microRNA.

[00441] In one particular embodiment, the invention provides a method of modulating the expression of a polypeptide by a cell, comprising providing to a cell a lipid particle that consists of or consists essentially of a cationic lipid of formula A, a neutral lipid, a sterol, a PEG of PEG-modified lipid, e.g., in a molar ratio of about 35-65% of cationic lipid of formula A, 3-12% of the neutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG or PEG- modified lipid, wherein the lipid particle is associated with a nucleic acid capable of modulating the expression of the polypeptide. In particular embodiments, the molar lipid ratio is approximately 60/7.5/31/1.5 or 57.5/7.5/31.5/3.5 (mol% LIPID A/DSPC/Chol/PEG- DMG). In another group of embodiments, the neutral lipid in these compositions is replaced with DPPC (dipalmitoylphosphatidylcholine), POPC, DOPE or SM.

[00442] In particular embodiments, the therapeutic agent is selected from an siRNA, a microRNA, an antisense oligonucleotide, and a plasmid capable of expressing an siRNA, a microRNA, an ABP, or an antisense oligonucleotide, and wherein the siRNA, microRNA, or antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide that encodes the polypeptide, or a complement thereof, such that the expression of the polypeptide is reduced.

[00443] In other embodiments, the nucleic acid is a plasmid that encodes the polypeptide or a functional variant or fragment thereof, such that expression of the polypeptide or the functional variant or fragment thereof is increased.

[00444] In related embodiments, the invention provides a method of treating a disease or disorder characterized by overexpression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition of the invention, wherein the therapeutic agent is selected from an siRNA, a microRNA, an antisense oligonucleotide, and a plasmid capable of expressing an siRNA, a microRNA, or an antisense oligonucleotide, and wherein the siRNA, microRNA, or antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide that encodes the polypeptide, or a complement thereof.

[00445] In one embodiment, the pharmaceutical composition comprises a lipid particle that consists of or consists essentially of Lipid A, DSPC, Choi and PEG-DMG, PEG-C- DOMG or PEG-DMA, e.g., in a molar ratio of about 35-65% of cationic lipid of formula A, 3-12% of the neutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG or PEG-modified lipid PEG-DMG, PEG-C-DOMG or PEG-DMA, wherein the lipid particle is associated with the therapeutic nucleic acid. In particular embodiments, the molar lipid ratio is

approximately 60/7.5/31/1.5 or 57.5/7.5/31.5/3.5 (mol% LIPID A/DSPC/Chol/PEG-DMG). In another group of embodiments, the neutral lipid in these compositions is replaced with DPPC, POPC, DOPE or SM.

[00446] In another related embodiment, the invention includes a method of treating a disease or disorder characterized by underexpression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition of the invention, wherein the therapeutic agent is a plasmid that encodes the polypeptide or a functional variant or fragment thereof.

[00447] 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.

PHARMACEUTICAL KITS

[00448] In certain embodiments, kits are provided for producing a single-dose

administration unit. In certain embodiments, the kit can contain a first container having a dried protein, a second container having a dsRNA, and/or a third container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes) are included. Instructions for using the kit can also be included with the kit. METHODS FOR INHIBITING EXPRESSION AND/OR ACTIVITY OF THE PCSK9 GENE AND/OR PROTEIN

[00449] In yet another aspect, the invention provides a method for inhibiting the expression of the PCSK9 gene in a mammal and/or blocking or inhibiting or decreasing the activity of the PCSK9 protein in a mammal. The method can include administering a composition of the invention to the mammal such that expression of the target PCSK9 gene is decreased for an extended duration, e.g., at least one week, two weeks, three weeks, or four weeks or longer. The method can include administering a composition of the invention to the mammal such that the activity of the target PCSK9 protein is decreased for an extended duration, e.g., at least one week, two weeks, three weeks, or four weeks or longer.

[00450] For example, in certain instances, expression of the PCSK9 gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a double-stranded oligonucleotide described herein. In some embodiments, the PCSK9 gene is suppressed by at least about 60%>, 70%>, or 80%> by administration of the double-stranded oligonucleotide. In some embodiments, the PCSK9 gene is suppressed by at least about 85%, 90%), or 95%) by administration of the double-stranded oligonucleotide. Table lb, Table 2b, and Table 5b of PCT/US07/68655 provide a wide range of values for inhibition of expression obtained in an in vitro assay using various PCSK9 dsR A molecules at various

concentrations (data not shown). In certain instances, activity of the PCSK9 protein is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an ABP described herein. In some embodiments, the PCSK9 activity is suppressed by at least about 60%>, 70%>, or 80%> by administration of the ABP. In some embodiments, the PCSK9 activity is suppressed by at least about 85%, 90%>, or 95% by administration of the ABP.

[00451] The effect of the decreased target PCSK9 gene expression and/or activity preferably results in a decrease in LDLc (low density lipoprotein cholesterol) levels in the blood, and more particularly in the serum, of the mammal. In some embodiments, LDLc levels are decreased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.

[00452] The method includes administering a composition containing a dsRNA, where the dsRNA has a nucleotide sequence that is complementary to at least a part of an R A transcript of the PCSK9 gene of the mammal to be treated. In some embodiments, the composition includes an ABP targeting PCSK9. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous,

intramuscular, subcutaneous, transdermal, and airway (aerosol) administration. In some embodiments, the compositions are administered by intravenous infusion or injection.

[00453] The methods and compositions described herein can be used to treat diseases and conditions that can be modulated by down regulating PCSK9 gene expression and/or activity. For example, the compositions described herein can be used to treat hyperlipidemia and other forms of lipid imbalance such as hypercholesterolemia, hypertriglyceridemia and the pathological conditions associated with these disorders such as heart and circulatory diseases. In some embodiments, a patient treated with a PCSK9 dsRNA is also administered a non- dsRNA therapeutic agent, such as an agent known to treat lipid disorders, e.g., an ABP.

[00454] In one embodiment, doses of RNA effector agent, e.g., siRNA and/or ABP are administered not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week. In another embodiment, the administrations can be maintained for one, two, three, or six months, or one year or longer.

[00455] In another embodiment, administration of RNA effector agent, e.g., siRNA and/or ABP can be provided when Low Density Lipoprotein cholesterol (LDLc) levels reach or surpass a predetermined minimal level, such as greater than 70mg/dL, 130 mg/dL, 150 mg/dL, 200 mg/dL, 300 mg/dL, or 400 mg/dL.

[00456] In one embodiment, the subject is selected, at least in part, on the basis of needing (as opposed to merely selecting a patient on the grounds of who happens to be in need of) LDL lowering, LDL lowering without lowering of HDL, ApoB lowering, or total cholesterol lowering without HDL lowering.

[00457] In one embodiment, the dsRNA does not activate the immune system, e.g., it does not increase cytokine levels, such as TNF-alpha or IFN-alpha levels. For example, when measured by an assay, such as an in vitro PBMC assay, such as described herein, the increase in levels of TNF-alpha or IFN-alpha, is less than 30%, 20%, or 10% of control cells treated with a control dsRNA, such as a dsRNA that does not target PCSK9.

[00458] In another embodiment, a composition containing a dsRNA featured in the invention, i.e., a dsRNA targeting PCSK9, is administered with a non-dsRNA therapeutic agent, such as an agent known to treat a lipid disorders, such as hypercholesterolemia, atherosclerosis or dyslipidemia. For example, a dsRNA featured in the invention can be administered with, e.g., an ABP targeting PCSK9 as described herein, an HMG-CoA reductase inhibitor (e.g., a statin), a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist (e.g., losartan potassium, such as Merck & Co. 's Cozaar®), an acylCoA cholesterol

acetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor, a cholesterol modulator, a bile acid modulator, a peroxisome proliferation activated receptor (PPAR) agonist, a gene -based therapy, a composite vascular protectant (e.g., AGI- 1067, from Atherogenics), a glycoprotein Ilb/IIIa inhibitor, aspirin or an aspirin- like compound, an IB AT inhibitor (e.g., S-8921 , from Shionogi), a squalene synthase inhibitor, or a monocyte chemoattractant protein (MCP)-I inhibitor. Exemplary HMG-CoA reductase inhibitors include atorvastatin (Pfizer's Lipitor®/Tahor/Sortis/Torvast/Cardyl), pravastatin (Bristol-Myers Squibb's Pravachol, Sankyo's Mevalotin/Sanaprav), simvastatin (Merck's Zocor®/Sinvacor, Boehringer Ingelheim's Denan, Banyu's Lipovas), lovastatin (Merck's Mevacor/Mevinacor, Bexal's Lovastatina, Cepa; Schwarz Pharma's Liposcler), fluvastatin (Novartis' Lescol®/Locol/Lochol, Fujisawa's Cranoc, Solvay's Digaril), cerivastatin

(Bayer's Lipobay/GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca's Crestor®), and pitivastatin (itavastatin/risivastatin) (Nissan Chemical, Kowa Kogyo, Sankyo, and Novartis). Exemplary fibrates include, e.g., bezafibrate (e.g., Roche's Befizal®/Cedur®/Bezalip®, Kissei's Bezatol), clofibrate (e.g., Wyeth's Atromid-S®), fenofibrate (e.g., Fournier's Lipidil/Lipantil, Abbott's Tricor®, Takeda's Lipantil, generics), gemfibrozil (e.g., Pfizer's Lopid/Lipur) and ciprofibrate (Sanofi-Synthelabo's Modalim®). Exemplary bile acid sequestrants include, e.g., cholestyramine (Bristol-Myers Squibb's Questran® and Questran Light™), colestipol (e.g., Pharmacia's Colestid), and colesevelam (Genzyme/Sankyo's WelChol™). Exemplary niacin therapies include, e.g., immediate release formulations, such as Aventis' Nicobid, Upsher-Smith's Niacor, Aventis' Nicolar, and Sanwakagaku's Perycit. Niacin extended release formulations include, e.g., Kos Pharmaceuticals' Niaspan and Upsher-Smith's SIo- Niacin. Exemplary antiplatelet agents include, e.g., aspirin (e.g., Bayer's aspirin), clopidogrel (Sanofi-Synthelabo/Bristol-Myers Squibb's Plavix), and ticlopidine (e.g., Sanofi-Synthelabo's Ticlid and Daiichi's Panaldine). Other aspirin-like compounds useful in combination with a dsRNA targeting PCSK9 include, e.g., Asacard (slow-release aspirin, by Pharmacia) and Pamicogrel (Kanebo/Angelini Ricerche/CEPA). Exemplary angiotensin-converting enzyme inhibitors include, e.g., ramipril (e.g., Aventis' Altace) and enalapril (e.g., Merck & Co.'s Vasotec). Exemplary acyl Co A cholesterol acetyltransferase (AC AT) inhibitors include, e.g., avasimibe (Pfizer), eflucimibe

(BioMsrieux Pierre Fabre/Eli Lilly), CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito). Exemplary cholesterol absorption inhibitors include, e.g., ezetimibe (Merck/Schering-Plough Pharmaceuticals Zetia®) and Pamaqueside (Pfizer). Exemplary CETP inhibitors include, e.g., Torcetrapib (also called CP-529414, Pfizer), JTT-705 (Japan Tobacco), and CETi-I (Avant Immunotherapeutics). Exemplary microsomal triglyceride transfer protein (MTTP) inhibitors include, e.g., implitapide (Bayer), R- 103757 (Janssen), and CP-346086 (Pfizer). Other exemplary cholesterol modulators include, e.g., NO-1886 (Otsuka/TAP

Pharmaceutical), CI- 1027 (Pfizer), and WAY- 135433 (Wyeth-Ayerst). Exemplary bile acid modulators include, e.g., HBS-107 (Hisamitsu/Banyu), Btg-511 (British Technology Group), BARI-1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), and AZD-7806 (AstraZeneca).₌ Exemplary peroxisome proliferation activated receptor (PPAR) agonists include, e.g., tesaglitazar (AZ-242) (AstraZeneca), Netoglitazone (MCC-555) (Mitsubishi/ Johnson & Johnson), GW-409544 (Ligand Pharniaceuticals/GlaxoSmithKline), GW-501516 (Ligand Pharmaceuticals/GlaxoSmithKline), LY-929 (Ligand Pharmaceuticals and Eli Lilly), LY- 465608 (Ligand Pharmaceuticals and Eli Lilly), LY-518674 (Ligand Pharmaceuticals and Eli Lilly), and MK-767 (Merck and Kyorin). Exemplary gene -based therapies include, e.g., AdGWEGF121.10 (GenVec), ApoAl (UCB Pharma/Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics), and ATP-binding cassette transporter- Al (ABCA1) (CV

Therapeutics/Incyte, Aventis, Xenon). Exemplary Glycoprotein Ilb/IIIa inhibitors include, e.g.,. roxifiban (also called DMP754, Bristol-Myers Squibb), Gantofiban (Merck

KGaA/Y amanouchi), and Cromafiban (Millennium Pharmaceuticals). Exemplary squalene synthase inhibitors include, e.g., BMS- 1884941 (Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer), CP-294838 (Pfizer), and TAK-475 (Takeda). An exemplary MCP-I inhibitor is, e.g., RS-504393 (Roche Bioscience). The anti-atherosclerotic agent BO-653 (Chugai Pharmaceuticals), and the nicotinic acid derivative Nyclin (Yamanouchi

Pharmacuticals) are also appropriate for administering in combination with a dsRNA featured in the invention. Exemplary combination therapies suitable for administration with a dsRNA targeting PCSK9 include, e.g., advicor (Niacin/lovastatin from Kos Pharmaceuticals), amlodipine/atorvastatin (Pfizer), and ezetimibe/simvastatin (e.g., Vytorin® 10/10, 10/20, 10/40, and 10/80 tablets by Merck/Schering-Plough Pharmaceuticals). Agents for treating hypercholesterolemia, and suitable for administration in combination with a dsRNA targeting PCSK9 include, e.g., lovastatin, niacin Altoprev® Extended-Release Tablets (Andrx Labs), lovastatin Caduet® Tablets (Pfizer), amlodipine besylate, atorvastatin calcium Crestor® Tablets (AstraZeneca), rosuvastatin calcium Lescol® Capsules (Novartis), fluvastatin sodium Lescol® (Reliant, Novartis), fluvastatin sodium Lipitor® Tablets (Parke-Davis), atorvastatin calcium Lofibra® Capsules (Gate), Niaspan Extended-Release Tablets (Kos), niacin

Pravachol Tablets (Bristol-Myers Squibb), pravastatin sodium TriCor® Tablets (Abbott), fenofibrate Vytorin® 10/10 Tablets (Merck/Schering-Plough Pharmaceuticals), ezetimibe, simvastatin WelChol™ Tablets (Sankyo), colesevelam hydrochloride Zetia® Tablets (Schering), ezetimibe Zetia® Tablets (Merck/Schering-Plough Pharmaceuticals), and ezetimibe Zocor® Tablets (Merck).

[00459] In one embodiment, a dsRNA targeting PCSK9 is administered in combination with an anti-PCSK9 antibody.

[00460] In one embodiment, the PCSK9 dsRNA is administered to the patient, and then the non-dsRNA agent is administered to the patient (or vice versa). In another embodiment, the PCSK9 dsRNA and the non-dsRNA therapeutic agent are administered at the same time.

[00461] In one aspect, the invention provides a method of inhibiting the expression and/or activity of the PCSK9 gene in a subject, e.g., a human. The method includes administering a dose of dsRNA, e.g., a dose sufficient to depress levels of PCSK9 mRNA for at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days; and administering a dose of ABP, wherein the ABP dose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the dsRNA dose is administered, thereby inhibiting the expression and/or activity of the PCSK9 gene in the subject. Additional doses of ABP and/or dsRNA can be administered when and where deemed helpful by one of skill in the art, either in combination or separately.

[00462] In one aspect, the invention provides a method of inhibiting the expression and/or activity of the PCSK9 gene in a subject, e.g., a human. The method includes administering a dose of ABP, and administering a dose of dsRNA, e.g., a dose sufficient to depress levels of PCSK9 mRNA for at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days; wherein the dsRNA dose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the ABP dose is administered, thereby inhibiting the expression and/or activity of the PCSK9 gene in the subject. Additional doses of ABP and/or dsRNA can be administered when and where deemed helpful by one of skill in the art, either in combination or separately.

[00463] In one aspect, the invention provides a method of inhibiting the expression and/or activity of the PCSK9 gene in a subject, e.g., a human. The method includes administering a dose of ABP and administering a dose of dsRNA in combination, thereby inhibiting the expression and/or activity of the PCSK9 gene in the subject. Additional doses of ABP and/or dsRNA can be administered when and where deemed helpful by one of skill in the art, either in combination or separately.

[00464] In another aspect, the invention features, a method of instructing an end user, e.g., a caregiver or a subject, on how to administer a dsRNA and/or ABP described herein. The method includes, optionally, providing the end user with one or more doses of the dsRNA and/or ABP, and instructing the end user to administer the dsRNA and/or ABP on a regimen described herein, thereby instructing the end user.

[00465] In yet another aspect, the invention provides a method of treating a patient by selecting a patient on the basis that the patient is in need of LDL lowering, LDL lowering without lowering of HDL, ApoB lowering, or total cholesterol lowering. The method includes administering to the patient a RNA effector agent, e.g., siRNA and/or ABP in an amount sufficient to lower the patient's LDL levels or ApoB levels, e.g., without

substantially lowering HDL levels.

[00466] In another aspect, the invention provides a method of treating a patient by selecting a patient on the basis that the patient is in need of lowered ApoB levels, and administering to the patient a RNA effector agent, e.g., siRNA and/or ABP targeting PCSK9 in an amount sufficient to lower the patient's ApoB levels. In one embodiment, the amount of PCSK9 is sufficient to lower LDL levels as well as ApoB levels. In another embodiment, administration of the PCSK9 RNA effector agent, e.g., siRNA and/or ABPdoes not affect the level of HDL cholesterol in the patient.

[00467] 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. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES

[00468] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[00469] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al, Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);

Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3^rd Ed. (Plenum Press) Vols A and B(l 992).

Example 1: Gene Walking of the PCSK9 gene

[00470] 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.

[00471] 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.

[00472] 19 mer stretches conserved in human and mouse, and human and rat PCSK9 mRNA sequences were identified in the first step, resulting in the selection of siRNAs crossreactive to human and mouse, and siRNAs crossreactive to human and rat targets

[00473] 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 crossreactive to mouse, rat, human and monkey are all listed in Tables 1 and 2.

[00474] 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.

[00475] For predicting siRNA-specific off-target potential, the following assumptions were made: 1) positions 2 to 9 (counting 5' to 3') of a strand (seed region) may contribute more to off-target potential than rest of sequence (non-seed and cleavage site region); 2) positions 10 and 11 (counting 5' to 3') of a strand (cleavage site region) may contribute more to off-target potential than non-seed region; 3) positions 1 and 19 of each strand are not relevant for off-target interactions; 4) an off-target score can be calculated for each gene and each strand, based on complementarity of siRNA strand sequence to the gene's sequence and position of mismatches ; 5) number of predicted off-targets as well as highest off-target score must be considered for off-target potential; 6) off-target scores are to be considered more relevant for off-target potential than numbers of off-targets; and 7) assuming potential abortion of sense strand activity by internal modifications introduced, only off-target potential of antisense strand will be relevant.

[00476] To identify potential off-target genes, 19mer candidate sequences were subjected to a homology search against publically available human mRNA sequences.

[00477] 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

[00478] The off-target score was calculated for considering assumption 1 to 3 as follows: Off-target score = number of seed mismatches * 10 + number of cleavage site mismatches * 1.2 + number of non-seed mismatches * 1

[00479] 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.

Example 2: dsRNA synthesis

[00480] Source of reagents

[00481] 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.

[00482] siRNA synthesis

[00483] Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μιηοΐε using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 A, 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).

[00484] Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and

concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, UnterschleiBheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85 - 90°C for 3 minutes and cooled to room temperature over a period of 3 - 4 hours. The annealed RNA solution was stored at -20 °C until use.

[00485] 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.

[00486] Synthesis of dsRNAs conjugated to Chol-p-(GalNAc)3 (N-acetyl galactosamine - cholesterol) (FIG. 16)and LCO(GalNAc)₃ (N-acetyl galactosamine - 3'-Lithocholic-oleoyl) (FIG. 17) is described in United States patent application number 12/328,528, filed on December 4, 2008, which is hereby incorporated by reference.

[00487] Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 1-2.

Example 3. PCSK9 siRNA screening in HuH7, HepG2, HeLa and Primary Monkey Hepatocytes Discovers Highly Active Sequences

[00488] Duplex siRNAs with sequences disclosed in Table 1 were assayed for activity in

HuH7, HepG2, HeLa and primary monkey Hepatocytes as described in PCT/US07/68655, filed on May 10, 2007, herein incorporated by reference (data not shown). The siR A AD- 9680 showed the following results:

[00489] Active sequences are those that have less than 90% transcript remaining after treatment with a high dose (ΙΟΟηΜ).

Example 4. In vivo Efficacy Screen of LNP-01 formulated PC SK9 siRNAs

[00490] 32 active PCSK9 siRNAs formulated in LNP-01 liposomes were tested in vivo in a mouse model. LNPOl is a lipidoid formulation formed from cholesterol, mPEG2000-C14 Glyceride, and dsRNA. The LNPOl formulation is useful for delivering dsRNAs to the liver.

[00491] The LNPOl formulaiton prodecure, bolus dosing, detection of PCSK9mRNA, and measurement of serum cholesterol were performed as described in International patent application no. PCT/US07/68655, herein incorporated by reference.

Results

[00492] The results are shown in FIG. 2 and FIG. 3. 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. Silencing of PCSK9 transcript also correlated with a lowering of total serum cholesterol in these animals (FIG. 4 and FIG. 5). The most efficacious siRNAs with respect to knocking down PCSK9 mRNAs also showed the most pronounced cholesterol lowering effects (compare FIGs. 2-3 and FIGs. 4-5). In addition there was a strong correlation between those molecules that were active in vitro and those active in vivo (compare FIG. 6 A and FIG. 6B).

[00493] Sequences containing different chemical modifications (as desdribed in Tables 1 and 2) were also screened in vitro and in vivo. As an example, less modified sequences AD- 9314 and AD-9318, and a more modified versions of that sequence AD-9314 ( AD-10792, AD- 10793, and AD- 10796); and AD-9318 (AD- 10794, AD- 10795, AD- 10797) were tested both in vitro (in primary monkey hepatocytes) or in vivo (AD-9314 and AD-10792) formulated in LNP-01. FIG. 7 shows that the parent molecules AD-9314 and AD-9318 and the modified versions were all active in vitro. FIG. 8 as an example shows that both the parent AD-9314 and the more highly modified AD- 10792 sequences were active in vivo displaying 50-60% silencing of endogenous PCSK9 in mice. FIG. 9 further exemplifies that activity of other chemically modified versions of AD-9314 and AD-10792.

[00494] AD-3511, a derivative of AD-10792, was as efficacious as 10792 (IC50 of -0.07- 0.2 nM) (data not shown). The sequences of the sense and antisense strands of AD-3511 are as follows:

Sense strand: 5'- GccuGGAGuuuAuucGGAAdTsdT SEQ ID

NO: 1521

Antisense strand: 5'- puUCCGAAuAAACUCcAGGCdTsdT SEQ ID NO: 1522

Example 5. PCSK9 Duration of Action Experiments.

Rats

[00495] Rats were treated via tail vein injection with 5mg/kg of LNP01-10792

(Formulated ALDP- 10792). Blood was drawn at the indicated time points (see Table 3) and the amount of total cholesterol compared to PBS treated animals was measured by standard means. AS shown in Table 3, total cholesterol levels decreased at day two -60% and returned to baseline by day 28. These data show that formulated versions of PCSK9 siRNAs lower cholesterol levels for extended periods of time.

Monkeys

[00496] Cynomolgus monkeys were treated with LNP01 formulated dsRNA AD-9680, AD-10792, and controls AD- 1955 and PBS and LDL-C levels were evaluated. A total of 19 cynomolgus monkeys were assigned to dose groups. Beginning on Day -11, animals were limit-fed twice-a-day according to the following schedule: feeding at 9 a.m., feed removal at 10 a.m., feeding at 4 p.m., feed removal at 5 p.m. On the first day of dosing all animals were dosed once via 30-minute intravenous infusion. The animals were evaluated for changes in clinical signs, body weight, and clinical pathology indices, including direct LDL and HDL cholesterol.

[00497] Venipuncture through the femoral vein was used to collect blood samples.

Samples were collected prior to the morning feeding (i.e., before 9 a.m.) and at

approximately 4 hours (beginning at 1 p.m.) after the morning feeding on Days -3, -1, 3, 4, 5, and 7 for Groups 1-7; on Day 14 for Groups 1, 4, and 6; on Days 18 and 21 for Group 1; and on Day 21 for Groups 4 and 6. At least two 1.0 ml samples were collected at each time point. [00498] No anticoagulant was added to the 1.0 ml serum samples, and the dry anticoagulant Ethylenediaminetetraacetic acid (K2) was added to each 1.0 ml plasma sample. Serum samples were allowed to stand at room temperature for at least 20 minutes to facilitate coagulation and then the samples were placed on ice. Plasma samples were placed on ice as soon as possible following sample collection. Samples were transported to the clinical pathology lab within 30 minutes for further processing.

[00499] Blood samples were processed to serum or plasma as soon as possible using a refrigerated centrifuge, per Testing Facility Standard operating procedure. Each sample was split into 3 approximately equal volumes, quickly frozen in liquid nitrogen, and placed at - 70°C. Each aliquot should have had a minimum of approximately 50 μί. If the total sample volume collected was under 150 μί, the residual sample volume went into the last tube. Each sample was labeled with the animal number, dose group, day of collection, date, nominal collection time, and study number(s). Serum LDL cholesterol was measured directly per standard procedures on a Beckman analyzer according to manufactures instructions.

[00500] The results are shown in Table 4. LNP01 - 10792 and LNP01 -9680 administered at 5 mg/kg decreased serum LDL cholesterol within 3 to 7 days following dose administration. Serum LDL cholesterol returned to baseline levels by Day 14 in most animals receiving LNP01-10792 and by Day 21 in animals receiving LNP01-9680. This data demonstrated a greater than 21 day duration of action for cholesterol lowering of LNP01 formulated ALDP- 9680.

Example 6. PCSK9 siRNAs cause decreased PCSK mRNA in liver extracts, and lower serum cholesterol levels.

[00501] To test if acute silencing of the PCSK9 transcript by a PCSK9 siRNA (and subsequent PCSK9 protein down-regulation), would result in acutely lower total cholesterol levels, siRNA molecule AD-la2 (AD- 10792) was formulated in an LNP01 lipidoid formulation. Sequences and modifications of these dsRNAs are shown in Table 5a.

Liposomal formulated siRNA duplex AD-la2 (LNP01-10792) was injected via tail vein in low volumes (-0.2 ml for mouse and ~1.0 ml for rats) at different doses into C57/BL6 mice or Sprague Dawley rats.

[00502] In mice, livers were harvested 48 hours post-injection, and levels of PCSK9 transcript were determined. In addition to liver, blood was harvested and subjected to a total cholesterol analysis. LNP01-la2 displayed a clear dose response with maximal PCSK9 message suppression (-60-70%) as compared to a control siRNA targeting luciferase

(LNPOl-ctrl) or PBS treated animals (FIG. 14A). The decrease of PCSK9 transcript at the highest dose translated into a -30% lowering of total cholesterol in mice (FIG. 14B). This level of cholesterol reduction is between that reported for heterozygous and homozygous PCSK9 knock-out mice (Rashid et al, Proc. Natl. Acad. Sci. USA 102:5374-9, 2005, epub April 1, 2005). Thus, lowering of PCSK9 transcript through an RNAi mechanism is capable of acutely decreasing total cholesterol in mice. Moreover the effect on the PCSK9 transcript persisted between 20-30 days, with higher doses displaying greater initial transcript level reduction, and subsequently more persistent effects.

[00503] Down-modulation of total cholesterol in rats has been historically difficult as cholesterol levels remain unchanged even at high doses of HMG-CoA reductase inhibitors. Interestingly, as compared to mice, rats appear to have a much higher level of PCSK9 basal transcript levels as measured by bDNA assays. Rats were dosed with a single injection of LNP01-a2 via tail vein at 1, 2.5 and 5 mg/kg. Liver tissue and blood were harvested 72 hours post-injection. LNP01-la2 exhibited a clear dose response effect with maximal 50-60% silencing of the PCSK9 transcript at the highest dose, as compared to a control luciferase siRNA and PBS (FIG. 10A). The mRNA silencing was associate with an acute ~50-60%> decrease of serum total cholesterol (FIGs. 10A and 10B) lasting 10 days, with a gradual return to pre-dose levels by ~3 weeks (FIG. 10B). This result demonstrated that lowering of PCSK9 via siRNA targeting had acute, potent and lasting effects on total cholesterol in the rat model system. To confirm that the transcript reduction observed was due to a siRNA mechanism, liver extracts from treated or control animals were subjected to 5' RACE, a method previously utilized to demonstrate that the predicted siRNA cleavage event occurs (Zimmermann et al. , Nature. 441 : 111 -4, 2006, Epub 2006 Mar 26). PCR amplification and detection of the predicted site specific mRNA cleavage event was observed in animals treated with LNP01-la2, but not PBS or LNPOl-ctrl control animals. (Frank-Kamanetsky et al. (2008) PNAS 105: 119715-11920) This result demonstrated that the effects of LNP01-la2 observed were due to cleavage of the PCSK9 transcript via an siRNA specific mechanism.

[00504] The mechanism by which PCSK9 impacts cholesterol levels has been linked to the number of LDLRs on the cell surface. Rats (as opposed to mice, NHP, and humans) control their cholesterol levels through tight regulation of cholesterol synthesis and to a lesser degree through the control of LDLR levels. To investigate whether modulation of LDLR was occurring upon RNAi therapeutic targeting of PCSK9, we quantified the liver LDLR levels (via western blotting) in rats treated with 5mg/kg LNP01-la2. As shown in FIG. 11, LNP01- la2 treated animals had a significant (~3-5 fold average) induction of LDLR levels 48 hours post a single dose of LNP01-la2 compared to PBS or LNPOl-ctrl control siRNA treated animals..

[00505] Assays were also performed to test whether reduction of PCSK9 changes the levels of triglycerides and cholesterol in the liver itself. Acute lowering of genes involved in VLDL assembly and secretion such as microsomal triglyceride transfer protein (MTP) or ApoB by genetic deletion, compounds, or siRNA inhibitors results in increased liver triglycerides (see, e.g., Akdim et ah, Curr. Opin. Lipidol. 18:397-400, 2007). Increased clearance of plasma cholesterol induced by PCSK9 silencing in the liver (and a subsequent increase in liver LDLR levels) was not predicted to result in accumulation of liver triglycerides. However, to address this possibility, liver cholesterol and triglyceride concentrations in livers of the treated or control animals were quantified. As shown in FIG. IOC, there was no statistical difference in liver TG levels or cholesterol levels of rats administered PCSK9 siRNAs compared to the controls. These results indicated that PCSK9 silencing and subsequent cholesterol lowering is unlikely to result in excess hepatic lipid accumulation.

Example 7. Additional modifications to siRNAs do not affect silencing and duration of cholesterol reduction in rats.

[00506] Phosphorothioate modifications at the 3' ends of both sense and antisense strands of a dsRNA can protect against exonucleases. 2'OMe and 2'F modifications in both the sense and antisense strands of a dsRNA can protect against endonucleases. AD-la2 (see

Table 5b) contains 2'OMe modifications on both the sense and antisense strands.

Experiments were performed to determine if the inherent stability (as measured by siRNA stability in human serum) or the degree or type of chemical modification (2'OMe versus 2'F or a mixture) was related to either the observed rat efficacy or the duration of silencing effects. Stability of siRNAs with the same AD-la2 core sequence, but containing different chemical modifications were created and tested for activity in vitro in primary Cyno monkey hepatocytes. A series of these molecules that maintained similar activity as measured by in vitro IC50 values for PCSK9 silencing (Table 5b), were then tested for their stability against exo and endonuclease cleavage in human serum. Each duplex was incubated in human serum at 37 °C (a time course), and subjected to HPLC analysis. The parent sequence AD-la2 had a T½ of ~7 hours in pooled human serum. Sequences AD-la3, AD-la5, and AD-la4, which were more heavily modified (see chemical modifications in Table 5) all had T ½'s greater than 24 hours. To test whether the differences in chemical modification or stability resulted in changes in efficacy, AD-la2, AD-la3, AD-la5, AD-la4, and an AD-control sequence were formulated and injected into rats. Blood was collected from animals at various days post-dose, and total cholesterol concentrations were measured. Previous experiments had shown a very tight correlation between the lowering of PCSK9 transcript levels and total cholesterol values in rats treated with LNP01-la2 (FIG. 10A). All four molecules were observed to decrease total cholesterol by -60% day 2 post-dose (versus PBS or control siRNA), and all of the molecules had equal effects on total cholesterol levels displaying similar magnitude and duration profiles. There was no statistical difference in the magnitude of cholesterol lowering and the duration of effect demonstrated by these molecules, regardless of their different chemistries or stabilities in human serum.

Example 8. LNP01-la2 and LNP01-3al silence human PCSK9 and circulating human PCSK9 protein in transgenic mice

[00507] The efficacy of LNP01-la2 (i.e., PCS-A2 or AD- 10792) and another molecule,

AD-3al (i.e., PCS-C2 or AD-9736) (which targets only human and monkey PCSK9 message), to silence the human PCSK9 gene was tested in vivo. A line of transgenic mice expressing human PCSK9 under the ApoE promoter was used (Lagace et ah, J Clin Invest.

116:2995-3005, 2006). Specific PCR reagents and antibodies were designed that detected the human but not the mouse transcripts and protein respectively. Cohorts of the humanized mice were injected with a single dose of LNP01-la2 (a.k.a. LNP-PCS-A2) or LNP01-3al

(a.k.a. LNP-PCS-C2), and 48 hours later both livers and blood were collected. A single dose of LNP01-la2 or LNP01-3al was able to decrease the human PCSK9 transcript levels by

>70% (FIG. 15 A), and this transcript down-regulation resulted in significantly lower levels of circulating human PCSK9 protein as measured by ELISA (FIG. 15B). These results demonstrated that both siRNAs were capable of silencing the human transcript and subsequently reducing the amount of circulating plasma human PCSK9 protein.

Example 9. Secreted PCSK9 levels are regulated by diet in NHP

[00508] In mice, PCSK9 mRNA levels are regulated by the transcription factor sterol regulatory element binding protein-2 and are reduced by fasting. In clinical practice, and standard NHP studies, blood collection and cholesterol levels are measured after an overnight fasting period. This is due in part to the potential for changes in circulating TGs to interfere with the calculation of LDLc values. Given the regulation of PCSK9 levels by fasting and feeding behavior in mice, experiments were performed to understand the effect of fasting and feeding in NHP.

[00509] Cyno monkeys were acclimated to a twice daily feeding schedule during which food was removed after a one hour period. Animals were fed from 9- 10am in the morning, after which food was removed. The animals were next fed once again for an hour between 5pm-6pm with subsequent food removal. Blood was drawn after an overnight fast (6pm until 9am the next morning), and again, 2 and 4 hours following the 9am feeding. PCSK9 levels in blood plasma or serum were determined by ELISA assay (see Methods). Interestingly, circulating PCSK9 levels were found to be higher after the overnight fasting and decreased 2 and 4 hours after feeding. This data was consistent with rodent models where PCSK9 levels were highly regulated by food intake. However, unexpectedly, the levels of PCSK9 went down the first few hours post-feeding. This result enabled a more carefully designed NHP experiment to probe the efficacy of formulated AD-la2 and another PCSK9 siRNA (AD-2al) that was highly active in primary Cyno hepatocytes.

Example 10. PCSK9 siRNAs reduce circulating LDLc ApoB, and PCSK9, but not HDLc in non-human primates (NHPs).

[00510] siRNAs targeting PCSK9 acutely lowered both PCSK9 and total cholesterol levels by 72 hours post-dose and lasted -21-30 days after a single dose in mice and rats. To extend these findings to a species whose lipoprotein profiles most closely mimic that of humans, further experiments were performed in the Cynomologous (Cyno) monkey model.

[00511] siRNA 1 (LNP01 - 10792) and siRNA 2 (LNP-01 -9680), both targeting PCSK9 were administered to cynomologous monkeys. As shown in FIG. 12, both siRNAs caused significant lipid lowering for up to 7 days post administration. siRNA 2 caused -50% lipid lowering for at least 7 days post-administration, and -60% lipid lowering at day 14 post- administration, and siRNA 1 caused -60% LDLc lowering for at least 7 days.

[00512] Male Cynos were first pre-screened for those that had LDLc of 40mg/dl or higher. Chosen animals were then put on a fasted/fed diet regime and acclimated for 11 days. At day -3 and -1 pre-dose, serum was drawn at both fasted and 4 hours post-fed time points and analyzed for total cholesterol (Tc), LDL (LDLc), HDL cholesterol (HDLc) as well as triglycerides (TG), and PCSK9 plasma levels. Animals were randomized based on their day - 3 LDLc levels. On the day of dosing (designated day 1), either 1 mg/kg or 5 mg/kg of LNP01-la2 and 5 mg/kg LNP01-2al were injected, along with PBS and 1 mg/kg LNPOl-ctrl as controls. All doses were well tolerated with no in-life findings. As the experiment progressed it became apparent (based on LDLc lowering) that the lower dose was not efficacious. We therefore dosed the PBS group animals on day 14 with 5mg/kg LNPOl-ctrl control siR A, which could then serve as an additional control for the high dose groups of 5 mg/kg LNP01-la2 and 5 mg/kg LNP01-2al . Initially blood was drawn from animals on days 3, 4, 5, and 7 post-dose and Tc, HDLc, LDLc, and TGs concentrations were measured.

Additional blood draws from the LNP01-la2, LNP01-2al high dose groups were carried out at day 14 and day 21 post-dose (as the LDLc levels had not returned to baseline by day 7).

[00513] As shown in FIG. 12A, a single dose of LNP01-la2 or LNP01-2al resulted in a statistically significant reduction of LDLc beginning at day 3 post-dose that returned to baseline over ~14 days ( for LNP01-la2 ) and ~ 21 days (LNP01-2al). This effect was not seen in either the PBS, the control siRNA groups, or the 1 mg/kg treatment groups. LNP01- 2al resulted in an average lowering of LDLc of 56% 72 hours post-dose, with 1 of 4 animals achieving nearly 70% LDLc, and all others achieving >50% LDLc decrease, as compared to pre-dose levels, (see FIG. 12A. As expected, the lowering of LDLc in the treated animals also correlated with a reduction of circulating ApoB levels as measured by serum ELISA (FIG. 12B). Interestingly, the degree of LDLc lowering observed in this study of Cyno monkey was greater than those that have been reported for high dose statins, as well as, for other current standard of care compounds used for hypercholesterolemia. The onset of action is also much more acute than that of statins with effects being seen as early as 48 hours post- dose.

[00514] Neither LNP01 - 1 a2 nor LNP01 -2al treatments resulted in a lowering of HDLc. In fact, both molecules resulted (on average) in a trend towards a decreased Tc/HDL ratio (FIG. 12C). In addition, circulating triglyceride levels, and with the exception of one animal, ALT and AST levels were not significantly impacted.

[00515] PCSK9 protein levels were also measured in treated and control animals. As shown in FIG. 11, LNP01-la2 and LNP01-2al treatment each resulted in trends toward decreased circulating PCSK9 protein levels versus pre-dose. Specifically, the more active siRNA LNP01-2al demonstrated significant reduction of circulating PCSK9 protein versus both PBS (day 3-21) and LNPOl-ctrl siRNA control (day 4, day 7).

Example 11. siRNA modifications immune responses to siRNAs

[00516] siRNAs were tested for activation of the immune system in primary human blood monocytes (hPBMC). Two control inducing sequences and the unmodified parental compound AD-lal was found to induce both IFN-alpha and TNF-alpha. However, chemically modified versions of this sequence (AD-la2, AD-la3, AD-la5, and AD-la4) as well as AD-2al were negative for both IFN-alpha and TNF-alpha induction in these same assays (see Table 5, and FIGs. 13A and 13B). Thus chemical modifications are capable of dampening both IFN-alpha and TNF-alpha responses to siRNA molecules. In addition, neither AD-la2, nor AD-2al activated IFN-alpha when formulated into liposomes and tested in mice.

Example 12. Evaluation of siRNA conjugates

[00517] AD- 10792 was conjugated to GalNAc)3/Cholesterol (FIG. 16) or

GalNAc)3/LCO (FIG. 17). The sense strand was synthesized with the conjugate on the 3' end. The conjugated siRNAs were assayed for effects on PCSK9 transcript levels and total serum cholesterol in mice using the methods described below.

[00518] Briefly, mice were dosed via tail injection with one of the 2 conjugated siRNAs or PBS on three consecutive days: day 0, day 1 and day 2 with a dosage of about 100, 50, 25 or 12.5 mg/kg. Each dosage group included 6 mice. 24 hour post last dosing mice were sacrificed and blood and liver samples were obtained, stored, and processed to determine PCSK9 mRNA levels and total serum cholesterol.

[00519] The results are shown in FIG. 18. Compared to control PBS, both siRNA conjugates demonstrated activity with an ED50 of 3 X 50 mg/kg for GalNAc)3/Cholesterol conjugated AD- 10792 and 3 X 100 mg/kg for GalNAc)3/LCO conjugated AD- 10792. The results indicate that Cholesterol conjugated siRNA with GalNAc are active and capable of silencing PCSK9 in the liver resulting in cholesterol lowering.

Bolus dosing

[00520] Bolus dosing of formulated siRNAs in C57/BL6 mice (6/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) and diluted with PBS to concentrations 1.0, 0.5, 0.25 and 0.125 mg/ml allowing the delivery of 100; 50; 25 and 12.5 mg/kg doses in 10 μΐ/g body weight. Mice were kept under an infrared lamp for

approximately 3 min prior to dosing to ease injection.

[00521] 24 hour post last dose mice were sacrificed by C02-asphyxiation. 0.2 ml blood was collected by retro-orbital bleeding and the liver was harvested and frozen in liquid nitrogen. Serum and livers were stored at -80°C. Frozen livers were grinded using 6850 Freezer/Mill Cryogenic Grinder (SPEX CentriPrep, Inc) and powders stored at -80°C until analysis. [00522] PCSK9 mR A levels were detected using the branched-DNA technology based kit from QuantiGene Reagent System (Panomics, USA) according to the protocol. 10-20mg of frozen liver powders was lysed in 600 μΐ of 0.16 μg/ml Proteinase K (Epicentre,

#MPPvK092) in Tissue and Cell Lysis Solution (Epicentre, #MTC096H) at 65oC for 3hours. Then 10 μΐ of the lysates were added to 90μ1 of Lysis Working Reagent (1 volume of stock Lysis Mixture in two volumes of water) and incubated at 52oC overnight on Genospectra capture plates with probe sets specific to mouse PCSK9 and mouse GAPDH. Probes sets for mouse PCSK9 and mouse GAPDH were purchased from Panomics, USA.. Chemo luminescence was read on a Victor2-Light (Perkin Elmer) as Relative light units. The ratio of PCSK9 mRNA to mGAPDH 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).

[00523] Total serum cholesterol in mouse serum was measured using the Total Cholesterol Assay (Wako, USA) according to manufacturer's instructions. Measurements were taken on a Victor2 1420 Multilabel Counter (Perkin Elmer) at 600 nm.

Example 13. Evaluation of SNALP-DLinDMA formulated siRNAs

[00524] Briefly, rats were dosed via tail injection with SNALP formulated siRNAs or PBS with a single dosage of about 0.3, 1.0, and 3.0 mg/kg of SNALP formulated AD-10792. Each dosage group included 5 rats. 72 hour post dosing rats were sacrificed and blood and liver samples were obtained, stored, and processed to determine PCSK9 mRNA and total serum cholesterol levels. The results are shown in FIG. 19. Compared to control PBS, SNALP formulated AD-10792 (FIG. 19A) had an ED50 of about 1.0 mg/kg for both lowering of PCSK9 transcript levels and total serum cholesterol levels. These results show that administration of SNALP formulated siRNA results in effective and efficient silencing of PCSK9 and subsequent lowering of total cholesterol in vivo.

Bolus dosing

[00525] Bolus dosing of formulated siRNAs in Sprague-Dawley rats (5/group, 170-190 g body weight, Charles River Laboratories, MA) was performed by tail vein injection using a 27G needle. SiRNAs were formulated in SNALP (and then dialyzed against PBS) and diluted with PBS to concentrations 0.066; 0.2 and 0.6 mg/ml allowing the delivery of 0.3; 1.0 and 3.0 mg/kg of SNALP formulated AD-10792 in 5 μΐ/g body weight. Rats were kept under an infrared lamp for approximately 3 min prior to dosing to ease injection. [00526] 72 hour post last dose rats were sacrificed by C02-asphyxiation. 0.2 ml blood was collected by retro-orbital bleeding and the liver was harvested and frozen in liquid nitrogen. Serum and livers were stored at -80°C. Frozen livers were grinded using 6850 Freezer/Mill Cryogenic Grinder (SPEX CentriPrep, Inc) and powders stored at -80°C until analysis.

[00527] PCSK9 mR A levels were detected using the branched-DNA technology based kit from QuantiGene Reagent System (Panomics, USA) according to the protocol. 10-20mg of frozen liver powders was lysed in 600 μΐ of 0.16 μg/ml Proteinase K (Epicentre,

#MPPvK092) in Tissue and Cell Lysis Solution (Epicentre, #MTC096H) at 65oC for 3hours. Then 10 μΐ of the lysates were added to 90μ1 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 rat PCSK9 and rat GAPDH. Probes sets for rat PCSK9 and rat GAPDH were purchased from Panomics, USA.. Chemo luminescence was read on a Victor2-Light (Perkin Elmer) as Relative light units. The ratio of rat PCSK9 mRNA to rat 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).

[00528] Total serum cholesterol in rat serum was measured using the Total Cholesterol Assay (Wako, USA) according to manufacturer's instructions. Measurements were taken on a Victor2 1420 Multilabel Counter (Perkin Elmer) at 600 nm.

Example 14. In vitro Efficacy screen of Mismatch walk of AD-9680 and AD- 14676

[00529] The effects of variations in sequence or modification on the effectiveness of AD- 9680 and AD- 14676 were assayed in HeLa cells. A number of variants were synthesized as shown in Table 6.

[00530] HeLa were plated in 96-well plates (8,000-10,000 cells/well) in 100 μΐ 10% fetal bovine serum in Dulbecco's Modified Eagle Medium (DMEM). When the cells reached approximately 50% confluence (~ 24 hours later) they were transfected with serial four-fold dilutions of siRNA starting at 10 nM. 0.4 μΐ of transfection reagent Lipofectamine™ 2000 (Invitrogen Corporation, Carlsbad, CA) was used per well and transfections were performed according to the manufacturer's protocol. Namely, the siRNA: Lipofectamine™ 2000 complexes were prepared as follows. The appropriate amount of siRNA was diluted in Opti- MEM I Reduced Serum Medium without serum and mixed gently. The Lipofectamine™ 2000 was mixed gently before use, then for each well of a 96 well plate 0.4 μΐ was diluted in 25 μΐ of Opti-MEM I Reduced Serum Medium without serum and mixed gently and incubated for 5 minutes at room temperature. After the 5 minute incubation, 1 μΐ of the diluted siRNA was combined with the diluted Lipofectamine™ 2000 (total volume is 26.4 μΐ). The complex was mixed gently and incubated for 20 minutes at room temperature to allow the siRNA: Lipofectamine™ 2000 complexes to form. Then 100 μΐ of 10% fetal bovine serum in DMEM was added to each of the siRNA:Lipofectamine™ 2000 complexes and mixed gently by rocking the plate back and forth. ΙΟΟμΙ of the above mixture was added to each well containing the cells and the plates were incubated at 37°C in a C02 incubator for 24 hours, then the culture medium was removed and 100 μΐ 10% fetal bovine serum in DMEM was added.

[00531] 24 hours post medium change medium was removed, cells were lysed and cell lysates assayed for PCSK9 mRNA silencing by bDNA assay (Panomics, USA) following the manufacturer's protocol. Chemo luminescence was read on a Victor2-Light (Perkin Elmer) as Relative light units. The ratio of human PCSK9 mRNA to human GAPDH mRNA in cell lysates was compared to that of cells treated with Lipofectamine™ 2000 only control.

[00532] FIG. 20 is dose response curves of a series of compounds related to AD-9680. FIG. 21 is a dose response curve of a series of compounds related to AD- 14676 (21 A) The results show that DFTs or mismatches in certain positions are able increase the activity (as evidenced by lower IC50 values) of both parent compounds. Without being bound by theory, it is hypothesized that destabilization of the sense strand through the introduction of mismatches, or DFT might result in quicker removal of the sense strand.

[00533]

Example 15. Dose response of SNALP-DlinDMA and XTC formulated AD- 10792 in rats

[00534] Rats were treated with AD-10792 in two different formulations: a SNALP formulation with DlinDMA and a LNP formulation with XTC. At day 3, total serum cholesterol and liver PCSK9 mRNA levels were determined. The experiment was performed using the methods described herein.

[00535] The results are shown in the graph of FIG. 22. Administration of XTC formulated AD-10792 results in a lower EC50 of 0.4 mg/kg compared to administration of SNALP- DlinDMA formulated AD-10792 with an EC50 of 1.0 mg/kg. Example 16. Maintenance of decrease in total cholesterol levels by lower dosage of AD-10792

[00536] Two different maintenance dosing regimens were investigated.

[00537] Rats were treated with 3 mg/kg bolus dose of SNALP-DlinDMA formulated AD- 10792. At day 2, total serum cholesterol levels were determined. This was followed by once a week dosing at 1.0 and 0.3 mg/kg for four weeks. Rats were bled one day prior to repeated dosing and total serum cholesterol levels were determined. The negative control was PBS.

[00538] The results are shown in the graph of FIG. 23. After 3 mg/kg bolus dose, total cholesterol levels decreased by 60% and were maintained at about 50% by repeated once a week 1.0 and 0.3 mg/kg dosing and come back to pre dose levels after repeated dosing is stopped.

[00539] A second maintenance dosing regimen was investigated. Rats were treated with 3 mg/kg bolus dose of SNALP-DlinDMA formulated AD-10792. At day 2, total serum cholesterol levels were determined. This was followed by once every 2 weeks dosing at 1.5 mg/kg for three weeks. Rats were bled one day prior to repeated dosing and total serum cholesterol levels were determined. The negative control was PBS.

[00540] The results are shown in the graph of FIG. 24. After 3 mg/kg bolus dose, total cholesterol levels decreased by 60%> and were maintained at about 50%> by repeated once every two weeks 1.5 mg/kg dosing and come back to pre dose levels after repeated dosing is stopped.

[00541] A 10 fold lower (than EC50), once a week, maintenance dose effectively maintains silencing with cholesterol levels returning to baseline by 15 days post last injection. Once in two weeks maintenance dosing of 1.5 mg/kg shows sinusoidal behavior.

[00542] The initial does of PCSK9 increased LDLR levels as reflected by the decrease in total serum cholesterol. This increase in LDLR levels increased the efficacy of the PCSK9 targeted siRNA as reflected by the lower dosage of subesequnet administration AD-10792.

[00543] Those skilled in the art are familiar with methods and compositions in addition to those specifically set out in the present disclosure which will allow them to practice this invention to the full scope of the claims hereinafter appended.

Example 17: Lowering serum cholesterol levels in mammals using anti-PCSK9 siRNA and/or anti-PCSK9 ABP.

[00544] Mammals (e.g., mice, rats, rodents, humans, guinea pigs) are used in the study.

Mammals are administered (e.g., intravenously) siRNA alone, ABP alone, siRNA and ABP in a single dose, siR A followed by ABP, ABP followed by siR A, siRNA control, ABP control, or saline control. In some instances the ABP is an anti-PCSK9 antibody. In some instances the ABP is formulated as described in U.S. Pat. App. Pub. 20090142352, herein incorporated by reference. In some instances the ABP is formulated as described in the pharmaceutical compositions section above. In some instances the siRNA is formulated as described in the pharmaceutical compositions section above. In some instances the ABP and the siRNA are formulated together in a single formulation. In some instances the ABP and the siRNA are formulated separately.

[00545] The siRNA targets PCSK9. In some instances the siRNA is AD-9680. In some instances the siRNA is AD- 10792. The siRNA can be any of those disclosed herein.

[00546] The ABP, e.g., antibody, is an anti-PCSK9 antibody. In some instances the antibody is 30A4, 3C4, 23B5, 25G4, 31H4, 27B2, 25A7, 27H5, 26H5, 31D1.20D10, 27E7, 30B9, 19H9, 26E10, 21B12, 17C2, 23G1, 13H1, 9C9, 9H6, 31A4, 1A12, 16F12, 22E2, 27A6, 28B12, 28D6, 31G11, 13B5, 31B12 and/or 3B6. In some instances the antibody is 2 IB 12. In some instances the antibody is 31H4. In some instances the antibody is 3C4.

[00547] Multiple rounds of doses are used where deemed useful. Mammals are monitored for serum cholesterol levels and/or PCSK9 mRNA levels and/or PCSK9 protein levels. Similar studies are performed with different treatment protocols and administration routes (e.g., intramuscular administration, etc.). The effectiveness of siRNAs and/or antibodies targeting PCSK9 is demonstrated by comparing the PCSK9 mRNA levels and/or the serum cholesterol of mammals treated with anti-PCSK9 siRNAs and/or anti-PCSK9 antibody to mammals treated with control formulations.

[00548] In an example, a human subject in need of treatment is selected or identified. The subject can be in need of LDL lowering, LDL lowering without lowering of HDL, ApoB lowering, or total cholesterol lowering. The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.

[00549] At time zero, a suitable first dose of AD-9680 and/or anti-PCSK9 ABP (e.g., 21B12, 31H4, or 3C4) is administered to the subject. AD-9680 and the ABP are formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated, e.g., by measuring LDL, ApoB, and/or total cholesterol levels. This measurement can be accompanied by a measurement of PCSK9 expression and/or activity in the subject, and/or the products of the successful targeting of PCSK9 mR A and/or protein. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.

[00550] After treatment, the subject's LDL, ApoB, or total cholesterol levels are lowered relative to the levels existing prior to the treatment, or relative to the levels measured in a similarly afflicted but untreated subject.

[00551] In another example, a rodent subject in need of treatment is selected or identified. The subject can be in need of LDL lowering, LDL lowering without lowering of HDL, ApoB lowering, or total cholesterol lowering. The identification of the subject can occur in a laboratory setting or elsewhere.

[00552] At time zero, a suitable first dose of AD- 10792 and/or a rodent anti-PCSK9 ABP is administered to the subject. AD-10792 and the ABP are formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated, e.g., by measuring LDL, ApoB, and/or total cholesterol levels. This measurement can be accompanied by a measurement of PCSK9 expression and/or activity in the subject, and/or the products of the successful targeting of PCSK9 mRNA and/or protein. Other relevant criteria can also be measured.

[00553] The number and strength of doses are adjusted according to the subject's needs. After treatment, the subject's LDL, ApoB, or total cholesterol levels are lowered relative to the levels existing prior to the treatment, or relative to the levels measured in a similarly afflicted but untreated subject.

Example 18. Inhibition of PCSK9 expression and/or activity in humans

[00554] A human subject is treated with a dsRNA targeted to a PCSK9 gene to inhibit expression of the PCSK9 gene and lower cholesterol levels for an extended period of time. The subject can also be treated with an ABP targeted to PCSK9 protein to inhibit activity of the PCSK9 protein and lower cholesterol levels for an extended period of time.

[00555] A subject in need of treatment is selected or identified. The subject can be in need of LDL lowering, LDL lowering without lowering of HDL, ApoB lowering, or total cholesterol lowering. The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.

[00556] At time zero, a suitable first dose of an anti-PCSK9 siRNA and/or anti-PCSK9 ABP is administered to the subject. The dsRNA and ABP are formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated, e.g., by measuring LDL, ApoB, and/or total cholesterol levels. This measurement can be accompanied by a measurement of PCSK9 expression and/or activity in said subject, and/or the products of the successful targeting of PCSK9 mRNA and/or protein. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.

[00557] After treatment, the subject's LDL, ApoB, or total cholesterol levels are lowered relative to the levels existing prior to the treatment, or relative to the levels measured in a similarly afflicted but untreated subject.

[00558] 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 also described in U.S. Pat. App. Pub. 20090142352, U.S. Serial No. 11/746,864, and U.S. Prov. App. 61/187,242 (filed on 6/15/2009), all of which are herein incorporated by reference in their entirety for all purposes. 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.

[00559] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

Tables

Table 1: dsRNA sequences targeted to PCSK9

position in

SEQ

human SEQ

Sense strand sequence (5 ^'-3 ^')' Antisense-strand sequence (5 ^'-3^')' ID Duplex name access. # ID NO:

NO:

NM 174936

138-156 CAGCCAGGAUUCCGCGCGCTsT 35 GCGCGCGGAAUCCUGGCUGT sT 36 AD-9523

138-156 cAGccAGGAuuccGcGcGcTsT 37 GCGCGCGGAAUCCUGGCUGT sT 38 AD-9649

185-203 AGCUCCUGCACAGUCCUCCTsT 39 GGAGGACUGUGCAGGAGCUT sT 40 AD-9569

185-203 AGcuccuGcAcAGuccuccTsT 41 GGAGGACUGUGcAGGAGCUT sT 42 AD-9695

205-223 CACCGCAAGGCUCAAGGCGTT 43 CGCCUUGAGCCUUGCGGUGTT 44 AD-15222

208-226 CGCAAGGCUCAAGGCGCCGTT 45 CGGCGCCUUGAGCCUUGCGTT 46 AD-15278

210-228 CAAGGCUCAAGGCGCCGCCTT 47 GGCGGCGCCUUGAGCCUUGTT 48 AD-15178

232-250 GUGGACCGCGCACGGCCUCTT 49 GAGGCCGUGCGCGGUCCACTT 50 AD-15308

233-251 UGGACCGCGCACGGCCUCUTT 51 AGAGGCCGUGCGCGGUCCATT 52 AD-15223

234-252 GGACCGCGCACGGCCUCUATT 53 UAGAGGCCGUGCGCGGUCCTT 54 AD-15309

235-253 GACCGCGCACGGCCUCUAGTT 55 CUAGAGGCCGUGCGCGGUCTT 56 AD-15279

236-254 ACCGCGCACGGCCUCUAGGTT 57 CCUAGAGGCCGUGCGCGGUTT 58 AD-15194

237-255 CCGCGCACGGCCUCUAGGUTT 59 ACCUAGAGGCCGUGCGCGGTT 60 AD-15310

238-256 CGCGCACGGCCUCUAGGUCTT 61 GACCUAGAGGCCGUGCGCGTT 62 AD-15311

239-257 GCGCACGGCCUCUAGGUCUTT 63 AGACCUAGAGGCCGUGCGCTT 64 AD-15392

240-258 CGCACGGCCUCUAGGUCUCTT 65 GAGACCUAGAGGCCGUGCGTT 66 AD-15312

248-266 CUCUAGGUCUCCUCGCCAGTT 67 CUGGCGAGGAGACCUAGAGTT 68 AD-15313

249-267 UCUAGGUCUCCUCGCCAGGTT 69 CCUGGCGAGGAGACCUAGATT 70 AD-15280

250-268 CUAGGUCUCCUCGCCAGGATT 71 UCCUGGCGAGGAGACCUAGTT 72 AD-15267

252-270 AGGUCUCCUCGCCAGGACATT 73 UGUCCUGGCGAGGAGACCUTT 74 AD-15314

258-276 CCUCGCCAGGACAGCAACCTT 75 GGUUGCUGUCCUGGCGAGGTT 76 AD-15315

300-318 CGUCAGCUCCAGGCGGUCCTsT 77 GGACCGCCUGGAGCUGACGT sT 78 AD-9624

300-318 cGucAGcuccAGGcGGuccTsT 79 GGACCGCCUGGAGCUGACGT sT 80 AD-9750

301-319 GUCAGCUCCAGGCGGUCCUTsT 81 AGGACCGCCUGGAGCUGACT sT 82 AD-9623

301-319 GucAGcuccAGGcGGuccuTsT 83 AGGACCGCCUGGAGCUGACT sT 84 AD-9749

370-388 GGCGCCCGUGCGCAGGAGGTT 85 CCUCCUGCGCACGGGCGCCTT 86 AD-15384

408-426 GGAGCUGGUGCUAGCCUUGTsT 87 CAAGGCUAGCACCAGCUCCT sT 88 AD-9607

408-426 GGAGcuGGuGcuAGccuuGTsT 89 cAAGGCuAGcACcAGCUCCT sT 90 AD-9733

411-429 GCUGGUGCUAGCCUUGCGUTsT 91 ACGCAAGGCUAGCACCAGCT sT 92 AD-9524

411-429 GcuGGuGcuAGccuuGcGuTsT 93 ACGcAAGGCuAGcACcAGCT sT 94 AD-9650

412-430 CUGGUGCUAGCCUUGCGUUTsT 95 AACGCAAGGCUAGCACCAGT sT 96 AD-9520

412-430 CUGGUGCUAGCCUUGCGUUTsT 97 AACGCAAGGCUAGCACCAGT sT 98 AD-9520

412-430 cuGGuGcuAGccuuGcGuuTsT 99 AACGcAAGGCuAGcAC cAGT sT 100 AD-9646

416-434 UGCUAGCCUUGCGUUCCGATsT 101 UCGGAACGCAAGGCUAGCAT sT 102 AD-9608

416-434 uGcuAGccuuGcGuuccGATsT 103 UCGGAACGcAAGGCuAGcAT sT 104 AD-9734

419-437 UAGCCUUGCGUUCCGAGGATsT 105 UCCUCGGAACGCAAGGCUAT sT 106 AD-9546

419-437 uAGccuuGcGuuccGAGGATsT 107 UCCUCGGAACGcAAGGCuAT sT 108 AD-9672

439-457 GACGGCCUGGCCGAAGCACTT 109 GUGCUUCGGCCAGGCCGUCTT 110 AD-15385

447-465 GGCCGAAGCACCCGAGCACTT 111 GUGCUCGGGUGCUUCGGCCTT 112 AD-15393

448-466 GCCGAAGCACCCGAGCACGTT 113 CGUGCUCGGGUGCUUCGGCTT 114 AD-15316

449-467 CCGAAGCACCCGAGCACGGTT 115 CCGUGCUCGGGUGCUUCGGTT 116 AD-15317

458-476 CCGAGCACGGAACCACAGCTT 117 GCUGUGGUUCCGUGCUCGGTT 118 AD-15318

484-502 CACCGCUGCGCCAAGGAUCTT 119 GAUCCUUGGCGCAGCGGUGTT 120 AD-15195

486-504 CCGCUGCGCCAAGGAUCCGTT 121 CGGAUCCUUGGCGCAGCGGTT 122 AD-15224

487-505 CGCUGCGCCAAGGAUCCGUTT 123 ACGGAUCCUUGGCGCAGCGTT 124 AD-15188

489-507 CUGCGCCAAGGAUCCGUGGTT 125 CCACGGAUCCUUGGCGCAGTT 126 AD-15225

500-518 AUCCGUGGAGGUUGCCUGGTT 127 CCAGGCAACCUCCACGGAUTT 128 AD-15281

509-527 GGUUGCCUGGCACCUACGUTT 129 ACGUAGGUGCCAGGCAACCTT 130 AD-15282

542-560 AGGAGACCCACCUCUCGCATT 131 UGCGAGAGGUGGGUCUCCUTT 132 AD-15319

543-561 GGAGACCCACCUCUCGCAGTT 133 CUGCGAGAGGUGGGUCUCCTT 134 AD-15226

544-562 GAGACCCACCUCUCGCAGUTT 135 ACUGCGAGAGGUGGGUCUCTT 136 AD-15271

549-567 CCACCUCUCGCAGUCAGAGTT 137 CUCUGACUGCGAGAGGUGGTT 138 AD-15283

552-570 CCUCUCGCAGUCAGAGCGCTT 139 GCGCUCUGACUGCGAGAGGTT 140 AD-15284

553-571 CUCUCGCAGUCAGAGCGCATT 141 UGCGCUCUGACUGCGAGAGTT 142 AD-15189

554-572 UCUCGCAGUCAGAGCGCACTT 143 GUGCGCUCUGACUGCGAGATT 144 AD-15227

555-573 CUCGCAGUCAGAGCGCACUTsT 145 AGUGCGCUCUGACUGCGAGT sT 146 AD-9547

555-573 cucGcAGucAGAGcGcAcuTsT 147 AGUGCGCUCUGACUGCGAGT sT 148 AD-9673

558-576 GCAGUCAGAGCGCACUGCCTsT 149 GGCAGUGCGCUCUGACUGCT sT 150 AD-9548

558-576 GcAGucAGAGcGcAcuGccTsT 151 GGcAGUGCGCUCUGACUGCT sT 152 AD-9674

606-624 GGGAUACCUCACCAAGAUCTsT 153 GAUCUUGGUGAGGUAUCCCT sT 154 AD-9529

606-624 GGGAuAccucAccAAGAucTsT 155 GAUCUUGGUGAGGuAUCCCT sT 156 AD-9655

659-677 UGGUGAAGAUGAGUGGCGAT s T 157 UCGCCACUCAUCUUCACCAT sT 158 AD-9605

659-677 uGGuGAAGAuGAGuGGcGATsT 159 UCGCcACUcAUCUUcACcAT sT 160 AD-9731

663-681 GAAGAUGAGUGGCGACCUGTsT 161 CAGGUCGCCACUCAUCUUCT sT 162 AD-9596

663-681 GAAGAuGAGuGGcGAccuGTsT 163 cAGGUCGCcACUcAUCUUCT sT 164 AD-9722 position in

SEQ

human SEQ

Sense strand sequence (5 ^'-3 ^')' Antisense-strand sequence (5 ^'-3^')' ID Duplex name access. # ID NO:

NO:

NM 174936

704-722 CCCAUGUCGACUACAUCGATsT 165 UCGAUGUAGUCGACAUGGGT sT 166 AD-9583

704-722 cccAuGucGAcuAcAucGATsT 167 UCGAUGuAGUCGAcAUGGGT sT 168 AD-9709

718-736 AUCGAGGAGGACUCCUCUGTsT 169 CAGAGGAGUCCUCCUCGAUT sT 170 AD-9579

718-736 AucGAGGAGGAcuccucuGTsT 171 cAGAGGAGUCCUCCUCGAUT sT 172 AD-9705

758-776 GGAACCUGGAGCGGAUUACTT 173 GUAAUCCGCUCCAGGUUCCTT 174 AD-15394

759-777 GAACCUGGAGCGGAUUACCTT 175 GGUAAUCCGCUCCAGGUUCTT 176 AD-15196

760-778 AACCUGGAGCGGAUUACCCTT 177 GGGUAAUCCGCUCCAGGUUTT 178 AD-15197

777-795 CCCUCCACGGUACCGGGCGTT 179 CGCCCGGUACCGUGGAGGGTT 180 AD-15198

782-800 CACGGUACCGGGCGGAUGATsT 181 UCAUCCGCCCGGUACCGUGT sT 182 AD-9609

782-800 cAcGGuAccGGGcGGAuGATsT 183 UcAUCCGCCCGGuACCGUGT sT 184 AD-9735

783-801 ACGGUACCGGGCGGAUGAATsT 185 UUCAUCCGCCCGGUACCGUT sT 186 AD-9537

783-801 AcGGuAccGGGcGGAuGAATsT 187 UUcAUCCGCCCGGuACCGUT sT 188 AD-9663

784-802 CGGUACCGGGCGGAUGAAUTsT 189 AUUCAUCCGCCCGGUACCGT sT 190 AD-9528

784-802 cGGuAccGGGcGGAuGAAuTsT 191 AUUcAUCCGCCCGGuACCGT sT 192 AD-9654

785-803 GGUACCGGGCGGAUGAAUATsT 193 UAUUCAUCCGCCCGGUACCT sT 194 AD-9515

785-803 GGuAccGGGcGGAuGAAuATsT 195 uAUUcAUCCGCCCGGuACCT sT 196 AD-9641

786-804 GUACCGGGCGGAUGAAUACTsT 197 GUAUUCAUCCGCCCGGUACT sT 198 AD-9514

786-804 GuAccGGGcGGAuGAAuAcTsT 199 GuAUUcAUCCGCCCGGuACT sT 200 AD-9640

788-806 ACCGGGCGGAUGAAUACCATsT 201 UGGUAUUCAUCCGCCCGGUT sT 202 AD-9530

788-806 AccGGGcGGAuGAAuAccATsT 203 UGGuAUUcAUCCGCCCGGUT sT 204 AD-9656

789-807 CCGGGCGGAUGAAUACCAGTsT 205 CUGGUAUUCAUCCGCCCGGT sT 206 AD-9538

789-807 ccGGGcGGAuGAAuAccAGTsT 207 CUGGuAUUcAUCCGCCCGGT sT 208 AD-9664

825-843 CCUGGUGGAGGUGUAUCUCTsT 209 GAGAUACACCUCCACCAGGT sT 210 AD-9598

825-843 ccuGGuGGAGGuGuAucucTsT 211 GAGAuAcACCUCcACcAGGT sT 212 AD-9724

826-844 CUGGUGGAGGUGUAUCUCCTsT 213 GGAGAUACACCUCCACCAGT sT 214 AD-9625

826-844 cuGGuGGAGGuGuAucuccTsT 215 GGAGAu Ac AC CUC c AC cAGT sT 216 AD-9751

827-845 UGGUGGAGGUGUAUCUCCUTsT 217 AGGAGAUACACCUCCACCAT sT 218 AD-9556

827-845 uGGuGGAGGuGuAucuccuTsT 219 AGGAGAuAcACCUCcACcAT sT 220 AD-9682

828-846 GGUGGAGGUGUAUCUCCUATsT 221 UAGGAGAUACACCUCCACCT sT 222 AD-9539

828-846 GGuGGAGGuGuAucuccuATsT 223 uAGGAGAuAcACCUCcACCT sT 224 AD-9665

831-849 GGAGGUGUAUCUCCUAGACTsT 225 GUCUAGGAGAUACACCUCCT sT 226 AD-9517

831-849 GGAGGuGuAucuccuAGAcTsT 227 GUCuAGGAGAuAcACCUCCT sT 228 AD-9643

833-851 AGGUGUAUCUCCUAGACACTsT 229 GUGUCUAGGAGAUACACCUT sT 230 AD-9610

833-851 AGGuGuAucuccuAGAcAcTsT 231 GUGUCuAGGAGAuAcACCUT sT 232 AD-9736

AfgGf uGfuAf uC fuCf cUfaG p-

833-851 233 gUfgUf cUfaGf gAfgAf uAf cAf cC f 234 AD- 14681 f aC faCf TsT

uT sT

AGGUf GUfAUf Cf Uf Cf Cf Uf A GUfGUf Cf Uf AGGAGAUf AC fACf Cf U

833-851 235 236 AD- 14691

GAC fACf TsT f T sT

p-

833-851 AgGuGuAuCuCcUaGaCaCTsT 237 gUfgUf cUfaGf gAfgAf uAf cAf cC f 238 AD- 14701 uT sT

GUfGUf Cf Uf AGGAGAUf AC fACf Cf U

833-851 AgGuGuAuCuCcUaGaCaCTsT 239 240 AD-14711 f T sT

AfgGf uGfuAf uC fuCf cUfaG

833-851 241 GUGUCuaGGagAUACAccuT sT 242 AD- 14721 f aC faCf TsT

AGGUf GUfAUf Cf Uf Cf Cf Uf A

833-851 243 GUGUCuaGGagAUACAccuT sT 244 AD- 14731

GAC fACf TsT

833-851 AgGuGuAuCuCcUaGaCaCTsT 245 GUGUCuaGGagAUACAccuT sT 246 AD- 14741

Gf cAf cC f cUf cAfuAf gGf cC p-

833-851 247 uC fcAf gGf cCfuAfuGf aGfgGf uGf 248 AD-15087 f uGfgAf TsT

cT sT

GC f AC fC fC fUfC f AUf AGGC f UfCfCfAGGC fC fUfAUfGAGGGUfGC

833-851 249 250 AD-15097

C f Uf GGATsT f T sT

p-

833-851 GcAcCcUcAuAgGcCuGgATsT 251 uC fcAf gGf cCfuAfuGf aGfgGf uGf 252 AD-15107 cT sT

UfCfCfAGGC fC fUfAUfGAGGGUfGC

833-851 GcAcCcUcAuAgGcCuGgATsT 253 254 AD-15117 f T sT

Gf cAf cC f cUf CAfuAf gGf cC

833-851 255 UCCAGgcCUauGAGGGugcT sT 256 AD-15127 f uGfgAf TsT

GC f AC fC fC fUfC f AUf AGGC f

833-851 257 UCCAGgcCUauGAGGGugcT sT 258 AD-15137

C fUf GGATsT

833-851 GcAcCcUcAuAgGcCuGgATsT 259 UCCAGgcCUauGAGGGugcT sT 260 AD-15147

836-854 UGUAUCUCCUAGACACCAGTsT 261 CUGGUGUCUAGGAGAUACAT sT 262 AD-9516

836-854 uGuAucuccuAGAcAccAGTsT 263 CUGGUGUCuAGGAGAuAcAT sT 264 AD-9642 position in

SEQ

human SEQ

uence (5^'-3^')' Antisense-strand sequence (5^'-3^')' ID Duplex name access. # Sense strand seq

ID NO:

NO:

NM 174936

840-858 UCUCCUAGACACCAGCAUATsT 265 UAUGCUGGUGUCUAGGAGATsT 266 AD-9562

840-858 UCUCCUAGAcAccAGcAuATsT 267 uAUGCUGGUGUCuAGGAGAT sT 268 AD-9688

Uf cUf cCfuAf gAf cAf cCfaG p-

840-858 269 uAfuGf cUfgGf uGfuCf uAfgGf aGf 270 AD- 14677 f cAf uAf TsT

aTsT

UfCfUfCfCfUfAGACfACfCf Uf AUfGCf Uf GGUf GUfCfUfAGGAGA

840-858 271 272 AD- 14687

AGCfAUf ATsT TsT

p-

840-858 UcUcCuAgAcAcCaGcAuATsT 273 uAfuGf cUfgGf uGfuCf uAfgGf aGf 274 AD- 14697 aTsT

Uf AUfGCf Uf GGUf GUfCfUfAGGAGA

840-858 UcUcCuAgAcAcCaGcAuATsT 275 276 AD- 14707

TsT

Uf cUf cCfuAafAf cAf cCfaG

840-858 277 UAUGCugGUguCUAGGagaTsT 278 AD-14717 f cAf uAf TsT

UfCfUfCfCfUfAGACfACfCf

840-858 279 UAUGCugGUguCUAGGagaTsT 280 AD- 14727

AGCfAUf AT sT

840-858 UcUcCuAgAcAcCaGcAuATsT 281 UAUGCugGUguCUAGGagaTsT 282 AD-14737

AfgGf cCfuGf gAfgUf uUfaU p-

840-858 f uCfgGf TsT 283 cCfgAf aUfaAf aCfuCf cAfgGf cCf 284 AD-15083 uTsT

AGGCf Cf Uf GGAGUf Uf Uf AUf CfCfGAAUfAAACfUfCfCf AGGCf Cf

840-858 285 286 AD-15093

UfCfGGTsT Uf TsT

p-

840-858 AgGcCuGgAgUuUaUuCgGTsT 287 cCfgAf aUfaAf aCfuCf cAfgGf cCf 288 AD-15103 uTsT

CfCfGAAUfAAACfUfCfCf AGGCf Cf

840-858 AgGcCuGgAgUuUaUuCgGTsT 289 290 AD-15113

Uf TsT

AfgGf cCfuGf gAf gUfuUfaU

840-858 291 CCGAAuaAAcuCCAGGccuTsT 292 AD-15123 f uCfgGf TsT

AGGCf Cf Uf GGAGUf Uf Uf AUf

840-858 293 CCGAAuaAAcuCCAGGccuTsT 294 AD-15133

UfCfGGTsT

840-858 AgGcCuGgAgUuUaUuCgGTsT 295 CCGAAuaAAcuCCAGGccuTsT 296 AD-15143

841-859 C UC CUAGAC AC CAGC AUAC TsT 297 GUAUGCUGGUGUCUAGGAGTsT 298 AD-9521

841-859 CUCCUAGAcAccAGcAuAcTsT 299 GuAUGCUGGUGUCuAGGAGT sT 300 AD-9647

842-860 UCCUAGACACCAGCAUACAT s T 301 UGUAUGCUGGUGUCUAGGATsT 302 AD-9611

842-860 UCCUAGAcAccAGcAuAcATsT 303 UGuAUGCUGGUGUCuAGGAT sT 304 AD-9737

843-861 CCUAGACACCAGCAUACAGTsT 305 CUGUAUGCUGGUGUCUAGGT sT 306 AD-9592

843-861 c cuAGAcAc cAGcAuAcAGT s T 307 CUGuAUGCUGGUGUCuAGGT sT 308 AD-9718

847-865 GACACCAGCAUACAGAGUGT s T 309 CACUCUGUAUGCUGGUGUCT sT 310 AD-9561

847-865 GAcAc cAGcAuAcAGAGuGT s T 311 cACUCUGuAUGCUGGUGUCTsT 312 AD-9687

855-873 CAUACAGAGUGACCACCGGTsT 313 CCGGUGGUCACUCUGUAUGT sT 314 AD-9636

855-873 cAuAcAGAGuGAc cAccGGT s T 315 CCGGUGGUcACUCUGuAUGTsT 316 AD-9762

860-878 AGAGUGACCACCGGGAAAUTsT 317 AUUUCCCGGUGGUCACUCUTsT 318 AD-9540

860-878 AGAGuGAccAccGGGAAAuTsT 319 AUUUCCCGGUGGUcACUCUTsT 320 AD-9666

861-879 GAGUGACCACCGGGAAAUCTsT 321 GAUUUCCCGGUGGUCACUCTsT 322 AD-9535

861-879 GAGuGAccAccGGGAAAucTsT 323 GAUUUCCCGGUGGUcACUCTsT 324 AD-9661

863-881 GUGACCACCGGGAAAUCGATsT 325 UCGAUUUCCCGGUGGUCACTsT 326 AD-9559

863-881 GuGAccAccGGGAAAucGATsT 327 UCGAUUUCCCGGUGGUcACTsT 328 AD-9685

865-883 GACCACCGGGAAAUCGAGGTsT 329 CCUCGAUUUCCCGGUGGUCTsT 330 AD-9533

865-883 GAccAccGGGAAAucGAGGTsT 331 CCUCGAUUUCCCGGUGGUCTsT 332 AD-9659

866-884 ACCACCGGGAAAUCGAGGGTsT 333 CCCUCGAUUUCCCGGUGGUTsT 334 AD-9612

866-884 AccAccGGGAAAucGAGGGTsT 335 CCCUCGAUUUCCCGGUGGUTsT 336 AD-9738

867-885 CCACCGGGAAAUCGAGGGCTsT 337 GCCCUCGAUUUCCCGGUGGTsT 338 AD-9557

867-885 ccAccGGGAAAucGAGGGcTsT 339 GCCCUCGAUUUCCCGGUGGTsT 340 AD-9683

875-893 AAAUCGAGGGCAGGGUCAUT s T 341 AUGACCCUGCCCUCGAUUUTsT 342 AD-9531

875-893 AAAucGAGGGcAGGGucAuTsT 343 AUGACCCUGCCCUCGAUUUTsT 344 AD-9657

AfaAf uCfgAf gGfgCf aGfgG p-

875-893 345 aUfgAf cCf cUf gCf cCf uCfgAf uUf 346 AD- 14673 f uCfaUf TsT

uTsT

AAAUf Cf GAGGGCf AGGGUf Cf AUfGACfCfCfUfGCfCfCfUfCfGAU

875-893 347 348 AD- 14683

AUf TsT fUfUfTsT

p-

875-893 AaAuCgAgGgCaGgGuCaUTsT 349 aUfgAf cCf cUf gCf cCf uCfgAf uUf 350 AD- 14693 uTsT

AUfGACfCfCfUfGCfCfCfUfCfGAU

875-893 AaAuCgAgGgCaGgGuCaUTsT 351 352 AD- 14703 fUfUfTsT position in

SEQ

human SEQ

Sense strand sequence (5 ^'-3 ^')' Antisense-strand sequence (5 ^'-3^')' ID Duplex name access. # ID NO:

NO:

NM 174936

AfaAf uC fgAf gGfgCf aGfgG

875-893 353 AUGACccUGccCUCGAuuuT sT 354 AD-14713 f uC faUf TsT

AAAUf Cf GAGGGC f AGGGUf C f

875-893 355 AUGACccUGccCUCGAuuuT sT 356 AD- 14723

AUf TsT

875-893 AaAuCgAgGgCaGgGuCaUTsT 357 AUGACccUGccCUCGAuuuT sT 358 AD-14733

C fgGf cAf cCf cUf cAf uAfgG p-

875-893 359 cAfgGf cC fuAf uGfaGf gGfuGf cC f 360 AD-15079 f cC fuGf TsT

gT sT

C fGGC fACfCfCfUfCfAUfAG CfAGGC fC fUfAUfGAGGGUfGCfCfG

875-893 361 362 AD-15089

GCf Cf Uf GT sT TsT

p-

875-893 CgGcAcCcUcAuAgGcCuGTsT 363 cAfgGf cC fuAf uGfaGf gGfuGf cC f 364 AD-15099 gT sT

CfAGGC fC fUfAUfGAGGGUfGCfCfG

875-893 CgGcAcCcUcAuAgGcCuGTsT 365 366 AD-15109

TsT

C fgGf cAf cCf cUf cAf uAfgG

875-893 367 CAGGCcuAUgaGGGUGccgT sT 368 AD-15119 f cC fuGf TsT

C fGGC fACfCfCfUfCfAUfAG

875-893 369 CAGGCcuAUgaGGGUGccgT sT 370 AD-15129

GCf Cf Uf GT sT

875-893 CgGcAcCcUcAuAgGcCuGTsT 371 CAGGCcuAUgaGGGUGccgT sT 372 AD-15139

877-895 AUCGAGGGCAGGGUCAUGGTsT 373 CCAUGACCCUGCCCUCGAUT sT 374 AD-9542

877-895 AucGAGGGcAGGGucAuGGTsT 375 CcAUGACCCUGCCCUCGAUT sT 376 AD-9668

878-896 cGAGGGcAGGGucAuGGucTsT 377 GACcAUGACCCUGCCCUCGT sT 378 AD-9739

880-898 GAGGGCAGGGUCAUGGUCATsT 379 UGACCAUGACCCUGCCCUCT sT 380 AD-9637

880-898 GAGGGcAGGGu cAuGGu CAT s T 381 UGACcAUGACCCUGCCCUCT sT 382 AD-9763

882-900 GGGCAGGGUCAUGGUCACCTsT 383 GGUGACCAUGACCCUGCCCT sT 384 AD-9630

882-900 GGGcAGGGucAuGGucAccTsT 385 GGUGACcAUGACCCUGCCCT sT 386 AD-9756

885-903 CAGGGUCAUGGUCACCGACTsT 387 GUCGGUGACCAUGACCCUGT sT 388 AD-9593

885-903 cAGGGucAuGGucAccGAcTsT 389 GUCGGUGACcAUGACCCUGT sT 390 AD-9719

886-904 AGGGUCAUGGUCACCGACUTsT 391 AGUCGGUGACCAUGACCCUT sT 392 AD-9601

886-904 AGGGucAuGGucAccGAcuTsT 393 AGUCGGUGACcAUGACCCUT sT 394 AD-9727

892-910 AUGGUCACCGACUUCGAGATsT 395 UCUCGAAGUCGGUGACCAUT sT 396 AD-9573

892-910 AuGGucAccGAcuucGAGATsT 397 UCUCGAAGUCGGUGACcAUT sT 398 AD-9699

899-917 CCGACUUCGAGAAUGUGCCTT 399 GGCACAUUCUCGAAGUCGGTT 400 AD-15228

921-939 GGAGGACGGGACCCGCUUCTT 401 GAAGCGGGUCCCGUCCUCCTT 402 AD-15395

993-1011 CAGCGGCCGGGAUGCCGGCTsT 403 GCCGGCAUCCCGGCCGCUGT sT 404 AD-9602

993-1011 cAGcGGccGGGAuGccGGcTsT 405 GCCGGcAUCCCGGCCGCUGT sT 406 AD-9728

1020-1038 GGGUGCCAGCAUGCGCAGCTT 407 GCUGCGCAUGCUGGCACCCTT 408 AD-15386

1038-1056 CCUGCGCGUGCUCAACUGCTsT 409 GCAGUUGAGCACGCGCAGGT sT 410 AD-9580

1038-1056 ccuGcGcGuGcucAAcuGcTsT 411 GcAGUUGAGcACGCGcAGGT sT 412 AD-9706

1040-1058 UGCGCGUGCUCAACUGCCATsT 413 UGGCAGUUGAGCACGCGCAT sT 414 AD-9581

1040-1058 uGcGcGuGcucAAcuGccATsT 415 UGGcAGUUGAGcACGCGcAT sT 416 AD-9707

1042-1060 CGCGUGCUCAACUGCCAAGTsT 417 CUUGGCAGUUGAGCACGCGT sT 418 AD-9543

1042-1060 cGcGuGcucAAcuGccAAGTsT 419 CUUGGcAGUUGAGcACGCGT sT 420 AD-9669

1053-1071 CUGCCAAGGGAAGGGCACGTsT 421 CGUGCCCUUCCCUUGGCAGT sT 422 AD-9574

1053-1071 cuGccAAGGGAAGGGcAcGTsT 423 CGUGCCCUUCCCUUGGcAGT sT 424 AD-9700

1057-1075 CAAGGGAAGGGCACGGUUATT 425 UAACCGUGCCCUUCCCUUGTT 426 AD-15320

1058-1076 AAGGGAAGGGCACGGUUAGTT 427 CUAACCGUGCCCUUCCCUUTT 428 AD-15321

1059-1077 AGGGAAGGGCACGGUUAGCTT 429 GCUAACCGUGCCCUUCCCUTT 430 AD-15199

1060-1078 GGGAAGGGCACGGUUAGCGTT 431 CGCUAACCGUGCCCUUCCCTT 432 AD-15167

1061-1079 GGAAGGGCACGGUUAGCGGTT 433 CCGCUAACCGUGCCCUUCCTT 434 AD-15164

1062-1080 GAAGGGCACGGUUAGCGGCTT 435 GCCGCUAACCGUGCCCUUCTT 436 AD-15166

1063-1081 AAGGGCACGGUUAGCGGCATT 437 UGCCGCUAACCGUGCCCUUTT 438 AD-15322

1064-1082 AGGGCACGGUUAGCGGCACTT 439 GUGCCGCUAACCGUGCCCUTT 440 AD-15200

1068-1086 CACGGUUAGCGGCACCCUCTT 441 GAGGGUGCCGCUAACCGUGTT 442 AD-15213

1069-1087 ACGGUUAGCGGCACCCUCATT 443 UGAGGGUGCCGCUAACCGUTT 444 AD-15229

1072-1090 GUUAGCGGCACCCUCAUAGTT 445 CUAUGAGGGUGCCGCUAACTT 446 AD-15215

1073-1091 UUAGCGGCACCCUCAUAGGTT 447 CCUAUGAGGGUGCCGCUAATT 448 AD-15214

1076-1094 GCGGCACCCUCAUAGGCCUTsT 449 AGGCCUAUGAGGGUGCCGCT sT 450 AD-9315

1079-1097 GCACCCUCAUAGGCCUGGATsT 451 UCCAGGCCUAUGAGGGUGCT sT 452 AD-9326

1085-1103 UCAUAGGCCUGGAGUUUAUTsT 453 AUAAACUCCAGGCCUAUGAT sT 454 AD-9318

1090-1108 GGCCUGGAGUUUAUUCGGATsT 455 UCCGAAUAAACUCCAGGCCT sT 456 AD-9323

1091-1109 GCCUGGAGUUUAUUCGGAATsT 457 UUCCGAAUAAACUCCAGGCT sT 458 AD-9314

1091-1109 GccuGGAGuuuAuucGGAATsT 459 UUCCGAAuAAACUCcAGGCT sT 460 AD- 10792

1091-1109 GccuGGAGuuuAuucGGAATsT 461 UUCCGAAUAACUCCAGGCTsT 462 AD- 10796

1093-1111 CUGGAGUUUAUUCGGAAAATsT 463 UUUUCCGAAUAAACUCCAGT sT 464 AD-9638 position in

SEQ

human SEQ

Sense strand sequence (5 ^'-3 ^')' Antisense-strand sequence (5 ^'-3^')' ID Duplex name access. # ID NO:

NO:

NM 174936

1093-1111 cuGGAGuuuAuucGGAAAATsT 465 UUUUCCGAAuAAACUCcAGT sT 466 AD-9764

1095-1113 GGAGUUUAUUCGGAAAAGCTsT 467 GCUUUUCCGAAUAAACUCCT sT 468 AD-9525

1095-1113 GGAGuuuAuucGGAAAAGcTsT 469 GCUUUUCCGAAuAAACUCCT sT 470 AD-9651

1096-1114 GAGUUUAUUCGGAAAAGCCTsT 471 GGCUUUUCCGAAUAAACUCT sT 472 AD-9560

1096-1114 GAGuuuAuucGGAAAAGccTsT 473 GGCUUUUCCGAAuAAACUCT sT 474 AD-9686

1100-1118 UUAUUCGGAAAAGCCAGCUTsT 475 AGCUGGCUUUUCCGAAUAAT sT 476 AD-9536

1100-1118 uuAuucGGAAAAGccAGcuTsT 477 AGCUGGCUUUUCCGAAuAAT sT 478 AD-9662

1154-1172 CCCUGGCGGGUGGGUACAGTsT 479 CUGUACCCACCCGCCAGGGT sT 480 AD-9584

1154-1172 cccuGGcGGGuGGGuAcAGTsT 481 CUGuACCcACCCGCcAGGGT sT 482 AD-9710

1155-1173 CCUGGCGGGUGGGUACAGCTT 483 GCUGUACCCACCCGCCAGGTT 484 AD-15323

1157-1175 UGGCGGGUGGGUACAGCCGTsT 485 CGGCUGUACCCACCCGCCAT sT 486 AD-9551

1157-1175 uGGcGGGuGGGuAcAGccGTsT 487 CGGCUGuACCcACCCGCcAT sT 488 AD-9677

1158-1176 GGCGGGUGGGUACAGCCGCTT 489 GCGGCUGUACCCACCCGCCTT 490 AD-15230

1162-1180 GGUGGGUACAGCCGCGUCCTT 491 GGACGCGGCUGUACCCACCTT 492 AD-15231

1164-1182 UGGGUACAGCCGCGUCCUCTT 493 GAGGACGCGGCUGUACCCATT 494 AD-15285

1172-1190 GCCGCGUCCUCAACGCCGCTT 495 GCGGCGUUGAGGACGCGGCTT 496 AD-15396

1173-1191 CCGCGUCCUCAACGCCGCCTT 497 GGCGGCGUUGAGGACGCGGTT 498 AD-15397

1216-1234 GUCGUGCUGGUCACCGCUGTsT 499 CAGCGGUGACCAGCACGACT sT 500 AD-9600

1216-1234 GucGuGcuGGucAccGcuGTsT 501 cAGCGGUGAC cAGcACGACT sT 502 AD-9726

1217-1235 UCGUGCUGGUCACCGCUGCTsT 503 GCAGCGGUGACCAGCACGAT sT 504 AD-9606

1217-1235 ucGuGcuGGucAccGcuGcTsT 505 GcAGCGGUGACcAGcACGAT sT 506 AD-9732

1223-1241 UGGUCACCGCUGCCGGCAATsT 507 UUGCCGGCAGCGGUGACCAT sT 508 AD-9633

1223-1241 uGGucAccGcuGccGGcAATsT 509 UUGCCGGcAGCGGUGACcAT sT 510 AD-9759

1224-1242 GGUCACCGCUGCCGGCAACTsT 511 GUUGCCGGCAGCGGUGACCT sT 512 AD-9588

1224-1242 GGucAccGcuGccGGcAAcTsT 513 GUUGCCGGcAGCGGUGACCT sT 514 AD-9714

1227-1245 CACCGCUGCCGGCAACUUCTsT 515 GAAGUUGCCGGCAGCGGUGT sT 516 AD-9589

1227-1245 cAccGcuGccGGcAAcuucTsT 517 GAAGUUGCCGGcAGCGGUGT sT 518 AD-9715

1229-1247 CCGCUGCCGGCAACUUCCGTsT 519 CGGAAGUUGCCGGCAGCGGT sT 520 AD-9575

1229-1247 ccGcuGccGGcAAcuuccGTsT 521 CGGAAGUUGCCGGcAGCGGT sT 522 AD-9701

1230-1248 CGCUGCCGGCAACUUCCGGTsT 523 CCGGAAGUUGCCGGCAGCGT sT 524 AD-9563

1230-1248 cGcuGccGGcAAcuuccGGTsT 525 CCGGAAGUUGCCGGcAGCGT sT 526 AD-9689

1231-1249 GCUGCCGGCAACUUCCGGGTsT 527 CCCGGAAGUUGCCGGCAGCT sT 528 AD-9594

1231-1249 GcuGccGGcAAcuuccGGGTsT 529 CCCGGAAGUUGCCGGcAGCT sT 530 AD-9720

1236-1254 CGGCAACUUCCGGGACGAUTsT 531 AUCGUCCCGGAAGUUGCCGT sT 532 AD-9585

1236-1254 cGGcAAcuuccGGGAcGAuTsT 533 AUCGUCCCGGAAGUUGCCGT sT 534 AD-9711

1237-1255 GGCAACUUCCGGGACGAUGTsT 535 CAUCGUCCCGGAAGUUGCCT sT 536 AD-9614

1237-1255 GGcAAcuuccGGGAcGAuGTsT 537 cAUCGUCCCGGAAGUUGCCT sT 538 AD-9740

1243-1261 UUCCGGGACGAUGCCUGCCTsT 539 GGCAGGCAUCGUCCCGGAAT sT 540 AD-9615

1243-1261 uuccGGGAcGAuGccuGccTsT 541 GGcAGGcAUCGUCCCGGAAT sT 542 AD-9741

1248-1266 GGACGAUGCCUGCCUCUACTsT 543 GUAGAGGCAGGCAUCGUCCT sT 544 AD-9534

1248-1266 GGACGAUGCCUGCCUCUACTsT 545 GUAGAGGCAGGCAUCGUCCT sT 546 AD-9534

1248-1266 GGAcGAuGccuGccucuAcTsT 547 GuAGAGGcAGGcAUCGUCCT sT 548 AD-9660

1279-1297 GCUCCCGAGGUCAUCACAGTT 549 CUGUGAUGACCUCGGGAGCTT 550 AD-15324

1280-1298 CUCCCGAGGUCAUCACAGUTT 551 ACUGUGAUGACCUCGGGAGTT 552 AD-15232

1281-1299 UCCCGAGGUCAUCACAGUUTT 553 AACUGUGAUGACCUCGGGATT 554 AD-15233

1314-1332 CCAAGACCAGCCGGUGACCTT 555 GGUCACCGGCUGGUCUUGGTT 556 AD-15234

1315-1333 CAAGACCAGCCGGUGACCCTT 557 GGGUCACCGGCUGGUCUUGTT 558 AD-15286

1348-1366 ACCAACUUUGGCCGCUGUGTST 559 CACAGCGGCCAAAGUUGGUT sT 560 AD-9590

1348-1366 AccAAcuuuGGccGcuGuGTsT 561 cAcAGCGGCcAAAGUUGGUT sT 562 AD-9716

1350-1368 CAACUUUGGCCGCUGUGUGTsT 563 CACACAGCGGCCAAAGUUGT sT 564 AD-9632

1350-1368 cAAcuuuGGccGcuGuGuGTsT 565 cAcAcAGCGGCcAAAGUUGT sT 566 AD-9758

1360-1378 CGCUGUGUGGACCUCUUUGTsT 567 CAAAGAGGUCCACACAGCGT sT 568 AD-9567

1360-1378 cGcuGuGuGGAccucuuuGTsT 569 cAAAGAGGUCcAcAcAGCGT sT 570 AD-9693

1390-1408 GACAUCAUUGGUGCCUCCATsT 571 UGGAGGCACCAAUGAUGUCT sT 572 AD-9586

1390-1408 GAcAucAuuGGuGccuccATsT 573 UGGAGGcACcAAUGAUGUCT sT 574 AD-9712

1394-1412 UCAUUGGUGCCUCCAGCGATsT 575 UCGCUGGAGGCACCAAUGAT sT 576 AD-9564

1394-1412 ucAuuGGuGccuccAGcGATsT 577 UCGCUGGAGGcACcAAUGAT sT 578 AD-9690

1417-1435 AGCACCUGCUUUGUGUCACTST 579 GUGACACAAAGCAGGUGCUT sT 580 AD-9616

1417-1435 AGcAccuGcuuuGuGucAcTsT 581 GUGAcAcAAAGcAGGUGCUT sT 582 AD-9742

1433-1451 CACAGAGUGGGACAUCACATT 583 UGUGAUGUCCCACUCUGUGTT 584 AD-15398

1486-1504 AUGCUGUCUGCCGAGCCGGTsT 585 CCGGCUCGGCAGACAGCAUT sT 586 AD-9617

1486-1504 AuGcuGucuGccGAGccGGTsT 587 CCGGCUCGGcAGAcAGcAUT sT 588 AD-9743

1491-1509 GUCUGCCGAGCCGGAGCUCTsT 589 GAGCUCCGGCUCGGCAGACT sT 590 AD-9635

1491-1509 GucuGccGAGccGGAGcucTsT 591 GAGCUCCGGCUCGGcAGACT sT 592 AD-9761

1521-1539 GUUGAGGCAGAGACUGAUCTsT 593 GAUCAGUCUCUGCCUCAACT sT 594 AD-9568 position in

SEQ

human SEQ

Sense strand sequence (5 ^'-3 ^')' Antisense-strand sequence (5 ^'-3^')' ID Duplex name access. # ID NO:

NO:

NM 174936

1521-1539 GuuGAGGcAGAGAcuGAucTsT 595 GAUcAGUCUCUGCCUcAACT sT 596 AD-9694

1527-1545 GCAGAGACUGAUCCACUUCTsT 597 GAAGUGGAUCAGUCUCUGCT sT 598 AD-9576

1527-1545 GcAGAGAcuGAuccAcuucTsT 599 GAAGUGGAUcAGUCUCUGCT sT 600 AD-9702

1529-1547 AGAGACUGAUCCACUUCUCTsT 601 GAGAAGUGGAUCAGUCUCUT sT 602 AD-9627

1529-1547 AGAGAcuGAuccAcuucucTsT 603 GAGAAGUGGAUcAGUCUCUT sT 604 AD-9753

1543-1561 UUCUCUGCCAAAGAUGUCATsT 605 UGACAUCUUUGGCAGAGAAT sT 606 AD-9628

1543-1561 uucucuGccAAAGAuGucATsT 607 UGAcAUCUUUGGcAGAGAAT sT 608 AD-9754

1545-1563 CUCUGCCAAAGAUGUCAUCTsT 609 GAUGACAUCUUUGGCAGAGT sT 610 AD-9631

1545-1563 cucuGccAAAGAuGucAucTsT 611 GAUGAcAUCUUUGGcAGAGT sT 612 AD-9757

1580-1598 CUGAGGACCAGCGGGUACUTsT 613 AGUACCCGCUGGUCCUCAGT sT 614 AD-9595

1580-1598 cuGAGGAccAGcGGGuAcuTsT 615 AGuACCCGCUGGUCCUcAGT sT 616 AD-9721

1581-1599 UGAGGACCAGCGGGUACUGTsT 617 CAGUACCCGCUGGUCCUCAT sT 618 AD-9544

1581-1599 uGAGGAccAGcGGGuAcuGTsT 619 cAGuACCCGCUGGUCCUcAT sT 620 AD-9670

1666-1684 ACUGUAUGGUCAGCACACUTT 621 AGUGUGCUGACCAUACAGUTT 622 AD-15235

1668-1686 UGUAUGGUCAGCACACUCGTT 623 CGAGUGUGCUGACCAUACATT 624 AD-15236

1669-1687 GUAUGGUCAGCACACUCGGTT 625 CCGAGUGUGCUGACCAUACTT 626 AD-15168

1697-1715 GGAUGGCCACAGCCGUCGCTT 627 GCGACGGCUGUGGCCAUCCTT 628 AD-15174

1698-1716 GAUGGCCACAGCCGUCGCCTT 629 GGCGACGGCUGUGGCCAUCTT 630 AD-15325

1806-1824 CAAGCUGGUCUGCCGGGCCTT 631 GGCCCGGCAGACCAGCUUGTT 632 AD-15326

1815-1833 CUGCCGGGCCCACAACGCUTsT 633 AGCGUUGUGGGCCCGGCAGT sT 634 AD-9570

1815-1833 cuGccGGGcccAcAAcGcuTsT 635 AGCGUUGUGGGCCCGGcAGT sT 636 AD-9696

1816-1834 UGCCGGGCCCACAACGCUUTsT 637 AAGCGUUGUGGGCCCGGCAT sT 638 AD-9566

1816-1834 uGccGGGcccAcAAcGcuuTsT 639 AAGCGUUGUGGGCCCGGcAT sT 640 AD-9692

1818-1836 CCGGGCCCACAACGCUUUUTsT 641 AAAAGCGUUGUGGGCCCGGT sT 642 AD-9532

1818-1836 ccGGGcccAcAAcGcuuuuTsT 643 AAAAGCGUUGUGGGCCCGGT sT 644 AD-9658

1820-1838 GGGCCCACAACGCUUUUGGTsT 645 CCAAAAGCGUUGUGGGCCCT sT 646 AD-9549

1820-1838 GGGcccAcAAcGcuuuuGGTsT 647 CcAAAAGCGUUGUGGGCCCT sT 648 AD-9675

1840-1858 GGUGAGGGUGUCUACGCCATsT 649 UGGCGUAGACACCCUCACCT sT 650 AD-9541

1840-1858 GGuGAGGGuGucuAcGccATsT 651 UGGCGuAGAcACCCUcACCT sT 652 AD-9667

1843-1861 GAGGGUGUCUACGCCAUUGTsT 653 CAAUGGCGUAGACACCCUCT sT 654 AD-9550

1843-1861 GAGGGuGucuAcGccAuuGTsT 655 cAAUGGCGuAGAcACCCUCT sT 656 AD-9676

1861-1879 GCCAGGUGCUGCCUGCUACTsT 657 GUAGCAGGCAGCACCUGGCT sT 658 AD-9571

1861-1879 GccAGGuGcuGccuGcuAcTsT 659 GuAGcAGGcAGcACCUGGCT sT 660 AD-9697

1862-1880 CCAGGUGCUGCCUGCUACCTsT 661 GGUAGCAGGCAGCACCUGGT sT 662 AD-9572

1862-1880 ccAGGuGcuGccuGcuAccTsT 663 GGuAGcAGGcAGcACCUGGT sT 664 AD-9698

2008-2026 ACCCACAAGCCGCCUGUGCTT 665 GCACAGGCGGCUUGUGGGUTT 666 AD-15327

2023-2041 GUGCUGAGGCCACGAGGUCTsT 667 GACCUCGUGGCCUCAGCACT sT 668 AD-9639

2023-2041 GuGcuGAGGccAcGAGGucTsT 669 GACCUCGUGGCCUcAGcACT sT 670 AD-9765

2024-2042 UGCUGAGGCCACGAGGUCATsT 671 UGACCUCGUGGCCUCAGCAT sT 672 AD-9518

2024-2042 UGCUGAGGCCACGAGGUCATsT 673 UGACCUCGUGGCCUCAGCAT sT 674 AD-9518

2024-2042 uGcuGAGGccAcGAGGucATsT 675 UGACCUCGUGGCCUcAGcAT sT 676 AD-9644

UfgCf uGfaGf gC f cAf cGfaG p-

2024-2042 677 uGfaCf cUf cGf uGfgCf cUf cAf gC f 678 AD- 14672 f gUf cAf TsT

aT sT

Uf GCf Uf GAGGCf Cf AC f GAGG UfGACfCfUfCfGUfGGC fC fUfC fAG

2024-2042 679 680 AD- 14682

UfC fATsT Cf AT sT

p-

2024-2042 UgCuGaGgCcAcGaGgUcATsT 681 uGfaCf cUf cGf uGfgCf cUf cAf gC f 682 AD- 14692 aT sT

UfGACfCfUfCfGUfGGC fC fUfC fAG

2024-2042 UgCuGaGgCcAcGaGgUcATsT 683 684 AD- 14702

Cf AT sT

UfgCf uGfaGf gC f cAf cGfaG

2024-2042 685 UGACCucGUggCCUCAgcaT sT 686 AD-14712 f gUf cAf TsT

Uf GCf Uf GAGGCf Cf AC f GAGG

2024-2042 687 UGACCucGUggCCUCAgcaT sT 688 AD- 14722

UfC fATsT

2024-2042 UgCuGaGgCcAcGaGgUcATsT 689 UGACCucGUggCCUCAgcaT sT 690 AD-14732

GfuGf gUf cAf gC fgGf cC fgG p-

2024-2042 691 cAfuCf cC fgGf cC fgCf uGfaCf cAf 692 AD-15078 f gAfuGf TsT

cT sT

GUf GGUf Cf AGCf GGCf Cf GGG CfAUfC fC fC fGGC fC fGCfUfGACfC

2024-2042 693 694 AD-15088

AUf GT sT fACf TsT

p-

2024-2042 GuGgUcAgCgGcCgGgAuGTsT 695 cAfuCf cC fgGf cC fgCf uGfaCf cAf 696 AD-15098 cT sT

CfAUfC fC fC fGGC fC fGCfUfGACfC

2024-2042 GuGgUcAgCgGcCgGgAuGTsT 697 698 AD-15108 fACf TsT position in

SEQ

human SEQ

Sense strand sequence (5 ^'-3 ^')' Antisense-strand sequence (5 ^'-3^')' ID Duplex name access. # ID NO:

NO:

NM 174936

GfuGf gUf cAf gC fgGf cC fgG

2024-2042 699 CAUCCcgGCcgCUGACcacT sT 700 AD-15118 f gAfuGf TsT

GUf GGUf Cf AGCf GGCf Cf GGG

2024-2042 701 CAUCCcgGCcgCUGACcacT sT 702 AD-15128

AUf GT sT

2024-2042 GuGgUcAgCgGcCgGgAuGTsT 703 CAUCCcgGCcgCUGACcacT sT 704 AD-15138

2030-2048 GGCCACGAGGUCAGCCCAATT 705 UUGGGCUGACCUCGUGGCCTT 706 AD-15237

2035-2053 CGAGGUCAGCCCAACCAGUTT 707 ACUGGUUGGGCUGACCUCGTT 708 AD-15287

2039-2057 GUCAGCCCAACCAGUGCGUTT 709 ACGCACUGGUUGGGCUGACTT 710 AD-15238

2041-2059 CAGCCCAACCAGUGCGUGGTT 711 CCACGCACUGGUUGGGCUGTT 712 AD-15328

2062-2080 CACAGGGAGGCCAGCAUCCTT 713 GGAUGCUGGCCUCCCUGUGTT 714 AD-15399

2072-2090 CCAGCAUCCACGCUUCCUGTsT 715 CAGGAAGCGUGGAUGCUGGT sT 716 AD-9582

2072-2090 ccAGcAuccAcGcuuccuGTsT 717 cAGGAAGCGUGGAUGCUGGT sT 718 AD-9708

2118-2136 AGUCAAGGAGCAUGGAAUCT s T 719 GAUUCCAUGCUCCUUGACUT sT 720 AD-9545

2118-2136 AGucAAGGAGcAuGGAAucTsT 721 GAUUCcAUGCUCCUUGACUT sT 722 AD-9671

AfgUf cAfaGf gAfgCf aUfgG p-

2118-2136 723 gAfuUf cC faUf gC fuCf cUfuGf aC f 724 AD- 14674 f aAfuCf TsT

uT sT

AGUf C f AAGGAGC f AUf GGAAU GAUfUfCfCfAUfGCfUfCfCfUfUfG

2118-2136 725 726 AD- 14684 f Cf TsT AC f Uf T sT

p-

2118-2136 AgUcAaGgAgCaUgGaAuCTsT 727 gAfuUf cC faUf gC fuCf cUfuGf aC f 728 AD- 14694 uT sT

GAUfUfCfCfAUfGCfUfCfCfUfUfG

2118-2136 AgUcAaGgAgCaUgGaAuCTsT 729 730 AD- 14704

AC f Uf T sT

AfgUf cAfaGf gAfgCf aUfgG

2118-2136 731 GAUUCcaUGcuCCUUGacuT sT 732 AD-14714 f aAfuCf TsT

AGUf C f AAGGAGC f AUf GGAAU

2118-2136 733 GAUUCcaUGcuCCUUGacuT sT 734 AD- 14724 f Cf TsT

2118-2136 AgUcAaGgAgCaUgGaAuCTsT 735 GAUUCcaUGcuCCUUGacuT sT 736 AD-14734

GfcGfgC faCf cC fuCfaUfaG p-

2118-2136 737 aGfgCf cUfaUf gAfgGf gUfgCf cGf 738 AD-15080 f gC f cUf TsT

cT sT

GCfGGCfAC fC fC fUfC fAUfA AGGC fC fUfAUfGAGGGUfGCfCfGC f

2118-2136 739 740 AD-15090

GGC f C fUf T sT TsT

p-

2118-2136 GcGgCaCcCuCaUaGgCcUTsT 741 aGfgCf cUfaUf gAfgGf gUfgCf cGf 742 AD-15100 cT sT

AGGC fC fUfAUfGAGGGUfGCfCfGC f

2118-2136 GcGgCaCcCuCaUaGgCcUTsT 743 744 AD-15110

TsT

GfcGfgC faCf cC fuCfaUfaG

2118-2136 745 AGGCCuaUGagGGUGCcgcT sT 746 AD-15120 f gC f cUf TsT

GCfGGCfAC fC fC fUfC fAUfA

2118-2136 747 AGGCCuaUGagGGUGCcgcT sT 748 AD-15130

GGC f C fUf T sT

2118-2136 GcGgCaCcCuCaUaGgCcUTsT 749 AGGCCuaUGagGGUGCcgcT sT 750 AD-15140

2122-2140 AAGGAGCAUGGAAUCCCGGTsT 751 CCGGGAUUCCAUGCUCCUUT sT 752 AD-9522

2122-2140 AAGGAGcAuGGAAucccGGTsT 753 CCGGGAUUCcAUGCUCCUUT sT 754 AD-9648

2123-2141 AGGAGCAUGGAAUCCCGGCTsT 755 GCCGGGAUUCCAUGCUCCUT sT 756 AD-9552

2123-2141 AGGAGcAuGGAAucccGGcTsT 757 GCCGGGAUUCcAUGCUCCUT sT 758 AD-9678

2125-2143 GAGCAUGGAAUCCCGGCCCTsT 759 GGGCCGGGAUUCCAUGCUCT sT 760 AD-9618

2125-2143 GAGcAuGGAAucccGGcccTsT 761 GGGCCGGGAUUCcAUGCUCT sT 762 AD-9744

2230-2248 GCCUACGCCGUAGACAACATT 763 UGUUGUCUACGGCGUAGGCTT 764 AD-15239

2231-2249 CCUACGCCGUAGACAACACTT 765 GUGUUGUCUACGGCGUAGGTT 766 AD-15212

2232-2250 CUACGCCGUAGACAACACGTT 767 CGUGUUGUCUACGGCGUAGTT 768 AD-15240

2233-2251 UACGCCGUAGACAACACGUTT 769 ACGUGUUGUCUACGGCGUATT 770 AD-15177

2235-2253 CGCCGUAGACAACACGUGUTT 771 ACACGUGUUGUCUACGGCGTT 772 AD-15179

2236-2254 GCCGUAGACAACACGUGUGTT 773 CACACGUGUUGUCUACGGCTT 774 AD-15180

2237-2255 CCGUAGACAACACGUGUGUTT 775 ACACACGUGUUGUCUACGGTT 776 AD-15241

2238-2256 CGUAGACAACACGUGUGUATT 777 UACACACGUGUUGUCUACGTT 778 AD-15268

2240-2258 UAGACAACACGUGUGUAGUTT 779 ACUACACACGUGUUGUCUATT 780 AD-15242

2241-2259 AGACAACACGUGUGUAGUCTT 781 GACUACACACGUGUUGUCUTT 782 AD-15216

2242-2260 GACAACACGUGUGUAGUCATT 783 UGACUACACACGUGUUGUCTT 784 AD-15176

2243-2261 ACAACACGUGUGUAGUCAGTT 785 CUGACUACACACGUGUUGUTT 786 AD-15181

2244-2262 CAACACGUGUGUAGUCAGGTT 787 CCUGACUACACACGUGUUGTT 788 AD-15243

2247-2265 CACGUGUGUAGUCAGGAGCTT 789 GCUCCUGACUACACACGUGTT 790 AD-15182

2248-2266 ACGUGUGUAGUCAGGAGCCTT 791 GGCUCCUGACUACACACGUTT 792 AD-15244

2249-2267 CGUGUGUAGUCAGGAGCCGTT 793 CGGCUCCUGACUACACACGTT 794 AD-15387 position in

SEQ

human SEQ

Sense strand sequence (5 ^'-3 ^')' Antisense-strand sequence (5 ^'-3^')' ID Duplex name access. # ID NO:

NO:

NM 174936

2251-2269 UGUGUAGUCAGGAGCCGGGTT 795 CCCGGCUCCUGACUACACATT 796 AD-15245

2257-2275 GUCAGGAGCCGGGACGUCATsT 797 UGACGUCCCGGCUCCUGACT sT 798 AD-9555

2257-2275 GucAGGAGccGGGAcGucATsT 799 UGACGUCCCGGCUCCUGACT sT 800 AD-9681

2258-2276 UCAGGAGCCGGGACGUCAGTST 801 CUGACGUCCCGGCUCCUGAT sT 802 AD-9619

2258-2276 UcAGGAGccGGGAcGucAGTsT 803 CUGACGUCCCGGCUCCUGAT sT 804 AD-9745

2259-2277 CAGGAGCCGGGACGUCAGCTsT 805 GCUGACGUCCCGGCUCCUGT sT 806 AD-9620

2259-2277 cAGGAGccGGGAcGucAGcTsT 807 GCUGACGUCCCGGCUCCUGT sT 808 AD-9746

2263-2281 AGCCGGGACGUCAGCACUATT 809 UAGUGCUGACGUCCCGGCUTT 810 AD-15288

2265-2283 CCGGGACGUCAGCACUACATT 811 UGUAGUGCUGACGUCCCGGTT 812 AD-15246

2303-2321 CCGUGACAGCCGUUGCCAUTT 813 AUGGCAACGGCUGUCACGGTT 814 AD-15289

2317-2335 GCCAUCUGCUGCCGGAGCCTsT 815 GGCUCCGGCAGCAGAUGGCT sT 816 AD-9324

2375-2393 CCCAUCCCAGGAUGGGUGUTT 817 ACACCCAUCCUGGGAUGGGTT 818 AD-15329

2377-2395 CAUCCCAGGAUGGGUGUCUTT 819 AGACACCCAUCCUGGGAUGTT 820 AD-15330

2420-2438 AGCUUUAAAAUGGUUCCGATT 821 UCGGAACCAUUUUAAAGCUTT 822 AD-15169

2421-2439 GCUUUAAAAUGGUUCCGACTT 823 GUCGGAACCAUUUUAAAGCTT 824 AD-15201

2422-2440 CUUUAAAAUGGUUCCGACUTT 825 AGUCGGAACCAUUUUAAAGTT 826 AD-15331

2423-2441 UUUAAAAUGGUUCCGACUUTT 827 AAGUCGGAACCAUUUUAAATT 828 AD-15190

2424-2442 UUAAAAUGGUUCCGACUUGTT 829 CAAGUCGGAACCAUUUUAATT 830 AD-15247

2425-2443 UAAAAUGGUUCCGACUUGUTT 831 ACAAGUCGGAACCAUUUUATT 832 AD-15248

2426-2444 AAAAUGGUUCCGACUUGUCTT 833 GACAAGUCGGAACCAUUUUTT 834 AD-15175

2427-2445 AAAUGGUUCCGACUUGUCCTT 835 GGACAAGUCGGAACCAUUUTT 836 AD-15249

2428-2446 AAUGGUUCCGACUUGUCCCTT 837 GGGACAAGUCGGAACCAUUTT 838 AD-15250

2431-2449 GGUUCCGACUUGUCCCUCUTT 839 AGAGGGACAAGUCGGAACCTT 840 AD-15400

2457-2475 CUCCAUGGCCUGGCACGAGTT 841 CUCGUGCCAGGCCAUGGAGTT 842 AD-15332

2459-2477 CCAUGGCCUGGCACGAGGGTT 843 CCCUCGUGCCAGGCCAUGGTT 844 AD-15388

2545-2563 GAACUCACUCACUCUGGGUTT 845 ACCCAGAGUGAGUGAGUUCTT 846 AD-15333

2549-2567 UCACUCACUCUGGGUGCCUTT 847 AGGCACCCAGAGUGAGUGATT 848 AD-15334

2616-2634 UUUCACCAUUCAAACAGGUTT 849 ACCUGUUUGAAUGGUGAAATT 850 AD-15335

2622-2640 CAUUCAAACAGGUCGAGCUTT 851 AGCUCGACCUGUUUGAAUGTT 852 AD-15183

2623-2641 AUUCAAACAGGUCGAGCUGTT 853 CAGCUCGACCUGUUUGAAUTT 854 AD-15202

2624-2642 UUCAAACAGGUCGAGCUGUTT 855 ACAGCUCGACCUGUUUGAATT 856 AD-15203

2625-2643 UCAAACAGGUCGAGCUGUGTT 857 CACAGCUCGACCUGUUUGATT 858 AD-15272

2626-2644 CAAACAGGUCGAGCUGUGCTT 859 GCACAGCUCGACCUGUUUGTT 860 AD-15217

2627-2645 AAACAGGUCGAGCUGUGCUTT 861 AGCACAGCUCGACCUGUUUTT 862 AD-15290

2628-2646 AACAGGUCGAGCUGUGCUCTT 863 GAGCACAGCUCGACCUGUUTT 864 AD-15218

2630-2648 CAGGUCGAGCUGUGCUCGGTT 865 CCGAGCACAGCUCGACCUGTT 866 AD-15389

2631-2649 AGGUCGAGCUGUGCUCGGGTT 867 CCCGAGCACAGCUCGACCUTT 868 AD-15336

2633-2651 GUCGAGCUGUGCUCGGGUGTT 869 CACCCGAGCACAGCUCGACTT 870 AD-15337

2634-2652 UCGAGCUGUGCUCGGGUGCTT 871 GCACCCGAGCACAGCUCGATT 872 AD-15191

2657-2675 AGCUGCUCCCAAUGUGCCGTT 873 CGGCACAUUGGGAGCAGCUTT 874 AD-15390

2658-2676 GCUGCUCCCAAUGUGCCGATT 875 UCGGCACAUUGGGAGCAGCTT 876 AD-15338

2660-2678 UGCUCCCAAUGUGCCGAUGTT 877 CAUCGGCACAUUGGGAGCATT 878 AD-15204

2663-2681 UCCCAAUGUGCCGAUGUCCTT 879 GGACAUCGGCACAUUGGGATT 880 AD-15251

2665-2683 CCAAUGUGCCGAUGUCCGUTT 881 ACGGACAUCGGCACAUUGGTT 882 AD-15205

2666-2684 CAAUGUGCCGAUGUCCGUGTT 883 CACGGACAUCGGCACAUUGTT 884 AD-15171

2667-2685 AAUGUGCCGAUGUCCGUGGTT 885 CCACGGACAUCGGCACAUUTT 886 AD-15252

2673-2691 CCGAUGUCCGUGGGCAGAATT 887 UUCUGCCCACGGACAUCGGTT 888 AD-15339

2675-2693 GAUGUCCGUGGGCAGAAUGTT 889 CAUUCUGCCCACGGACAUCTT 890 AD-15253

2678-2696 GUCCGUGGGCAGAAUGACUTT 891 AGUCAUUCUGCCCACGGACTT 892 AD-15340

2679-2697 UCCGUGGGCAGAAUGACUUTT 893 AAGUCAUUCUGCCCACGGATT 894 AD-15291

2683-2701 UGGGCAGAAUGACUUUUAUTT 895 AUAAAAGUCAUUCUGCCCATT 896 AD-15341

2694-2712 ACUUUUAUUGAGCUCUUGUTT 897 AC AAGAGC UC AAUAAAAGUT T 898 AD-15401

2700-2718 AUUGAGCUCUUGUUCCGUGTT 899 CACGGAACAAGAGCUCAAUTT 900 AD-15342

2704-2722 AGCUCUUGUUCCGUGCCAGTT 901 CUGGCACGGAACAAGAGCUTT 902 AD-15343

2705-2723 GCUCUUGUUCCGUGCCAGGTT 903 CCUGGCACGGAACAAGAGCTT 904 AD-15292

2710-2728 UGUUCCGUGCCAGGCAUUCTT 905 GAAUGCCUGGCACGGAACATT 906 AD-15344

2711-2729 GUUCCGUGCCAGGCAUUCATT 907 UGAAUGCCUGGCACGGAACTT 908 AD-15254

2712-2730 UUCCGUGCCAGGCAUUCAATT 909 UUGAAUGCCUGGCACGGAATT 910 AD-15345

2715-2733 CGUGCCAGGCAUUCAAUCCTT 911 GGAUUGAAUGCCUGGCACGTT 912 AD-15206

2716-2734 GUGCCAGGCAUUCAAUCCUTT 913 AGGAUUGAAUGCCUGGCACTT 914 AD-15346

2728-2746 CAAUCCUCAGGUCUCCACCTT 915 GGUGGAGACCUGAGGAUUGTT 916 AD-15347

2743-2761 CACCAAGGAGGCAGGAUUCTsT 917 GAAUCCUGCCUCCUUGGUGT sT 918 AD-9577

2743-2761 c Ac cAAGGAGG cAGGAu u c T s T 919 GAAUCCUGCCUCCUUGGUGT sT 920 AD-9703

position in

SEQ

human SEQ

Sense strand sequence (5 ^'-3 ^')' Antisense-strand sequence (5 ^'-3^')' ID Duplex name access. # ID NO:

NO:

NM 174936

3051-3069 AGGACUGACUCGGCAGUGUTT 1021 ACACUGCCGAGUCAGUCCUTT 1022 AD-15362

3052-3070 GGACUGACUCGGCAGUGUGTT 1023 CACACUGCCGAGUCAGUCCTT 1024 AD-15192

3074-3092 UGGUGCAUGCACUGUCUCATT 1025 UGAGACAGUGCAUGCACCATT 1026 AD-15256

3080-3098 AUGCACUGUCUCAGCCAACTT 1027 GUUGGCUGAGACAGUGCAUTT 1028 AD-15363

3085-3103 CUGUCUCAGCCAACCCGCUTT 1029 AGCGGGUUGGCUGAGACAGTT 1030 AD-15364

3089-3107 CUCAGCCAACCCGCUCCACTsT 1031 GUGGAGCGGGUUGGCUGAGT sT 1032 AD-9604

3089-3107 cucAGccAAcccGcuccAcTsT 1033 GUGGAGCGGGUUGGCUGAGT sT 1034 AD-9730

3093-3111 GCCAACCCGCUCCACUACCTsT 1035 GGUAGUGGAGCGGGUUGGCT sT 1036 AD-9527

3093-3111 GccAAcccGcuccAcuAccTsT 1037 GGuAGUGGAGCGGGUUGGCT sT 1038 AD-9653

3096-3114 AACCCGCUCCACUACCCGGTT 1039 CCGGGUAGUGGAGCGGGUUTT 1040 AD-15365

3099-3117 CCGCUCCACUACCCGGCAGTT 1041 CUGCCGGGUAGUGGAGCGGTT 1042 AD-15294

3107-3125 CUACCCGGCAGGGUACACATT 1043 UGUGUACCCUGCCGGGUAGTT 1044 AD-15173

3108-3126 UACCCGGCAGGGUACACAUTT 1045 AUGUGUACCCUGCCGGGUATT 1046 AD-15366

3109-3127 ACCCGGCAGGGUACACAUUTT 1047 AAUGUGUACCCUGCCGGGUTT 1048 AD-15367

3110-3128 CCCGGCAGGGUACACAUUCTT 1049 GAAUGUGUACCCUGCCGGGTT 1050 AD-15257

3112-3130 CGGCAGGGUACACAUUCGCTT 1051 GCGAAUGUGUACCCUGCCGTT 1052 AD-15184

3114-3132 GCAGGGUACACAUUCGCACTT 1053 GUGCGAAUGUGUACCCUGCTT 1054 AD-15185

3115-3133 CAGGGUACACAUUCGCACCTT 1055 GGUGCGAAUGUGUACCCUGTT 1056 AD-15258

3116-3134 AGGGUACACAUUCGCACCCTT 1057 GGGUGCGAAUGUGUACCCUTT 1058 AD-15186

3196-3214 GGAACUGAGCCAGAAACGCTT 1059 GCGUUUCUGGCUCAGUUCCTT 1060 AD-15274

3197-3215 GAACUGAGCCAGAAACGCATT 1061 UGCGUUUCUGGCUCAGUUCTT 1062 AD-15368

3198-3216 AACUGAGCCAGAAACGCAGTT 1063 CUGCGUUUCUGGCUCAGUUTT 1064 AD-15369

3201-3219 UGAGCCAGAAACGCAGAUUTT 1065 AAUCUGCGUUUCUGGCUCATT 1066 AD-15370

3207-3225 AGAAACGCAGAUUGGGCUGTT 1067 CAGCCCAAUCUGCGUUUCUTT 1068 AD-15259

3210-3228 AACGCAGAUUGGGCUGGCUTT 1069 AGCCAGCCCAAUCUGCGUUTT 1070 AD-15408

3233-3251 AGCCAAGCCUCUUCUUACUTsT 1071 AGUAAGAAGAGGCUUGGCUT sT 1072 AD-9597

3233-3251 AGccAAGccucuucuuAcuTsT 1073 AGuAAGAAGAGGCUUGGCUT sT 1074 AD-9723

AfgCf cAfaGf cC fuCf uUf cU p-

3233-3251 1075 aGfuAf aGfaAf gAfgGf cUfuGf gC f 1076 AD- 14680 f uAf cUf TsT

uT sT

AGC fC fAAGCfCfUfCfUfUfC AGUf AAGAAGAGGC fUfUfGGC fUfT s

3233-3251 1077 1078 AD- 14690 f Uf Uf AC fUfT sT T

p-

3233-3251 AgCcAaGcCuCuUcUuAcUTsT 1079 aGfuAf aGfaAf gAfgGf cUfuGfgC f 1080 AD- 14700 uT sT

AGUf AAGAAGAGGC fUfUfGGC fUfT s

3233-3251 AgCcAaGcCuCuUcUuAcUTsT 1081 1082 AD-14710

T

AfgCf cAfaGf cC fuCf uUf cU

3233-3251 1083 AGUAAgaAGagGCUUGgcuT sT 1084 AD- 14720 f uAf cUf TsT

AGC fC fAAGCfCfUfCfUfUfC

3233-3251 1085 AGUAAgaAGagGCUUGgcuT sT 1086 AD-14730 f Uf Uf AC fUfT sT

3233-3251 AgCcAaGcCuCuUcUuAcUTsT 1087 AGUAAgaAGagGCUUGgcuT sT 1088 AD- 14740

UfgGf uUf cCf cUfgAf gGfaC p-

3233-3251 1089 gC fuGf gUf cCf uC faGf gGfaAf cC f 1090 AD-15086 f cAfgCf TsT

aT sT

UfGGUfUfC fC fC fUfGAGGAC GC fUfGGUfC fC fUfC fAGGGAAC fC f

3233-3251 1091 1092 AD-15096 f Cf AGCf TsT AT sT

p-

3233-3251 UgGuUcCcUgAgGaCcAgCTsT 1093 gC fuGf gUf cCf uC faGf gGfaAf cC f 1094 AD-15106 aT sT

GC fUfGGUfC fC fUfC fAGGGAAC fC f

3233-3251 UgGuUcCcUgAgGaCcAgCTsT 1095 1096 AD-15116

AT sT

UfgGf uUf cCf cUfgAf gGfaC

3233-3251 1097 GCUGGucCUcaGGGAAccaT sT 1098 AD-15126 f cAfgCf TsT

UfGGUfUfC fC fC fUfGAGGAC

3233-3251 1099 GCUGGucCUcaGGGAAccaT sT 1100 AD-15136 f Cf AGCf TsT

3233-3251 UgGuUcCcUgAgGaCcAgCTsT 1101 GCUGGucCUcaGGGAAccaT sT 1102 AD-15146

3242-3260 UCUUCUUACUUCACCCGGCTT 1103 GCCGGGUGAAGUAAGAAGATT 1104 AD-15260

3243-3261 CUUCUUACUUCACCCGGCUTT 1105 AGCCGGGUGAAGUAAGAAGTT 1106 AD-15371

3244-3262 UUCUUACUUCACCCGGCUGTT 1107 CAGCCGGGUGAAGUAAGAATT 1108 AD-15372

3262-3280 GGGCUCCUCAUUUUUACGGTT 1109 CCGUAAAAAUGAGGAGCCCTT 1110 AD-15172

3263-3281 GGCUCCUCAUUUUUACGGGTT 1111 CCCGUAAAAAUGAGGAGCCTT 1112 AD-15295

3264-3282 GCUCCUCAUUUUUACGGGUTT 1113 ACCCGUAAAAAUGAGGAGCTT 1114 AD-15373

3265-3283 CUCCUCAUUUUUACGGGUATT 1115 UACCCGUAAAAAUGAGGAGTT 1116 AD-15163

3266-3284 UCCUCAUUUUUACGGGUAATT 1117 UUACCCGUAAAAAUGAGGATT 1118 AD-15165

3267-3285 CCUCAUUUUUACGGGUAACTT 1119 GUUACCCGUAAAAAUGAGGTT 1120 AD-15374 position in

SEQ

human SEQ

Sense strand sequence (5 ^'-3 ^')' Antisense-strand sequence (5 ^'-3^')' ID Duplex name access. # ID NO:

NO:

NM 174936

3268-3286 CUCAUUUUUACGGGUAACATT 1121 UGUUACCCGUAAAAAUGAGTT 1122 AD-15296

3270-3288 CAUUUUUACGGGUAACAGUTT 1123 ACUGUUACCCGUAAAAAUGTT 1124 AD-15261

3271-3289 AUUUUUACGGGUAACAGUGTT 1125 CACUGUUACCCGUAAAAAUTT 1126 AD-15375

3274-3292 UUUACGGGUAACAGUGAGGTT 1127 CCUCACUGUUACCCGUAAATT 1128 AD-15262

3308-3326 CAGACCAGGAAGCUCGGUGTT 1129 CACCGAGCUUCCUGGUCUGTT 1130 AD-15376

3310-3328 GACCAGGAAGCUCGGUGAGTT 1131 CUCACCGAGCUUCCUGGUCTT 1132 AD-15377

3312-3330 CCAGGAAGCUCGGUGAGUGTT 1133 CACUCACCGAGCUUCCUGGTT 1134 AD-15409

3315-3333 GGAAGCUCGGUGAGUGAUGTT 1135 CAUCACUCACCGAGCUUCCTT 1136 AD-15378

3324-3342 GUGAGUGAUGGCAGAACGATT 1137 UCGUUCUGCCAUCACUCACTT 1138 AD-15410

3326-3344 GAGUGAUGGCAGAACGAUGTT 1139 CAUCGUUCUGCCAUCACUCTT 1140 AD-15379

3330-3348 GAUGGCAGAACGAUGCCUGTT 1141 CAGGCAUCGUUCUGCCAUCTT 1142 AD-15187

3336-3354 AGAACGAUGCCUGCAGGCATT 1143 UGCCUGCAGGCAUCGUUCUTT 1144 AD-15263

3339-3357 ACGAUGCCUGCAGGCAUGGTT 1145 CCAUGCCUGCAGGCAUCGUTT 1146 AD-15264

3348-3366 GCAGGCAUGGAACUUUUUCTT 1147 GAAAAAGUUCCAUGCCUGCTT 1148 AD-15297

3356-3374 GGAACUUUUUCCGUUAUCATT 1149 UGAUAACGGAAAAAGUUCCTT 1150 AD-15208

3357-3375 GAACUUUUUCCGUUAUCACTT 1151 GUGAUAACGGAAAAAGUUCTT 1152 AD-15209

3358-3376 AACUUUUUCCGUUAUCACCTT 1153 GGUGAUAACGGAAAAAGUUTT 1154 AD-15193

3370-3388 UAUCACCCAGGCCUGAUUCTT 1155 GAAUCAGGCCUGGGUGAUATT 1156 AD-15380

3378-3396 AGGCCUGAUUCACUGGCCUTT 1157 AGGCCAGUGAAUCAGGCCUTT 1158 AD-15298

3383-3401 UGAUUCACUGGCCUGGCGGTT 1159 CCGCCAGGCCAGUGAAUCATT 1160 AD-15299

3385-3403 AUUCACUGGCCUGGCGGAGTT 1161 CUCCGCCAGGCCAGUGAAUTT 1162 AD-15265

3406-3424 GCUUCUAAGGCAUGGUCGGTT 1163 CCGACCAUGCCUUAGAAGCTT 1164 AD-15381

3407-3425 CUUCUAAGGCAUGGUCGGGTT 1165 CCCGACCAUGCCUUAGAAGTT 1166 AD-15210

3429-3447 GAGGGCCAACAACUGUCCCTT 1167 GGGACAGUUGUUGGCCCUCTT 1168 AD-15270

3440-3458 ACUGUCCCUCCUUGAGCACTsT 1169 GUGCUCAAGGAGGGACAGUT sT 1170 AD-9591

3440-3458 AcuGucccuccuuGAGcAcTsT 1171 GUGCUcAAGGAGGGAcAGUT sT 1172 AD-9717

3441-3459 CUGUCCCUCCUUGAGCACCTsT 1173 GGUGCUCAAGGAGGGACAGT sT 1174 AD-9622

3441-3459 cuGucccuccuuGAGcAccTsT 1175 GGUGCUcAAGGAGGGAcAGT sT 1176 AD-9748

3480-3498 ACAUUUAUCUUUUGGGUCUTsT 1177 AGACCCAAAAGAUAAAUGUT sT 1178 AD-9587

3480-3498 AcAuuuAucuuuuGGGucuTsT 1179 AGAC C c AAAAGAuAAAUGUT s T 1180 AD-9713

Af cAf uUfuAf uC fuUf uUfgG p-

3480-3498 1181 aGfaCf cC faAf aAfgAf uAfaAf uGf 1182 AD- 14679 f gUf cUf TsT

uT sT

ACf AUfUfUfAUf Cf Uf Uf Uf U AGAC f C f C f AAAAGAU f AAAUf GU f T s

3480-3498 1183 1184 AD- 14689 f GGGUfC fUfT sT T

p-

3480-3498 AcAuUuAuCuUuUgGgUcUTsT 1185 aGfaCf cC faAf aAfgAf uAfaAf uGf 1186 AD- 14699 uT sT

AGAC f C f C f AAAAGAU f AAAUf GU f T s

3480-3498 AcAuUuAuCuUuUgGgUcUTsT 1187 1188 AD- 14709

T

Af cAf uUfuAf uC fuUf uUfgG

3480-3498 1189 AGACCcaAAagAUAAAuguT sT 1190 AD-14719 f gUf cUf TsT

ACf AUfUfUfAUf Cf Uf Uf Uf U

3480-3498 1191 AGACCcaAAagAUAAAuguT sT 1192 AD- 14729 f GGGUfC fUfT sT

3480-3498 AcAuUuAuCuUuUgGgUcUTsT 1193 AGACCcaAAagAUAAAuguT sT 1194 AD-14739

GfcCfaUfcUfgC fuGf cC fgG p-

3480-3498 1195 gGf cUf cC fgGf cAfgCf aGfaUf gGf 1196 AD-15085 f aGf cCf TsT

cT sT

GCfCfAUfC fUfGCfUfGC fC f GGCfUfCfCfGGCfAGCfAGAUfGGC f

3480-3498 1197 1198 AD-15095

GGAGC f C f T sT TsT

p-

3480-3498 GcCaUcUgCuGcCgGaGcCTsT 1199 gGf cUf cC f gGf cAfgCf aGfaUf gGf 1200 AD-15105 cT sT

GGCfUfCfCfGGCfAGCfAGAUfGGC f

3480-3498 GcCaUcUgCuGcCgGaGcCTsT 1201 1202 AD-15115

TsT

GfcCfaUfcUfgC fuGf cC fgG

3480-3498 1203 GGCUCauGCagCAGAUggcT sT 1204 AD-15125 f aGf cCf TsT

GCfCfAUfC fUfGCfUfGC fC f

3480-3498 1205 GGCUCauGCagCAGAUggcT sT 1206 AD-15135

GGAGC f C f T sT

3480-3498 GcCaUcUgCuGcCgGaGcCTsT 1207 GGCUCauGCagCAGAUggcT sT 1208 AD-15145

3481-3499 CAUUUAUCUUUUGGGUCUGTsT 1209 CAGACCCAAAAGAUAAAUGT sT 1210 AD-9578

3481-3499 cAuuuAucuuuuGGGucuGTsT 1211 cAGACCcAAAAGAuAAAUGT sT 1212 AD-9704

3485-3503 UAUCUUUUGGGUCUGUCCUTsT 1213 AGGACAGACCCAAAAGAUAT sT 1214 AD-9558

3485-3503 uAucuuuuGGGucuGuccuTsT 1215 AGGAcAGACCcAAAAGAuAT sT 1216 AD-9684

3504-3522 CUCUGUUGCCUUUUUACAGTsT 1217 CUGUAAAAAGGCAACAGAGT sT 1218 AD-9634

3504-3522 cucuGuuGccuuuuuAcAGTsT 1219 CUGuAAAAAGGcAAcAGAGT sT 1220 AD-9760

position in

SEQ

human SEQ

(5^'-3^')' Antisense-strand sequence (5^'-3^')' ID Duplex name access. # Sense strand sequence

ID NO:

NO:

NM 174936

3570-3588 UCUGGGUUUUGUAGCAUUUTsT 1295 AAAUGCUACAAAACCCAGATsT 1296 AD-9629

3570-3588 ucuGGGuuuuGuAGcAuuuTsT 1297 AAAUGCuAcAAAACCcAGATsT 1298 AD-9755

3613-3631 AUAAAAACAAACAAACGUUTT 1299 AACGUUUGUUUGUUUUUAUTT 1300 AD-15412

3617-3635 AAACAAACAAACGUUGUCCTT 1301 GGACAACGUUUGUUUGUUUTT 1302 AD-15211

3618-3636 AACAAACAAACGUUGUCCUTT 1303 AGGACAACGUUUGUUUGUUTT 1304 AD-15300

U, C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g: corresponding -O- methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding 2'-deoxy-2'-fluoro ribonucleotide;

where nucleotides are written in sequence, they are connected by 3 '-5' phosphodiester groups; nucleotides with interjected "s" are connected by 3'-0-5'-0 phosphorothiodiester groups; unless denoted by prefix "p-", oligonucleotides are devoid of a 5 '-phosphate group on the 5 '-most nucleotide; all oligonucleotides bear 3 '-OH on the 3 '-most nucleotide

Table 2. Sequences of modified dsRNA targeted to PCSK9

SEQ SEQ

Duplex

Sense strand sequence (5 '-3 ')' ID Antisense-strand sequence (5 '-3 ')' ID number

NO: NO:

AD- 12338 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1375 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1376

AD-12339 GcCuGgAgUuUaUuCgGaA 1377 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1378

AD- 12340 GccuGGAGuuuAuucGGAA 1379 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1380

AD- 12341 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1381 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT 1382

AD- 12342 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1383 UUCCGAAuAAACUCcAGGCTsT 1384

AD- 12343 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1385 uUcCGAAuAAACUccAGGCTsT 1386

AD- 12344 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1387 UUCCGAAUAAACUCCAGGCTsT 1388

AD-12345 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1389 UUCCGAAUAAACUCCAGGCscsu 1390

AD- 12346 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1391 UUCCGaaUAaaCUCCAggcscsu 1392

AD-12347 GCCUGGAGUUUAUUCGGAATsT 1393 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT 1394

AD-12348 GccuGGAGuuuAuucGGAATsT 1395 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT 1396

AD-12349 GcCuGgnAgUuUaUuCgGaATsT 1397 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT 1398

AD-12350 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTTab 1399 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTTab 1400

AD-12351 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1401 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu 1402

AD-12352 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1403 UUCCGaaUAaaCUCCAggcscsu 1404

AD-12354 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1405 UUCCGAAUAAACUCCAGGCscsu 1406

AD-12355 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1407 UUCCGAAuAAACUCcAGGCTsT 1408

AD-12356 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1409 uUcCGAAuAAACUccAGGCTsT 1410

GmocCmouGmogAmogUmouUmoaUmouCm

AD-12357

o 1411 UUCCGaaUAaaCUCCAggc

gGmoaA 1412

GmocCmouGmogAmogUmouUmoaUmouCm

AD-12358 1413 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc

ogGmoaA 1414

GmocCmouGmogAmogUmouUmoaUmouCm

AD-12359 1415 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu

ogGmoaA 1416

GmocCmouGmogAmogUmouUmoaUmouCm

AD- 12360

ogGmoaA 1417 UUCCGAAUAAACUCCAGGCscsu 1418

GmocCmouGmogAmogUmouUmoaUmouCm

AD- 12361

ogGmoaA 1419 UUCCGAAuAAACUCcAGGCTsT 1420

GmocCmouGmogAmogUmouUmoaUmouCm

AD- 12362

ogGmoaA 1421 uUcCGAAuAAACUccAGGCTsT 1422

GmocCmouGmogAmogUmouUmoaUmouCm

AD- 12363

ogGmoaA 1423 UUCCGaaUAaaCUCCAggcscsu 1424

GmocCmouGmogAmogUmouUmoaUmouCm

AD- 12364

ogGmoaATsT 1425 UUCCGaaUAaaCUCCAggcTsT 1426

GmocCmouGmogAmogUmouUmoaUmouCm

AD-12365 CcAGGCTsT

OgGmoaATsT 1427 UUCCGAAuAAACU 1428

GmocCmouGmogAmogUmouUmoaUmouCm

AD- 12366 CCAGGCTsT

OgGmoaATsT 1429 UUCCGAAUAAACU 1430

GmocmocmouGGAGmoumoumouAmoumou

AD- 12367 CAggcTsT

mocGGAATsT 1431 UUCCGaaUAaaCUC 1432

GmocmocmouGGAGmoumoumouAmoumou

AD-12368

mocGGAATsT 1433 UUCCGAAuAAACUCcAGGCTsT 1434

GmocmocmouGGAGmoumoumouAmoumou

AD- 12369

mocGGAATsT 1435 UUCCGAAUAAACUCCAGGCTsT 1436

GmocmocmouGGAGmoumoumouAmoumou

AD- 12370

mocGGAATsT 1437 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1438

GmocmocmouGGAGmoumoumouAmoumou P-

AD- 12371

mocGGAATsT 1439 UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1440

GmocmocmouGGAGmoumoumouAmoumou

AD- 12372 1441 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu

mocGGAATsT 1442

GmocmocmouGGAGmoumoumouAmoumou

AD- 12373 1443 UUCCGAAUAAACUCCAGGCTsT

mocGGAATsT 1444

AD- 12374 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1445 UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1446

AD-12375 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1447 UUCCGAAUAAACUCCAGGCTsT 1448

AD- 12377 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1449 uUcCGAAuAAACUccAGGCTsT 1450

AD-12378 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1451 UUCCGaaUAaaCUCCAggcscsu 1452 SEQ SEQ

Duplex

Sense strand sequence (5 '-3 ')' ID Antisense-strand sequence (5 '-3 ')' ID number

NO: NO:

AD- 12379 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1453 UUCCGAAUAAACUCCAGGCscsu 1454

AD-12380 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1455 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu 1456

AD-12381 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1457 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT 1458

AD-12382 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1459 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1460

AD- 12383 GCCUGGAGUUUAUUCGGAATsT 1461 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1462

AD-12384 GccuGGAGuuuAuucGGAATsT 1463 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1464

AD-12385 GcCuGgnAgUuUaUuCgGaATsT 1465 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1466

AD-12386 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1467 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1468

AD-12387 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1469 UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1470

AD-12388 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1471 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1472

AD-12389 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1473 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu 1474

AD- 12390 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1475 UUCCGAAUAAACUCCAGGCscsu 1476

AD- 12391 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1477 UUCCGaaUAaaCUCCAggc 1478

AD- 12392 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1479 UUCCGAAUAAACUCCAGGCTsT 1480

AD- 12393 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1481 UUCCGAAuAAACUCcAGGCTsT 1482

AD- 12394 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1483 uUcCGAAuAAACUccAGGCTsT 1484

GmocCmouGmogAmogUmouUmoaUmouCm P-

AD-12395

ogGmoaATsT 1485 UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1486

GmocCmouGmogAmogUmouUmoaUmouCm P-

AD- 12396

ogGmoaA 1487 UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1488

P-

AD- 12397 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1489 UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1490

P-

AD-12398 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT 1491 UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1492

P-

AD-12399 GcCuGgnAgUuUaUuCgGaATsT 1493 UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1494

P-

AD- 12400 GCCUGGAGUUUAUUCGGAATsT 1495 UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1496

P-

AD- 12401 GccuGGAGuuuAuucGGAATsT 1497 UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1498

P-

AD- 12402 GccuGGAGuuuAuucGGAA 1499 UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1500

P-

AD- 12403 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1501 UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1502

AD-9314 GCCUGGAGUUUAUUCGGAATsT 1503 UUCCGAAUAAACUCCAGGCTsT 1504

AD- 10794 ucAuAGGccuGGAGuuuAudTsdT 1525 AuAAACUCcAGGCCuAUGAdTsdT 1526

AD-10795 ucAuAGGccuGGAGuuuAudTsdT 1527 AuAAACUccAGGcCuAuGAdTsdT 1528

AD- 10797 ucAuAGGccuGGAGuuuAudTsdT 1529 AUAAACUCCAGGCCUAUGAdTsdT 1530

Table 3. Cholesterol levels of rats treated with LNPOl-10792

Dosage of 5 mg/kg, n=6 rats per group

16 0.635 ± 0.107

18 0.704 ± 0.060

21 0.775 ± 0.102

28 0.815 ± 0.103

Table 4. Serum LDL-C levels of cynomolgus monkeys treated with LNP formulated dsRNAs

Serum LDL-C (relative to

Table 5a: Modified dsRNA targeted to PCSK9

U, C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g: corresponding 2'- O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding 2'-deoxy-2'-fluoro ribonucleotide; where nucleotides are written in sequence, they are connected by 3 '-5' phosphodiester groups; nucleotides with interjected "s" are connected by 3'-0-5'-0 phosphorothiodiester groups; unless denoted by prefix "p-", oligonucleotides are devoid of a 5 '-phosphate group on the 5 '-most nucleotide; all oligonucleotides bear 3 '-OH on the 3 '-most nucleotide. Table 5b: Silencing activity of modified dsRNA in monkey hepatocytes

Table 6: dsRNA targeted to PCSK9: mismatches and modifications

SEQ ID

Duplex # Strand Sequence 5' to 3'

NO:

AS 1552 AAGcAAAAcAGGUCuAGAATsT

S 1553 UfuCfuA¾AfcCfuGfuUfuU¾CfuUfTsT

AD-14676

AS 1554 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

S 1555 UfuCfuAfgAfcCuGfuUfuUfgCfuUfTsT

AD-3276

AS 1556 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

S 1557 UfuCfuAfgAfcCfUGfuUfuUfgCfuUfTsT

AD-3277

AS 1558 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

S 1559 UfuCfuAfgAfcCUGfuUfuUfgCfuUfTsT

AD-3278

AS 1560 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

S 1561 UfuCfuAfgAfcYluGfuUfuUfgCfuUfTsT

AD-3279

AS 1562 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

S 1563 UfuCfuAfgAfcYlUGfuUfuUfgCfuUfTsT

AD-3280

AS 1564 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

S 1565 UfuCfuAfgAfcCiYlGfuUfuUfgCfuUiTsT

AD-3281

AS 1566 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

S 1567 UfuCfuAfgAfcCYlGfuUfuUfgCfuUfTsT

AD-3282

AS 1568 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

S 1569 UfuCfuAfgAfcCfuYluUfuUfgCfuUfTsT

AD-3283

AS 1570 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

S 1571 UfuCfuAfgAfcCUYluUfuUfgCfuUfTsT

AD-3284

AS 1572 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT

Strand: S/Sense; AS/Antisense

U, C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g: corresponding 2'-0- methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding 2'-deoxy-2'-fluoro ribonucleotide; Yl corresponds to DFT difluorotoluyl ribo(or deoxyribo)nucleotide; where nucleotides are written in sequence, they are connected by 3 '-5' phosphodiester groups; nucleotides with interjected "s" are connected by 3'-0-5 '-0 phosphorothiodiester groups; unless denoted by prefix "p-", oligonucleotides are devoid of a 5 '-phosphate group on the 5 '-most nucleotide; all oligonucleotides bear 3'-OH on the 3 '-most nucleotide.

Table 7: Amino acid sequence of the mature form of human PCSK9

QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVWLKEETHL

SQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDS

SVFAQSIPW LERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFEN

VPEEDGTRFHRQASKCDSHGTHLAGWSGRDAGVAKGASMRSLRVLNCQGKGTVSGT

LIGLEFIRKSQLVQPVGPLWLLPLAGGYSRVLNAACQRLARAGWLVTAAGNFRDDAC

LYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGEDIIGASSDCSTCFVSQS

GTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVA

ALPPSTHGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERME

AQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHV

LTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPA

PQGQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCWRSRDVSTTGSTSEEAVTAV

AICCRSRHLAQASQELQ (SEQ ID NO: 1573) Table 8: Nucleic acid and amino acid sequences of PCSK9

The start and stop codons in the nucleotide sequence are shown in bold

Nucleotide cagcgacgtc gaggcgctca tggttgcagg cgggcgccgc cgttcagttc agggtctgag cctggaggag tgagccaggc agtgagactg gctcgggcgg gccgggacgc gtcgttgcag cagcggctcc cagctcccag ccaggattcc gcgcgcccct tcacgcgccc tgctcctgaa cttcagctcc tgcacagtcc tccccaccgc aaggctcaag gcgccgccgg cgtggaccgc gcacggcctc taggtctcct cgccaggaca gcaacctctc ccctggccct catgggcacc gtcagctcca ggcggtcctg gtggccgctg ccactgctgc tgctgctgct gctgctcctg ggtcccgcgg gcgcccgtgc gcaggaggac gaggacggcg actacgagga gctggtgcta gccttgcgtt ccgaggagga cggcctggcc gaagcacccg agcacggaac cacagccacc ttccaccgct gcgccaagga tccgtggagg ttgcctggca cctacgtggt ggtgctgaag gaggagaccc acctctcgca gtcagagcgc actgcccgcc gcctgcaggc ccaggctgcc cgccggggat acctcaccaa gatcctgcat gtcttccatg gccttcttcc tggcttcctg gtgaagatga gtggcgacct gctggagctg gccttgaagt tgccccatgt cgactacatc gaggaggact cctctgtctt tgcccagagc atcccgtgga acctggagcg gattacccct ccacggtacc gggcggatga ataccagccc cccgacggag gcagcctggt ggaggtgtat ctcctagaca ccagcataca gagtgaccac cgggaaatcg agggcagggt catggtcacc gacttcgaga atgtgcccga ggaggacggg acccgcttcc acagacaggc cagcaagtgt gacagtcatg gcacccacct ggcaggggtg gtcagcggcc gggatgccgg cgtggccaag ggtgccagca tgcgcagcct gcgcgtgctc aactgccaag ggaagggcac ggttagcggc accctcatag gcctggagtt tattcggaaa agccagctgg tccagcctgt ggggccactg gtggtgctgc tgcccctggc gggtgggtac agccgcgtcc tcaacgccgc ctgccagcgc ctggcgaggg ctggggtcgt gctggtcacc gctgccggca acttccggga cgatgcctgc ctctactccc cagcctcagc tcccgaggtc atcacagttg gggccaccaa tgcccaagac cagccggtga ccctggggac tttggggacc aactttggcc gctgtgtgga cctctttgcc ccaggggagg acatcattgg tgcctccagc gactgcagca cctgctttgt gtcacagagt gggacatcac aggctgctgc ccacgtggct ggcattgcag ccatgatgct gtctgccgag ccggagctca ccctggccga gttgaggcag agactgatcc acttctctgc caaagatgtc atcaatgagg cctggttccc tgaggaccag cgggtactga cccccaacct ggtggccgcc ctgcccccca gcacccatgg ggcaggttgg cagctgtttt gcaggactgt atggtcagca cactcggggc ctacacggat ggccacagcc gtcgcccgct gcgccccaga tgaggagctg ctgagctgct ccagtttctc caggagtggg aagcggcggg gcgagcgcat ggaggcccaa gggggcaagc tggtctgccg ggcccacaac gcttttgggg gtgagggtgt ctacgccatt gccaggtgct gcctgctacc ccaggccaac tgcagcgtcc acacagctcc accagctgag gccagcatgg ggacccgtgt ccactgccac caacagggcc acgtcctcac aggctgcagc tcccactggg aggtggagga ccttggcacc cacaagccgc ctgtgctgag gccacgaggt cagcccaacc agtgcgtggg ccacagggag gccagcatcc acgcttcctg ctgccatgcc ccaggtctgg aatgcaaagt caaggagcat ggaatcccgg cccctcagga gcaggtgacc gtggcctgcg aggagggctg gaccctgact ggctgcagtg ccctccctgg gacctcccac gtcctggggg cctacgccgt agacaacacg tgtgtagtca ggagccggga cgtcagcact acaggcagca ccagcgaagg ggccgtgaca gccgttgcca tctgctgccg gagccggcac ctggcgcagg cctcccagga gctccagtga cagccccatc ccaggatggg tgtctgggga gggtcaaggg ctggggctga gctttaaaat ggttccgact tgtccctctc tcagccctcc atggcctggc acgaggggat ggggatgctt ccgcctttcc ggggctgctg gcctggccct tgagtggggc agcctccttg cctggaactc actcactctg ggtgcctcct ccccaggtgg aggtgccagg aagctccctc cctcactgtg gggcatttca ccattcaaac aggtcgagct gtgctcgggt gctgccagct gctcccaatg tgccgatgtc cgtgggcaga atgactttta ttgagctctt gttccgtgcc aggcattcaa tcctcaggtc tccaccaagg aggcaggatt cttcccatgg ataggggagg gggcggtagg ggctgcaggg acaaacatcg ttggggggtg agtgtgaaag gtgctgatgg ccctcatctc cagctaactg tggagaagcc cctgggggct ccctgattaa tggaggctta gctttctgga tggcatctag ccagaggctg gagacaggtg cgcccctggt ggtcacaggc tgtgccttgg tttcctgagc cacctttact ctgctctatg The start and stop codons in the nucleotide sequence are shown in bold

ccaggctgtg ctagcaacac ccaaaggtgg cctgcgggga gccatcacct aggactgact cggcagtgtg cagtggtgca tgcactgtct cagccaaccc gctccactac ccggcagggt acacattcgc acccctactt cacagaggaa gaaacctgga accagagggg gcgtgcctgc caagctcaca cagcaggaac tgagccagaa acgcagattg ggctggctct gaagccaagc ctcttcttac ttcacccggc tgggctcctc atttttacgg gtaacagtga ggctgggaag gggaacacag accaggaagc tcggtgagtg atggcagaac gatgcctgca ggcatggaac tttttccgtt atcacccagg cctgattcac tggcctggcg gagatgcttc taaggcatgg tcgggggaga gggccaacaa ctgtccctcc ttgagcacca gccccaccca agcaagcaga catttatctt ttgggtctgt cctctctgtt gcctttttac agccaacttt tctagacctg ttttgctttt gtaacttgaa gatatttatt ctgggttttg tagcattttt attaatatgg tgacttttta aaataaaaac aaacaaacgt tgtcct (SEQ ID NO: 1574)

Amino Acid MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLA

LRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVWLKEETHLSQSERTARRLQAQA ARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQSIPWNLER ITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTRFHR QASKCDSHGTHLAGWSGRDAGVAKGASMRSLRVLNCQGKGTVSGTLIGLEFIRKSQL VQPVGPLWLLPLAGGYSRVLNAACQRLARAGWLVTAAGNFRDDACLYSPASAPEVI TVGATNAQDQPVTLGTLGTNFGRCVDLFAPGEDI IGASSDCS CFVSQSG SQAAAHV AGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTHG AGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLV CRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHW EVEDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTV ACEEGWTLTGCSALPGTSHVLGAYAVDNTCWRSRDVSTTGSTSEGAVTAVAICCRSR HLAQASQELQ (SEQ ID NO: 1575)

Table 9: Exemplary IgG2 heavy chain constant domain of an anti-PC SK9 antibody of the present invention; exemplary IgG4 heavy chain constant domain of an anti-PC SK9 antibody of the present invention; exemplary kappa light chain constant domain of an anti-PCSK9 antibody of the present invention; and an exemplary lambda light chain constant domain of an anti-PC SK9 antibody of the present invention

Human lgG2:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT.

VPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC VWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLP APIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:1576)

Human lgG4:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSvyT

VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NQ:1577)

Human lambda:

QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETITPSKQSNNKYAASSY LSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NQ: 1578)

Human kappa:

TVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NQ:1579)

TABLE 10: Amino Acid Sequences of Exemplary PCSK9 Antibodies

Heavy Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val lie Trp Tyr Asp Gly Ser Asp Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Thr Gly Pro Leu Lys Leu Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:1581)

C4 Light Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr lie Thr Cys Arg

Ala Ser Gin Arg lie Ser Asn Tyr Leu Ser Trp Tyr Leu Gin Lys Pro Gly He Ala Pro Lys Leu Leu He Tyr Ala Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Tin- Leu Thr He Ser Ser Leu Gin Ser Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Tyr Ser Thr Pro Leu He Phe Gly Gly Gly Thr Lys Val Glu He Lys (SEQ ID NO:1582)

Heavy Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin Thr Leu Ser Leu Thr Cys Thr

Val Ser Gly Gly Ser He Ser Ser Ser Asp Tyr Tyr Trp Ser Trp He Arg Gin His Pro Gly Lys Gly Leu Glu Trp He Gly Tyr He Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg He Thr He Ser Val Asp Thr Ser Lys Asn Leu Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly Val Thr Thr Tyr Tyr Tyr Ala Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 1583)

3B5 Light Asp He Leu Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr He Thr Cys Arg

Ala Ser Gin Ser He Ser Ser Tyr Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Val Leu He Tyr Ala Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Asn Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Tyr Ser Ser Pro He Thr Phe Gly Gin Gly Thr Arg Leu Glu He Lys (SEQ ID NO:1584)

Heavy Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Asn Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr He Ser Gly Ser Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Lys Phe Val Leu Met Val Tyr Ala Met Leu Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO: 1585)

5G4 Light Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr He Thr Cys Arg

Ala Ser Gin Ser He Ser He Tyr Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Tyr Leu Leu He Tyr Ala Ala Ala Ser Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Tyr Ser Ala Pro He Thr Phe Gly Gin Gly Thr Arg Leu Glu He Lys (SEQ ID NO: 1586)

Heavy Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Asn Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr He Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Lys Phe Val Leu Met Val Tyr Ala Met Leu Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO: 1587)

1H4 Light Gin Ser Val Leu Thr Gin Pro Pro Ser Val Ser Gly Ala Pro Gly Gin Arg Val Thr He Ser Cys Thr Gly

Ser Ser Ser Asn He Gly Ala Gly Tyr Asp Val His Trp Tyr Gin Gin Leu Pro Gly Thr Ala Pro Lys Leu Leu He Ser Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala He Thr Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gin Ser Tyr Asp Ser Ser Leu Ser Gly Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1588)

Heavy Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser Met Asn Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser He Ser Ser Ser Ser Ser Tyr He Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg Asp Tyr Asp Phe Trp Ser Ala Tyr Tyr Asp Ala Phe Asp Val Trp Gly Gin Gly Thr Met Val Thr Val Ser Ser (SEQ ID NO: 1589)

7B2 Light Gin Ser Val Leu Thr Gin Pro Pro Ser Val Ser Gly Ala Pro Gly Gin Arg Val Thr He Ser Cys Thr Gly

Ser Ser Ser Asn He Gly Ala His Tyr Asp Val His Trp Tyr Gin Gin Val Pro Gly Thr Ala Pro Lys Leu Leu He Tyr Gly Asn Thr Tyr Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala He Thr Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gin Ser Tyr Asp Asn Ser Leu Ser Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO: 1590)

Heavy Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin Thr Leu Ser Leu Thr Cys Thr

Val Ser Gly Gly Ser He Ser Ser Gly Gly Tyr Tyr Trp Ser Trp He Arg Gin His Pro Gly Lys Gly Leu Glu Trp He Gly Tyr He Tyr Asn Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Asp Thr Ala Met Val Pro Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO:1591)

5A7 Light Gin Ser Ala Leu Thr Gin Pro Ala Ser Val Ser Gly Ser Pro Gly Gin Ser He Thr He Ser Cys Thr Gly

Thr Ser Ser Asp Val Gly Arg Tyr Asn Ser Val Ser Trp Tyr Gin His His Pro Gly Lys Ala Pro Lys Val Met He Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Thr Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr He Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO: 1592)

Heavy Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys

Ala Ser Gly Tyr Thr Phe Pro Ser Tyr Gly lie Ser Tip Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Tip Met Gly Tip lie Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Glu Lys Leu Gin Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu Val Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Phe Tyr Cys Ala Arg Gly Tyr Val Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 1593)

H5 Light Gin Ser Ala Leu Thr Gin Pro Ala Ser Val Ser Gly Ser Pro Gly Gin Ser lie Thr lie Ser Cys Thr Gly

Thr Ser Ser Asp Val Gly Gly Tyr Asn Ser Val Ser Tip Tyr Gin Gin His Pro Gly Lys Pro Pro Lys Leu Met lie Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser lie Arg Phe Ser Gly Ser Lys Ser Gly Asn Tin- Ala Ser Leu Thr He Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Thr Ser Thr Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO: 1594)

Heavy Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Arg Pro Gly Ala Ser Val Lys Val Ser Cys Lys

Ala Ser Gly Tyr Thr Leu Thr Ser Tyr Gly lie Ser Tip Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Tip Met Gly Tip lie Ser Val Tyr Asn Gly Asn Thr Asn Tyr Ala Gin Lys Val Gin Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Arg Ser Leu Ser Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 1595)

H5 Light Gin Ser Ala Leu Thr Gin Pro Ala Ser Val Ser Gly Ser Pro Gly Gin Ser lie Thr lie Ser Cys Thr Gly

Thr Ser Ser Asp Val Gly Gly Tyr Asn Ser Val Ser Tip Tyr Gin Gin His Pro Gly Lys Pro Pro Lys Leu Met lie Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser lie Arg Phe Ser Gly Ser Lys Ser Gly Asn Tin- Ala Ser Leu Thr He Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Thr Ser Thr Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO: 1596)

Heavy Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys

Ala Ser Gly Tyr Thr Leu Thr Ser Tyr Gly He Ser Tip Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Tip Met Gly Tip He Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gin Lys Val Gin Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 1597)

D1 Light Gin Ser Ala Leu Thr Gin Pro Ala Ser Val Ser Gly Ser Pro Gly Gin Ser He Thr He Ser Cys Thr Gly

Thr Ser Ser Asp Val Gly Gly Tyr Asn Ser Val Ser Tip Tyr Gin Gin His Pro Gly Lys Pro Pro Lys Leu Met He Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Tin- Ala Ser Leu Thr He Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Thr Ser Thr Ser Met Val Phe Gly Gly Gly Thr Lys Leu Ala Val Leu (SEQ ID NO: 1598)

Heavy Gin He Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala

Ser Gly Tyr Thr Leu Thr Ser Tyr Gly He Ser Tip Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Tip Met Gly Tip He Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gin Lys Val Gin Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys Ala Arg Gly Tyr Gly Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 1599)

D10 Light Gin Ser Ala Leu Thr Gin Pro Ala Ser Val Ser Gly Ser Pro Gly Gin Ser He Thr He Ser Cys Thr Gly

Thr Ser Ser Asp Val Gly Gly Tyr Asn Ser Val Ser Tip Tyr Gin Gin Tyr Pro Gly Lys Pro Pro Lys Leu Lys He Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr He Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Thr Ser Thr Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1600)

Heavy Gin He Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala

Ser Gly Tyr Pro Leu Thr Ser Tyr Gly He Ser Tip Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Tip Met Gly Tip He Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gin Lys Val Gin Gly Ser Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:1601)

E7 Light Gin Ser Ala Leu Thr Gin Pro Ala Ser Val Ser Gly Ser Pro Gly Gin Ser He Thr He Ser Cys Thr Gly

Thr Ser Ser Asp Val Gly Gly Tyr Asn Ser Val Ser Tip Tyr Gin Gin His Pro Gly Lys Pro Pro Lys Leu Met He Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Tin- Ala Ser Leu Thr He Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Thr Ser Thr Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1602)

Heavy Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Leu Lys Val Ser Cys Lys

Ala Ser Gly Tyr Ser Leu Thr Ser Tyr Gly He Ser Tip Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Tip Met Gly Tip He Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gin Lys Val Gin Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Val Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 1603)

B9 Light Gin Ser Ala Leu Thr Gin Pro Ala Ser Val Ser Gly Ser Pro Gly Gin Ser He Thr He Ser Cys Thr Gly

Thr Ser Ser Asp Val Gly Gly Tyr Asn Ser Val Ser Tip Tyr Gin Gin His Pro Gly Lys Pro Pro Lys Leu Met He Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr He Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Thr Ser Thr Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1604)

Heavy Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys

Ala Ser Gly Tyr Pro Leu Thr Ser Tyr Gly He Ser Tip Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Tip Met Gly Tip He Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gin Lys Val Gin Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 1605)

H9 Light Gin Ser Ala Leu Thr Gin Pro Ala Ser Val Ser Gly Ser Pro Gly Gin Ser He Thr He Ser Cys Thr Gly

Thr Asn Ser Asp Val Gly Gly Tyr Asn Ser Val Ser Tip Tyr Gin Gin His Pro Gly Lys Pro Pro Lys Leu Met He Tyr Glu Val Ser Asn Arg Pro Ser Gly He Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr He Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Thr Ser Thr Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1606)

Heavy Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys

Ala Ser Gly Tyr Ala Leu Thr Ser Tyr Gly He Ser Tip Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Tip Met Gly Tip He Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gin Lys Val Gin Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 1607)

E10 Light Gin Ser Ala Leu Thr Gin Pro Ala Ser Val Ser Gly Ser Pro Gly Gin Ser He Thr He Ser Cys Thr Gly

Thr Ser Ser Asp Val Gly Gly Tyr Asn Ser Val Ser Tip Tyr Gin Gin His Pro Gly Lys Ala Pro Lys Leu Met He Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Tin- Ala Ser Leu Thr He Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Tyr Thr Ser Thr Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1608)

Heavy Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys

Ala Ser Gly Tyr Thr Leu Thr Ser Tyr Gly He Ser Tip Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Tip Met Gly Tip Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gin Lys Leu Gin Gly Arg Gly Thr Met Thr Thr Asp Pro Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 1609)

B12 Light Gin Ser Ala Leu Thr Gin Pro Ala Ser Val Ser Gly Ser Pro Gly Gin Ser He Thr He Ser Cys Thr Gly

Thr Ser Ser Asp Val Gly Gly Tyr Asn Ser Val Ser Tip Tyr Gin Gin His Pro Gly Lys Ala Pro Lys Leu Met He Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Tin- Ala Ser Leu Thr He Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Tyr Thr Ser Thr Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1610)

Heavy Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys

Ala Ser Gly Tyr Thr Leu Thr Ser Tyr Gly He Ser Tip Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Tip Met Gly Tip Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gin Lys Leu Gin Gly Arg Gly Thr Met Thr Thr Asp Pro Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 1611)

C2 Light Gin Ser Ala Leu Thr Gin Pro Ala Ser Val Ser Gly Ser Pro Gly Gin Ser He Thr He Ser Cys Thr Gly

Thr Ser Ser Asp Val Gly Ala Tyr Asn Ser Val Ser Tip Tyr Gin Gin His Pro Gly Lys Ala Pro Lys Arg Met He Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Tin- Ala Ser Leu Thr He Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Thr Asn Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1612)

Heavy Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys

Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Gly He Ser Tip Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Tip Met Gly Tip Val Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gin Lys Phe Gin Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Val Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:1613)

G1 Light Gin Ser Ala Leu Thr Gin Pro Ala Ser Val Ser Gly Ser Pro Gly Gin Ser He Thr He Ser Cys Thr Gly

Thr Ser Ser Asp Val Gly Gly Tyr Asn Ser Val Ser Tip Tyr Gin Gin His Pro Gly Lys Ala Pro Lys Leu Met He Tyr Glu Val Thr Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr He Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Tyr Thr Ser Thr Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1614)

Heavy Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys

Ala Ser Gly Tyr Thr Leu Thr Ser Tyr Gly He Ser Tip Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Tip Met Gly Tip Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gin Lys Leu Gin Gly Arg Gly Thr Met Thr Thr Asp Pro Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 1615)

H1 Light Leu Ser Ala Leu Thr Gin Pro Ala Ser Val Ser Gly Ser Pro Gly Gin Ser He Thr He Ser Cys Thr Gly

Thr Ser Ser Asp Val Gly Asn Tyr Asn Leu Val Ser Tip Tyr Gin Gin Tyr Ser Gly Lys Ala Pro Lys Leu Met He Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr He Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Ser Ser Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1616)

Heavy Gin Val Gin Leu Gin Gin Ser Gly Pro Gly Leu Val Lys Pro Ser Gin Thr Leu Ser Leu Thr Cys Ala

He Ser Gly Asp Ser Val Ser Ser Asn Ser Ala Ala Trp Asn Trp He Arg Gin Ser Pro Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Lys Asn Tyr Ser Val Ser Val Lys Ser Arg He Thr He Asn Pro Asp Thr Ser Lys Asn Gin Phe Ser Leu Gin Leu Asn Ser Val Thr Pro Gly Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly Pro Thr Ala Ala Phe Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO:1617)

C9 Light Gin Ser Val Leu Thr Gin Pro Pro Ser Ala Ser Gly Thr Pro Gly Gin Arg Val Thr He Ser Cys Ser Gly

Ser Ser Ser Asn He Gly Ser Lys Thr Val Asn Trp Tyr Gin Gin Val Pro Gly Thr Ala Pro Lys Leu Leu He Tyr Arg Asn Asn Gin Arg Pro Leu Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala He Ser Gly Leu Gin Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1618)

Heavy Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Leu Arg Leu Ser Cys Val

Val Ser Gly Phe Thr Phe Ser Ser Tyr Trp Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Asn He Lys Gin Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Ser Asn Trp Gly Phe Ala Phe Asp He Trp Gly Gin Gly Thr Met Val Thr Val Ser Ser (SEQ ID NO:1619)

H6 Light Gin Ser Val Leu Thr Gin Pro Pro Ser Ala Ser Gly Pro Pro Gly Gin Arg Val Thr He Ser Cys Ser Gly

Ser Ser Ser Asn He Gly Ser Asn Thr Val Asn Trp Tyr Gin Gin Leu Pro Gly Thr Ala Pro Lys Leu Leu He Tyr Ser Asn Asn Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala He Ser Gly Leu Gin Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1620)

Heavy Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

Ala Ser Gly Phe Thr Phe Ser Arg Tyr Trp Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Asn He Lys His Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Ser Asn Trp Gly Phe Ala Phe Asp Val Trp Gly His Gly Thr Met Val Thr Val Ser Ser (SEQ ID NO:1621)

1A4 Light Gin Ser Val Leu Thr Gin Pro Pro Ser Ala Ser Gly Thr Pro Gly Gin Arg Val Thr He Ser Cys Ser Gly

Ser Ser Ser Asn He Gly Ser Asn Thr Val Asn Trp Tyr Gin Gin Leu Pro Gly Thr Ala Pro Lys Leu Leu He Tyr Ser Asn Asn Gin Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala He Ser Gly Leu Gin Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Val Trp Asp Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1622)

Heavy Gin Val Gin Leu Gin Gin Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala

Val Tyr Gly Gly Ser Phe Ser Ala Tyr Tyr Trp Asn Trp He Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp He Gly Glu He Asn His Ser Gly Arg Thr Asp Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Lys Gin Phe Ser Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gin Leu Val Pro Phe Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO: 1623)

A12 Light Gin Ser Val Leu Thr Gin Pro Pro Ser Ala Ser Gly Thr Pro Gly Gin Arg Val Thr He Ser Cys Ser Gly

Ser Ser Ser Asn He Gly Ser Lys Thr Val Asn Trp Tyr Gin Gin Phe Pro Gly Thr Ala Pro Lys Leu Leu He Tyr Ser Asn Asn Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala He Ser Gly Leu Gin Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn Trp Val Phe Gly Ala Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1624)

Heavy Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

Ala Ser Gly Leu Thr Phe Ser Asn Phe Trp Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Asn He Lys Gin Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Ser Cys Thr Arg Glu Ser Asn Trp Gly Phe Ala Phe Asp He Trp Gly Gin Gly Thr Met Val Thr Val Ser Ser (SEQ ID NO:1625)

6F12 Light Gin Ser Val Leu Thr Gin Pro Pro Ser Val Ser Ala Ala Pro Gly Gin Lys Val Thr He Ser Cys Ser Gly

Ser Ser Ser Asn He Gly Asn Asn Phe Val Ser Trp Tyr Gin Gin Leu Pro Gly Thr Ala Pro Lys Leu Leu He Tyr Asp Tyr Asn Lys Arg Pro Ser Gly He Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly He Thr Gly Leu Gin Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu Ser Ala Tyr Val Phe Gly Thr Gly Thr Arg Val Thr Val Leu (SEQ ID NO:1626)

Heavy Gin Val His Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

Ala Ser Gly Phe Thr Phe Asn Ser Phe Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu He Trp Ser Asp Gly Ser Asp Glu Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala He Ala Ala Leu Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:1627) E2 Light Gin Ser Val Leu Thr Gin Pro Pro Ser Val Ser Ala Ala Pro Gly Gin Lys Val Thr lie Ser Cys Ser Gly Ser Ser Ser Asn He Gly Asn Asn Phe Val Ser Tip Tyr Gin Gin Leu Pro Gly Thr Ala Pro Lys Leu Leu He Tyr Asp Tyr Asn Lys Arg Pro Ser Gly He Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly He Thr Gly Leu Gin Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Tip Asp Ser Ser Leu Ser Gly Tyr Val Phe Gly Thr Gly Thr Arg Val Thr Val Leu (SEQ ID NO:1628)

Heavy Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met His Tip Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Tip Val Ala Leu He Tip Asn Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala He Ala Ala Leu Tyr Tyr Tyr Tyr Gly Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:1629)

A6 Light Gin Ser Val Leu Thr Gin Pro Pro Ser Val Ser Ala Ala Pro Gly Gin Lys Val Thr He Ser Cys Ser Gly

Ser Ser Ser Asn He Gly Asn Asn Phe Val Ser Tip Tyr Gin Gin Phe Pro Gly Thr Ala Pro Lys Leu Leu He Tyr Asp Tyr Asn Lys Arg Pro Ser Gly He Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly He Thr Gly Leu Gin Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Tip Asp Ser Ser Leu Ser Ser Tyr Val Phe Gly Thr Gly Thr Arg Val Thr Val Leu (SEQ ID NO:1630)

Heavy Gin Val His Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

Ala Ser Gly Phe Thr Phe Asn Ser Phe Gly Met His Tip Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Tip Val Ala Leu He Tip Ser Asp Gly Ser Asp Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala He Ala Ala Leu Tyr Tyr Tyr Tyr Gly Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 1631)

B12 Light Gin Ser Val Leu Thr Gin Pro Pro Ser Val Ser Ala Ala Pro Gly Gin Lys Val Thr He Ser Cys Ser Gly

Ser Ser Ser Asn He Gly Asn Asn Phe Val Ser Tip Tyr Gin Gin Leu Pro Gly Thr Ala Pro Lys Leu Leu He Tyr Asp Tyr Asn Lys Arg Pro Ser Gly He Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly He Thr Gly Leu Gin Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Tip Asp Ser Ser Leu Ser Gly Tyr Val Phe Gly Thr Gly Thr Arg Val Thr Val Leu (SEQ ID NO:1632)

Heavy Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met His Tip Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Tip Val Ala Leu He Tip Asn Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala He Ala Ala Leu Tyr Tyr Tyr Tyr Gly Met Asp Val Tip Gly His Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:1633)

D6 Light Gin Ser Val Leu Thr Gin Pro Pro Thr Val Ser Ala Ala Pro Gly Gin Lys Val Thr He Ser Cys Ser Gly

Ser Ser Ser Asn He Gly Asn Asn Phe Val Ser Tip Tyr Gin Gin Leu Pro Gly Thr Ala Pro Lys Leu Leu He Tyr Asp Tyr Asn Lys Arg Pro Ser Gly He Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly He Thr Gly Leu Gin Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Tip Asp Ser Ser Leu Ser Gly Tyr Val Phe Gly Thr Gly Thr Arg Val Thr Val Leu (SEQ ID NO:1634)

Heavy Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met His Tip Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Tip Val Ala Leu He Tip Asn Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala He Ala Ala Leu Tyr Tyr Tyr Tyr Gly Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:1635)

G11 Light Gin Ser Val Leu Thr Gin Pro Pro Ser Val Ser Ala Ala Pro Gly Gin Lys Val Thr He Ser Cys Ser Gly

Ser Ser Ser Asn He Gly Asn Asn Phe Val Ser Tip Tyr Gin Gin Leu Pro Gly Thr Ala Pro Lys Leu Leu He Tyr Asp Ser Asn Lys Arg Pro Ser Gly He Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Asp He Thr Gly Leu Gin Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Tip Asp Ser Ser Leu Ser Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu (SEQ ID NO:1636)

Heavy Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His Tip Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Tip Val Ala He He Tip Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Gly Gly Leu Ala Ala Arg Pro Gly Gly Met Asp Val Tip Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:1637)

B5 Light Gin Ser Val Leu Thr Gin Pro Pro Ser Val Ser Ala Ala Pro Gly Gin Lys Val Thr He Ser Cys Ser Gly

Ser Asn Ser Asn He Gly Asn Asn Tyr Val Ser Tip Tyr Gin Gin Leu Pro Gly Thr Ala Pro Lys Leu Leu He Tyr Asp Asn Asn Lys Arg Pro Ser Gly He Pro Asp Arg Phe Ser Gly Ser Asn Ser Gly Thr Ser Ala Thr Leu Gly He Thr Gly Leu Gin Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Tip Asp Ser Ser Leu Ser Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1638)

Heavy Glu Val Gin Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Tip Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Tip Val Ser Thr He Ser Gly Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Glu Val Gly Ser Pro Phe Asp Tyr Tip Gly Gin Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO: 1639)

1B12 Light Ser Tyr Glu Leu Thr Gin Pro Pro Ser Val Ser Val Ser Pro Gly Gin Thr Ala Arg lie Thr Cys Ser Gly

Asp Lys Leu Gly Asp Lys Tyr Ala Cys Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Val Leu Val He Tyr Gin Asn Thr Lys Trp Pro Leu Gly He Pro Glu Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Val Thr Leu Thr He Ser Gly Thr Gin Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gin Ala Trp Asp Ser Ser Thr Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO:1640)

Heavy Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala

Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ala He He Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Gly Gly Leu Ala Ala Arg Pro Gly Gly Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:1641)

B6 Light Gin Pro Val Leu Thr Gin Pro Leu Phe Ala Ser Ala Ser Leu Gly Ala Ser Val Thr Leu Thr Cys Tin- Leu Ser Ser Gly Tyr Ser Ser Tyr Glu Val Asp Trp Tyr Gin Gin Arg Pro Gly Lys Gly Pro Arg Phe Val Met Arg Val Asp Thr Gly Gly He Val Gly Ser Lys Gly Glu Gly He Pro Asp Arg Phe Ser Val Leu Gly Ser Gly Leu Asn Arg Tyr Leu Thr He Lys Asn He Gin Glu Glu Asp Glu Ser Asp Tyr His Cys Gly Ala Asp His Gly Ser Gly Thr Asn Phe Val Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu (SEQ ID NO: 1642)

Heavy Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys

Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Gly He Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met Gly Trp He Ser Thr Tyr Asn Gly Asn Thr Asn Tyr Ala Gin Lys Val Gin Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Thr Arg Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO: 1643)

Claims

1. A method of lowering serum cholesterol in a subject, said method comprising: administering to said subject an effective amount of: an antigen binding protein that selectively binds to and inhibits a PCSK9 protein; and an RNA effector agent which inhibits the expression of a PCSK9 gene in a cell, wherein administration of said antigen binding protein and said RNA effector agent lowers serum cholesterol levels in said subject.

2. The method of claim 1, wherein the antigen binding protein is selected from the group consisting of 2 IB 12, 31H4, and 3C4 and wherein the RNA effector agent is a dsRNA comprising an antisense strand which consists of the nucleotide sequence of SEQ ID NO: 1228 and a substantially complementary sense strand thereof.

3. A method for treating or preventing a condition associated with an elevated serum

cholesterol level in a subject, comprising administering to said subject in need thereof an effective amount of an antigen binding protein that selectively binds and inhibits a PCSK9 protein, and an RNA effector agent which inhibits the expression of a PCSK9 gene in a cell, wherein administration of said antigen binding protein and said RNA effector agent lowers serum cholesterol levels in said subject.

4. A method for treating or preventing a condition associated with an elevated serum

cholesterol level in a subject, comprising administering to said subject in need thereof an effective amount of an antigen binding protein that selectively binds to and inhibits a PCSK9 protein, an RNA effector agent which inhibits the expression of a human PCSK9 gene in a cell and a chemical agent that elevates the availability of LDLR protein, thereby lowering serum cholesterol levels in said subject.

5. The method of claim 4, wherein said chemical agent is a statin.

6. The method of claim 5, wherein the statin is selected from the group consisting of

atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, and some combination thereof.

7. The method of any one of claims 1-6, wherein said antigen binding protein binds to

PCSK9 with a Kd that is less than ΙΟΟρΜ.

8. The method of claim 7, wherein said antigen binding protein binds to PCSK9 with a Kd that is less than ΙΟρΜ.

9. The method of claim 8, wherein said antigen binding protein binds to PCSK9 with a Kd that is less than 5pM.

10. The method of any one of claims 1-9, wherein said PCSK9 protein comprises an amino acid sequence which is 90% or more identical to the amino acid sequence shown in Table 8.

11. The method of claim 10, wherein said PCSK9 protein comprises the amino acid sequence shown in Table 8.

12. The method of any one of claims 1-11, wherein said antigen binding protein is an

antibody.

13. The method of claim 12, wherein said antibody is a humanized antibody.

14. The method of claim 12, wherein said antibody is a human antibody.

15. The method of claim 12, wherein said antibody binds to an epitope within residues 31- 449 of the amino acid sequence shown in Table 8.

16. The method of claim 12, wherein said antibody is selected from the group consisting of 30A4, 3C4, 23B5, 25G4, 31H4, 27B2, 25A7, 27H5, 26H5, 31D1.20D10, 27E7, 30B9, 19H9, 26E10, 21B12, 17C2, 23G1, 13H1, 9C9, 9H6, 31A4, 1A12, 16F12, 22E2, 27A6, 28B12, 28D6, 31G11, 13B5, 31B12 and 3B6.

17. The method of claim 12, wherein said antibody is 21B12.

18. The method of claim 12, wherein said antibody is 31H4.

19. The method of claim 12, wherein said antibody is 3C4.

20. The method of any one of claims 1-19, wherein said R A effector agent is an siRNA selected from the group consisting of the siRNAs of Tables 1 and 2.

21. The method of any one of claims 1-19, wherein said RNA effector agent binds to

nucleotide residues 3530-3548 of the nucleotide sequence shown in Table 8.

22. The method of any one of claims 1-19, wherein said RNA effector agent binds to at least 15 contiguous nucleotides of nucleotide sequence in SEQ ID NO: 1523.

23. The method of any one of claims 1-22, wherein said RNA effector agent is a dsRNA

comprising a first sequence and a second sequence that are complementary to each other.

24. The method of claim 23, wherein said dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence having at least 15 contiguous nucleotides of SEQ ID NO: 1228.

25. The method of claim 24, wherein the second sequence comprises SEQ ID NO: 1228.

26. The method of claim 24, wherein the antisense strand consists of SEQ ID NO: 1228.

27. The method of claim 24, wherein the second sequence comprises SEQ ID NO: 1228 and wherein the first sequence comprises SEQ ID NO: 1227.

28. The method of claim 23, wherein said dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence, wherein the first sequence is selected from the group consisting of SEQ ID NO: 1227, SEQ ID NO: 1229, SEQ ID NO: 1231, SEQ ID NO: 1233, SEQ ID NO: 1235, SEQ ID NO: 1237, SEQ ID NO: 1239, SEQ ID NO: 1241, SEQ ID NO: 1243, SEQ ID NO: 1245, SEQ ID NO: 1247, SEQ ID NO: 1249, SEQ ID NO: 1251, SEQ ID NO: 1253, SEQ ID NO: 1255, and SEQ ID NO: 1257 and wherein the second sequence is selected from the group consisting of SEQ ID NO: 1228, SEQ ID NO: 1230, SEQ ID NO: 1232, SEQ ID NO: 1234, SEQ ID NO: 1236, SEQ ID NO: 1238, SEQ ID NO: 1240, SEQ ID NO: 1242, SEQ ID NO: 1244, SEQ ID NO: 1246, SEQ ID NO: 1248, SEQ ID NO: 1250, SEQ ID NO: 1252, SEQ ID NO: 1254, SEQ ID NO: 1256, and SEQ ID NO: 1258.

29. The method of any one of claims 1-28, wherein said RNA effector agent is administered in a delivery vehicle.

30. The method of claim 29, wherein the delivery vehicle is a vector which expresses said RNA effector agent.

31. The method of claim 29, wherein the delivery vehicle is a lipid formulation.

32. The method of any one of claims 23-27, wherein said dsRNA comprises at least one

modified nucleotide.

33. The method of claim 32, wherein said, modified nucleotide is chosen from the group of: a 2'-0-methyl modified nucleotide, a nucleotide comprising a S'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative of dodecanoic acid bisdecylamide group.

34. The method of claim 32, wherein said modified nucleotide is chosen from the group of: a 2'-0-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.

35. The method of any one of claims 3-34, wherein said condition is hypercholesterolemia, atherosclerosis, or dyslipidemia.

36. The method of any one of claims 1-35, wherein said antigen binding agent and said RNA effector agent are administered concurrently.

37. The method of any one of claims 1-35, wherein said antigen binding agent and said RNA effector agent are administered separately.

38. The method of any one of claims 1-37, wherein said RNA effector agent inhibits PCSK9 gene expression by at least 20% or by at least 80%.

39. The method of any one of claims 1-38, wherein said RNA effector agent lowers serum LDL cholesterol in said subject by at least 20%.

40. A method for lowering serum cholesterol levels in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an antibody selected from the group consisting of 21B12, 31H4 and 3C4 and a dsRNA comprising an antisense strand which consists of the nucleotide sequence of SEQ ID NO: 1230 and a substantially complementary sense strand thereof, wherein said administration of said antibody and said dsRNA lowers serum cholesterol levels in said subject.

41. A pharmaceutical composition for reducing serum cholesterol levels in a subject, said pharmaceutical composition comprising an effective amount of an antigen binding protein that selectively binds to and inhibits a PCSK9 protein, an RNA effector agent which inhibits the expression of a PCSK9 gene in a cell, and a pharmaceutically acceptable carrier.

42. The pharmaceutical composition of claim 41, wherein said antigen binding protein is an antibody selected from the group consisting of 21B12, 31H4 and 3C4, and said RNA effector agent is a dsRNA comprising a sense strand which consists of the nucleotide sequence of SEQ ID NO: 1229 and an antisense strand which consists of the nucleotide sequence of SEQ ID NO: 1230.

43. The pharmaceutical composition of claim 41, wherein said pharmaceutically acceptable carrier comprises a SNALP lipid formulation, a XTC lipid, a LNP01 lipid formulation, a MC3 lipid, a Lipid Formula A lipid, and/or a ALNY100 lipid.

44. A pharmaceutical kit for treating or preventing a condition associated with an elevated serum cholesterol level in a subject comprising, an antigen binding protein that binds to and inhibits a PCSK9 protein, an RNA effector agent which inhibits the expression of a human PCSK9 gene in a cell and a label or packaging insert containing instructions for use.

45. The pharmaceutical kit of claim 44, wherein said antigen binding protein and said RNA effector agent are contained in separate intravenous pharmaceutical dosage forms.

46. The pharmaceutical kit of claim 44 or 45, wherein the antigen binding protein is an antibody selected from the group consisting of 21B12, 31H4 and 3C4, and said RNA effector agent is a dsRNA comprising a sense strand which consists of the nucleotide sequence of SEQ IDNO: 1229 and an antisense strand which consists of the nucleotide sequence of SEQ ID NO: 1230.